What if researchers could survey bat infections without drawing blood from the bats themselves? This study shows how the bats’ own tiny, blood-feeding flies can reveal widespread and genetically diverse Polychromophilus parasites. Polychromophilus is the only one of nine haemosporidian genera that infect bats with a global distribution that also includes temperate zones.

Of the five described Polychromophilus species, Polychromophilus melanipherus is mainly found in miniopterid bats, while Polychromophilus murinus primarily infects vespertilionid bats. Both species have also been reported in other bat families, including rhinolophids, but overall patterns of host specificity and exposure remain poorly understood.

The study examined Polychromophilus infections in multiple species of nycteribiid bat flies collected from Miniopterus schreibersii and Rhinolophus ferrumequinum in Serbia and Bosnia and Herzegovina. Its aim was to characterize the prevalence and genetic diversity of Polychromophilus infections in this system.

Molecular screening of flies provides an efficient way to quantify the opportunity and frequency of parasite transmission without more invasive blood sampling in bats. The study defined spillover as infection of a fly with the Polychromophilus species not normally associated with the bat host from which that fly was collected.

Figure one maps the nine sampling sites across Serbia and Bosnia and Herzegovina, distinguishing sites used by Miniopterus schreibersii, Rhinolophus ferrumequinum, or both species. The inset photographs identify the two bats and four bat-fly taxa examined.

This geographic context matters because the subsequent parasite-DNA screening of entire fly specimens is tied to these specific roost locations and host associations. Nine roosting sites were sampled: eight in Serbia and one in Bosnia and Herzegovina.

Four sites were shared by Miniopterus schreibersii and Rhinolophus ferrumequinum, four were used only by Miniopterus schreibersii, and one only by Rhinolophus ferrumequinum. Sampling took place during summer and autumn in 2017 and 2018, and captured bats were released immediately after processing.

Bat flies were collected with fine-toothed forceps, stored individually in ninety-nine percent ethanol, and identified to species level both morphologically and genetically. A total of 215 bat flies were examined for Polychromophilus parasite DNA: 150 from Miniopterus schreibersii and 65 from Rhinolophus ferrumequinum.

Sequences without ambiguous bases were used to construct median-joining haplotype networks for cytb, cox1, and a concatenation of both fragments. The study compared its haplotypes with all existing Polychromophilus melanipherus sequences in GenBank for both cytb and cox1.

The sequences were trimmed to improve overlap with existing sequences: cytb included 479 base pairs from 119 sequences, and cox1 included 768 base pairs from 51 sequences. Polychromophilus DNA was detected in 33 of the 215 screened bat flies, or fifteen percent, including individuals of all four bat fly species and both bat host species.

Infections were recorded at six of the nine sampling locations. Overall prevalence was low, with fewer than five infections per site, except at Dardagani, where 17 of 20 bat flies were infected. Nearly all infections were detected in flies collected from Miniopterus schreibersii, with only one infection detected in a fly collected from Rhinolophus ferrumequinum.

Table one maps Polychromophilus DNA-positive bat flies across nine sampling sites, separating results by fly species and host species while also reporting mixed infections and overall prevalence. In total, thirty-three of two hundred fifteen flies were positive, spanning all four fly species and both hosts, with infections at six sites.

Dardagani stands out in the table with seventeen of twenty positive flies, while the species totals show twenty-one of one hundred thirty-three Nycteribia schmidlii and eight of fourteen Penicillidia conspicua positive. For cytb, 13 samples could be unambiguously aligned, yielding four haplotypes.

For cox1, five haplotypes were found across 17 samples without ambiguous sites. The topology of both networks was identical except for the additional fifth haplotype in cox1. The concatenated dataset, using samples with both unambiguous cytb and cox1 sequences, contained four haplotypes across 11 of the 33 positive samples.

Figure two shows haplotype networks for Polychromophilus melanipherus using cytb sequences in panel a and cox1 sequences in panel b. Each circle is a haplotype, colored by the bat fly species, and connecting lines represent single-base changes unless labeled otherwise; the network includes four cytb haplotypes and five cox1 haplotypes.

The nearly identical topology across markers, together with the lack of consistent clustering by bat fly species or location, supports the authors’ conclusion that these parasite haplotypes are broadly shared rather than strongly structured. Figure three shows haplotype networks for Polychromophilus melanipherus using cytb on the left and cox1 on the right.

Each labeled circle is a haplotype, colors indicate geographic origins, and connecting lines represent single-base changes unless a number in parentheses indicates additional changes. The authors found no geographic structuring and no consistent clustering by bat-fly species, suggesting that the same haplotypes are distributed across locations and hosts.

Polychromophilus melanipherus was detected in fifteen percent of the 215 bat flies examined, and positives were found in all four sampled fly species. All but one positive detection came from bat flies collected from Miniopterus schreibersii, and infections were present at six of the nine sampling sites.

Sequencing showed that nearly half of the positive samples, 15 of 33, represented mixed infections with multiple Polychromophilus melanipherus haplotypes. The results suggest that Miniopterus schreibersii bats and their associated nycteribiid flies are frequent hosts of Polychromophilus melanipherus in Serbia and Bosnia and Herzegovina.

The results provide new insights into the prevalence, distribution, and genetic diversity of Polychromophilus parasites in European bats and their nycteribiid vectors. The study reports a single case of spillover of Polychromophilus melanipherus infection in a Phthiridium biarticulatum fly collected from a Rhinolophus ferrumequinum host.

Using bat flies for the non-invasive study of Polychromophilus infections proved very efficient and represents an alternative for large-scale investigations without invasively collecting blood from bats. Bat flies provided an efficient, non-invasive window into Polychromophilus infections: most positives came from Miniopterus schreibersii, but one fly from Rhinolophus ferrumequinum revealed spillover, showing why this approach matters for large-scale surveillance.