Finding the Right Host in the Darkness of the Cave—New Insights into the Ecology and Spatio-temporal Dynamics of Hyperparasitic Fungi (Arthrorhynchus nycteribiae, Laboulbeniales)

Abstract

The aim of this study was to determine the presence of the hyperparasitic fungus Arthrorhynchus nycteribiae and to analyze its spatio-temporal pattern in the two bat flies (Penicillidia conspicua and P. dufourii) parasitizing on bats. We collected 612 samples of bat flies from 400 bats in 20 caves in the Central Balkans. Hyperparasite was identified based on morphological and molecular analyses of rDNA genes (LSU and SSU). A. nycteribiae was reported for the first time in Bosnia and Herzegovina and Montenegro, and confirmed in Serbia. Of the 20 sites examined, we found A. nycteribiae at 11 sites. The prevalence of A. nycteribiae infection in the bats examined was approximately 17%. Miniopterus schreibersii harbored the highest number of bat flies and was the only bat species hosting the infected bat flies of the species P. conspicua. Our results showed significant differences in infection patterns during the different seasons: the highest prevalence of bat flies with hyperparasitic fungi was found in the summer season (23%) and the lowest in spring (2%). Female bat fly hosts showed a significantly higher prevalence of infection than male bat flies. This study makes an important contribution to the knowledge of the distribution of A. nycteribiae and to the understanding of complex parasite-host relationships in the poorly studied areas of the Central Balkans.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00248-025-02521-4.

Introduction

Bats (Chiroptera) are the second largest group of mammals, widely distributed in various habitats, with over 1400 described species [1]. Their wide distribution is facilitated by numerous adaptations, such as the ability to fly and a primarily nocturnal lifestyle [2]. Different bat species use various structures for shelter (caves, crevices, human structures, trees), where they can live solitarily or form some of the largest mammalian colonies in the world [3]. Bats are regularly parasitized by a number of ectoparasites [2], including bat flies [4, 5].

Bat flies (Diptera: Nycteribiidae, Streblidae, and Mystacinobiidae) are characteristic bat ectoparasites from the class Insecta [6]. The family Nycteribiidae consists of wingless flies whose adult forms live on bats and feed on their blood [6]. The only time an adult leaves its host is when the female is looking for a suitable place to lay her larvae [7]. The first three larval stages (from the egg to the third larval stage) take place inside the female. The female leaves the host when the larva has reached the third stage and looks for a suitable place in the cave substrate or on the cave walls to lay her larvae. After birth, the larvae immediately enter the pupal stage. The time an individual spends in this stage varies from species to species. Usually, an adult emerges from the pupa after 3–4 weeks and immediately seeks out a host [8]. A female can produce 15 larvae over a period of 3 months [7]. The family Nycteribiidae includes around 275 species that are widely distributed, although the members of this family are more numerous in the eastern hemisphere [9].

The Laboulbeniomycetes is the class of perithecial fungi within Ascomycota division and Pezizomycotina subdivision that encompasses micromycetes obligatorily associated with arthropods either for dispersal (order Pyxidiophorales) or as biotrophs (orders Laboulbeniales and Herpomycetales) [10]. Laboulbeniales (beetle hangers) are a microscopically small, highly specialized group of fungi, obligate ectoparasites of three subphyla within the clade Mandibulata of Arthropoda phylum: Chelicerata (parasiting only members of the class Acariformes), Myriapoda, and Hexapoda [11]. Laboulbeniales are very specific in their choice of hosts and spend their entire life cycle on an arthropod host [12]. These fungi are notable for their (1) obligate biotrophic nature; (2) strictly determinate development from a two-celled ascospore into a complex thallus; (3) bilateral symmetry; and (4) absence of germ tubes, hyphae, and conidia [13]. The thallus consists of three parts: a receptacle that attaches to the host, a structure that bears spores—perithecia, and antheridia that produce sperm. Taxonomic characters of Laboulbeniales are based on the architecture of the cells of the parenchymal thallus, i.e., the visible part of the fungus outside the host [12]. These fungi produce ascospores, which are formed in the perithecium and released by pressure, typically triggered by the contact between hosts [14]. About 80% of described species of Laboulbeniales have been found on species of the order Coleoptera and only 10% on Diptera. Laboulbeniales that use Diptera as their host are divided into eight genera, where three genera are exclusive ectoparasites of wingless flies: Arthrorhynchus, Gloeandromyces, and Nycteromyces. Two Arthrorhynchus species are associated with nycteribiid flies in Europe [11].

The presence and complex relationship of hyperparasitic fungi–bat fly (primary host)–bat (secondary host), including Arthrorhynchus species, has been studied by several authors in Europe [10, 11, 15–19]. However, the area of the Central Balkans has been only recently studied in a systematic manner from the point of view of bat ectoparasites and their associations with their hosts [4, 20, 21]. According to the available literature, there are only a few records of the species A. nycteribiae recorded for Serbia, with one record from 1857 and a few records from the 1970 s [11]. To the best of our knowledge, no studies have been conducted since then to confirm the presence and ecology of this fungus species in the region of the Central Balkans. Pejić et al. [22] report only the presence of the genus Arthrorhynchus on its known host, Penicillidia conspicua, but without any details.

The aim of this study was (1) to determine the presence of the hyperparasitic fungus Arthrorhynchus nycteribiae using rDNA barcoding and (2) to analyze the spatio-temporal pattern of occurrence of this fungus in the two bat flies species Penicillidia conspicua and P. dufourii parasitizing different bat species in the Central Balkans.

Materials and Methods

Collection of Bat Flies

The fieldwork was conducted in 2013–2015 during the period of activity of bats (April–October), with extensive study being suspended in late May through June during the period of advanced pregnancy, delivery, and the first phase of nursing the young bats. We examined bat flies collected from bats at 20 localities (Fig. 1) in Serbia, Montenegro, and Bosnia and Herzegovina. Bats were caught using mist nets or harp traps placed at the entrances of the roost. Captured bats were placed separately in clean cotton bags to avoid cross-contamination. Each individual bat was identified to the species level [23]. The number of individual bats examined per species at each locality is given in Supplementary Table 1.

Fig. 1.

Map with localities (Legend: 1. Bogova vrata cave, 2. Bogovinska cave, 3. Canetova cave, 4. Degurićka cave, 5. Dubočka cave, 6. Dudićeva cave, 7. Hadži Prodanova cave, 8. Kađenica cave, 9. Lazareva river, 10. Ogorelička cave, 11. Petnička cave, 12. Petrlaška cave, 13. Ravanička cave, 14. Šalitrena cave, 15. Sesalačka cave, 16. Sokolovica cave, 17. Grbavci cave, 18. Magara cave, 19. Vilina cave, 20. Sokolačka cave)

The entire body of each bat host was carefully visually inspected for the presence of bat flies (Insecta; Diptera: Nycteribiidae, Streblidae). All bat flies found were removed with tweezers and placed in tubes with 70% ethanol and labeled appropriately (locality, date, bat specimen code). All bats were released at the same place where they were caught, immediately after checking for the presence of ectoparasites. All bat flies were identified, based on morphology, to the species level [24–26]. All identifications were made using a stereo microscope (Zeiss SteREO Discovery V8). Voucher specimens of all bat fly species are deposited in the collections of the Institute of Zoology at the Faculty of Biology, University of Belgrade.

Morphological Identification of Fungi

Bat flies were screened for the presence of ectoparasitic fungi. The position and density of thalli were examined using a stereomicroscope Nikon SMZ 745 T equipped with Dual Sight 1000 camera. After examination, thalli were removed from the host at the point of attachment (foot or haustorium) with an entomological pin and slide-mounted with glycerol. For detailed microscopy analyses of thalli structures, light microscope Zeiss Axio Imager M.1 was used. Laboulbeniaceae monograph by Thaxter [27] was used for preliminary identification.

Molecular Identification and Phylogenetic Analysis of Fungi

Fungal thalli were carefully removed from heavily infected insects under a stereomicroscope using an entomological pin and tweezers. Thalli were transferred with a micropipette to a bashing bead tube which is supplied in the DNA extraction kit. Fungal DNA was extracted using Quick-DNA Fungal/Bacterial Miniprep Kit (ZYMO RESEARCH USA, Irvine, CA, USA). Primer combinations for PCR amplification of small (SSU) and large subunits of nuclear rRNA genes (LSU) are given in Supplementary Table 2 (with references [28–31]).

PCR amplification was performed as follows: denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 1 min, 50 °C for 45 s, and 72 °C for 90 min, and a final extension step of 72 °C for 10 min [13]. The resulting PCR products were separated by electrophoresis and purified (Purification Kit, Qiagen, USA) and then sent for sequencing (Macrogene, Netherlands). Sequences were compared with other related sequences from the NCBI database using the BLAST algorithm (BLAST + 2.7.1 of the NCBI) for parasite identification. Obtained sequences of Laboulbeniales were deposited to NCBI GenBank (Supplementary Table 3). A multilocus (SSU and LSU) phylogenetic tree was constructed using MEGA11 software [32]. The alignment of our sequences with sequences obtained from the NCBI database was done using the ClustalW algorithm, and the phylogenetic tree was built using a neighbor-joining phylogeny model (1000 bootstrap replicas). Kimura 2 parameter model was determined as the best for estimating genetic distances between tested sequences.

Data Analysis

To determine whether there are differences in the occurrence of fungal infections between different bat species and sexes (bat flies hosts), locations, and seasons, we used a non-parametric similarity analysis (ANOSIM using Euclidean similarity with N = 9999 permutations) with pairwise comparisons based on a step-down sequential Bonferroni procedure. Variations in the presence of infected bat flies were compared using a repeated measure permutational multivariate analysis of variance (PERMANOVA with N = 9999 permutations of the Euclidean distance matrix).

The data were further analyzed using multivariate statistical analysis. Following the suggestion of Lepš and Šmilauer [33], we chose linear redundancy analysis (RDA), a multivariate direct gradient analysis [34]. The analysis was performed using correlation matrices, and the resulting RDA ordination plot is presented as a biplot with the following symbolism: (1) bat flies species are represented as arrows (four elements for two species and two sexes), and (2) the localities, bat hosts, and the presence of fungal infection are represented as environmental classes (yellow circle symbols for bat hosts, smaller blue and orange triangles for each locality, and larger orange/blue triangles as samples with or without fungal infection). Using the bat fly species abundance data and the recorded environmental variables, this analysis helped to identify the patterns that determine the distribution of the recorded species. Ordination analysis was performed using CANOCO 5.15 [35]. The significance of the two canonical axes was tested using a permutation test. Additional post hoc tests (ANOSIM with 9999 permutations with pairwise comparisons based on a step-down sequential Bonferroni procedure and PERMANOVA with 9999 permutations of the Euclidean distance matrix) were performed on the scores of the two significant RDA axes of the qualitative variables to confirm the trends observed in the visual interpretation of the resulting ordinations. All tests were performed using the software program PAST (Paleontological Statistics software program, Ver. 4.08) [36]. For all analyses, p < 0.05 was set as the significance level.

Results

Morphological Identification

Fungal infection was documented only on Penicillidia conspicua individuals sampled from Miniopterus schreibersii from 11 investigated sites in the Central Balkans. Both male and female individuals were infected with “beetle hangers.” All body parts of P. conspicua showed the symptoms of fungal infection in the form of thallus growth directly on bat fly cuticles. However, most of the thalli were detected on the dorsal and ventral parts of the abdomen (Fig. 2).

Fig. 2.

Penicillidia conspicua individuals infected with Arthrorhynchus nycteribiae collected from Miniopterus schreibersii. Thalli on the ventral side of male individuals (a–d); female individual with ventrally (e) and dorsally (f) positioned thalli. The thalli are marked with an arrow

Based on morphological criteria, the bat fly ectoparasite was identified as A. nycteribiae (Fig. 3a). Elongated cell III, which carries the appendage (Fig. 3c), and the perithecial tip with four lobes, each with three conspicuous smaller lobes (Fig. 3b), are the main distinctive characteristics that separate A. nycteribiae from other species within the genus (A. eucampsipodae, A. cyclopodiae, and A. acrandros).

Fig. 3.

Arthrorhynchus nycteribiae, a single thallus; rc, receptaculum; c3, cell III; tg, trichogyne; pe, perithecia; pt, perithecial tip. b Detail of perithecial tip. c Detail of the basal part of the thallus. Scale bar 20 µm

Molecular Identification and Phylogenetic Analysis

Identification based on molecular criteria using SSU and LSU gene markers confirmed morphological identification (GenBank accession numbers: LSU, PQ041203 and SSU, PQ041201 for BEOFB0970000; LSU, PQ047148 and SSU, PQ041200 for BEOFB0970001). The neighbor-joining multilocus cladogram of the isolates is shown in Fig. 4. Our isolates are grouped together with A. nycteribiae in a well-supported clade. Species of the genera Arthrorhynchus, Hesperomyces, Prolixandromyces, Nycteromyces, Rickia, Fanniomyces, and Gloeandromyces are also grouped together in a separate clade (Laboulbeniales), compared to the species of the genus Herpomyces (Herpomycetales).

Fig. 4.

Multilocus (SSU and LSU) neighbor-joining tree of the Laboulbeniomycetes isolates

Spatio-temporal Dynamics of Arthrorhynchus nycteribiae and Their Hosts

Our dataset of examined bat flies consisted of 612 specimens belonging to two species: Penicillidia conspicua (Pcon, 455) and P. dufourii (Pduf, 157). The bat flies were collected from 400 bat individuals belonging to four species: M. schreibersii, Myotis capaccinii, Myotis myotis, and Myotis blythii. Of the total number of bats examined, only six individuals did not have a single ectoparasite fly on them, all of which belonged to the species Miniopterus schreibersii. On the other hand, M. schreibersii hosted the most bat flies (531, on 343 bats with 349 bats sampled), followed by M. capaccinii (33, on 23 bats with 23 bats sampled), M. myotis (24, on 16 bats with 16 bats sampled), and M. blythii (24, on 12 bats with 12 bats sampled). A detailed overview of the number of bats examined and bat flies detected is shown in Table 1. The number of bat flies per species and per host species at each locality is given in Supplementary Table 4.

Table 1.

A detailed overview of the number of bats examined and bat flies detected

Bat species	Number of bats	Number of bats with bat flies	Number of bat flies	Average number of bat flies per bat species	Total number of bat flies per species
					Penicillidia conspicua	Penicillidia dufourii
Miniopterus schreibersii	349	343	531	1.55	452	79
Myotis blythii	12	12	24	2	2	22
Myotis capaccinii	23	23	33	1.43	1	32
Myotis myotis	16	16	24	1.5	0	24
Total	400	394	612	1.55	455	157

Of the total of 612 bat flies, we found 78 bat flies infected with A. nycteribiae, which corresponds to a prevalence of 13%. All 78 bat flies infected with the fungi belonged to a single species, P. conspicua, all of which were collected from a single bat host species, M. schreibersii. The prevalence of A. nycteribiae infection in M. schreibersii was 17.25%. However, in two other bat species—M. blythii and M. capaccinii—three uninfected specimens of P. conspicua were found, so that the overall prevalence of A. nycteribiae infection in P. conspicua was 17.14% in all bat species studied. Out of 20 examined localities, we found A. nycteribiae at 11 localities in the Central Balkans (Table 2). ANOSIM found a weak significant difference in the presence of hyperparasitic fungi at different localities (R = 0.04, p = 0.02). However, PERMANOVA analysis supported these results, finding a significant effect of location (F = 3.32, p = 0.00).

Table 2.

Localities with Arthrorhynchus nycteribiae presence and its prevalence on total bat flies collected per locality. The map with the geographical location of the localities and the presence of A. nycteribiae can be found in Supplementary Fig. 1

Name of the locality	Infected bat flies	Uninfected bat flies	Total bat flies	Prevalence
Serbia
Bogova vrata cave (1)	0	6	6	0%
Bogovinska cave (2)	6	17	23	26.09%
Canetova cave (3)	12	39	51	23.53%
Degurićka cave (4)	1	81	82	1.22%
Dubočka cave (5)	0	9	9	0%
Dudićeva cave (6)	3	12	15	20%
Hadži Prodanova cave (7)	0	12	12	0%
Kađenica cave (8)	0	6	6	0%
Lazareva river (9)	0	2	2	0%
Ogorelička cave (10)	0	9	9	0%
Petnička cave (11)	1	18	19	5.26%
Petrlaška cave (12)	0	2	2	0%
Ravanička cave (13)	20	82	102	19.61%
Šalitrena cave (14)	11	150	161	6.83%
Sesalačka cave (15)	10	51	61	16.39%
Sokolovica cave (16)	12	8	20	60%
Montenegro
Grbavci cave (17)	1	13	14	7.14%
Magara cave (18)	0	3	3	0%
Vilina cave (19)	0	7	7	0%
Bosnia and Herzegovina
Sokolačka cave (20)	1	7	8	12.50%
Total number/average prevalence	78	533	612	12.75%

The highest prevalence of bat flies with hyperparasitic fungi in the sample examined was found in summer (23%), slightly lower in autumn (11%), and the lowest in spring (2%) (Fig. 5). ANOSIM identified significant differences in the presence of hyperparasitic fungi in bat flies in the different seasons (R = 0.03, p = 0.00). The PERMANOVA analysis supported these results, finding a significant effect of season (F = 13.58, p = 0.00).

Fig. 5.

The proportion of infected and uninfected bat flies by season (P. con, P. conspicua; P. duf., P. dufourii)

Although female bat hosts had a slightly higher prevalence of infected bat flies compared to male bat hosts (13% and 12%, respectively), ANOSIM and PERMANOVA showed that there was no significant effect of bat hosts on infection patterns (R = − 0.05, p = 0.94; F = 1.95, p = 0.20, respectively). However, the results show that among the infected P. conspicua flies, females (PconF) were infected almost twice as often as males (PconM) (21% and 11%, respectively) (Fig. 6). ANOSIM identified these differences as significant (R = 0.15, p = 0.00), and PERMANOVA analysis confirmed these results (F = 29.56, p = 0.00).

Fig. 6.

The proportion of infected and uninfected P. conspicua flies by sex (PconM, P. conspicua males; PconF, P. conspicua females)

The ordination of the environmental factors and species on the space defining the first two canonical axes in the biplot of the redundancy analysis is shown in Fig. 7. The cumulative total proportion of the first two axes comprises 19.55% of the total variance (RDA 1, 17.83%; RDA 2, 1.72%). The RDA with the two extracted gradients was statistically significant (F = 4.5, p = 0.00). As observed, the bat species and localities are arranged such that the first RDA axis separates Miniopterus schreibersii from other bat species. ANOSIM detected significant differences in this pattern (R = 0.2, p = 0.00). Subsequent pairwise comparisons showed that Miniopterus schreibersii differed significantly from the other bat species. PERMANOVA analysis confirmed these results and a significant effect of bat host species (F = 44.19, p = 0.00). ANOSIM failed to detect significant differences in the scoring patterns for the presence of fungal infection (positive vs. negative samples) (R = − 0.05, p = 0.88), while the PERMANOVA analysis showed a significant effect of infection (F = 29.1, p = 0.00). In addition, the first axis separated the sites in the sense that the uninfected sites generally had higher ordination scores than the infected sites. However, ANOSIM and PERMANOVA revealed that this pattern was not significant (F = 3.05, p = 0.07; R = 0.13, p = 0.08, respectively). Furthermore, two bat fly species are arranged such that the first RDA axis separates P. dufourii with positive ordination scores from P. conspicua with negative ordination scores. These results suggest that the first, dominant gradient predicts both the position of the bat species in relation to the presence of infection and the position of the sites in the same context. The second RDA axis separates Myotis myotis from Myotis capaccinii and Myotis blythii, but ANOSIM and PERMANOVA revealed no significance (R = 0.02, p = 0.21; F = 2.20, p = 0.11, respectively). However, this pattern is most likely a consequence of the species-specific relationship between parasitic flies and their bat hosts. For instance, M. myotis was only parasitized by P. dufourii. In addition, the second RDA axis separates males and females of the two bat fly species. The length of the species arrow, together with the angle in relation to an axis, also indicates the relative contribution of that species to the axes shown in the triplot. The length of the arrow is also a measure of the dominance and contribution of the individual species to the extracted gradients. This is consistent with our findings of the largest number of positive findings in female P. conspicua flies.

Fig. 7.

Redundancy analysis biplot: bat species (yellow circles), localities positive for A. nycteribiae (orange down triangles), localities negative for A. nycteribiae (blue up triangles), bat flies species (arrows) ordination represented on a diagram defined by the first two canonical axes (bat flies species: PconM, P. conspicua males; PconF, P. conspicua females; PdufM, P. dufourii males; PdufF, P. dufourii females; bat species: Mcapa, Myotis capaccinii; Mblyt, Myotis blythii; Mmyot, Myotis myotis; Minschr, Miniopterus schreibersii)

Discussion

Morphological and Molecular Identification of Arthrorhynchus nycteribiae

Based on morphological analysis along with SSU/LSU barcoding, the ectoparasite of P. conspicua was identified as A. nycteribiae. BLAST analysis is based on very few ribosomal DNA sequences. Namely, there are only ten entries in the NCBI database regarding A. nycteribiae nucleotide sequences belonging to four different isolates (6 LSU, and 4 SSU; 6 from Hungary, and 4 from Bulgaria). All A. nycteribiae entries in the NCBI database are detected on P. conspicua as a host, and, as an isolation source, two bat species are listed: M. schreibersii (eight entries) and R. euryale (two entries). Conducting molecular analyses on Laboulbeniales is much more problematic compared to other groups of fungi. The most difficult steps in the molecular identification of beetle hangers are DNA extraction and PCR amplification [13]. Microscopic thalli no longer than 300 µm in length, strongly attached to the host’s exoskeleton, require micro-manipulation techniques and specific tools for detachment. Also, Laboulbeniales are not cultivable fungi, and the impossibility to grow them in axenic cultures is an obstacle to obtaining sufficient amounts of DNA for further analyses. The presence of melanin in the cell walls of Laboulbeniales interferes with PCR amplification by pigment affinity to bind to the DNA polymerase [37]. In research presented here, the detachment of numerous thalli from heavily infected female individuals of P. conspicua along with the usage of a specific DNA isolation kit containing bashing beads for cell disruption proved to be successful. PCR amplification was carried out as per the recommendations of Haelewaters et al. [13]. Although the correct identification of host species, such as P. conspicua in the case presented in this research, often facilitates the identification of ectoparasitic fungi based on published host-parasite lists [26, 38, 39], due to fortuitous infections, it is highly recommended to confirm pathogen identification by morphology and/or DNA sequence comparisons.

Host Specificity and Parasite Prevalence

Our results show that the ectoparasitic fungus A. nycteribiae was detected in a primary host species, P. conspicua, with an average prevalence of 17.14% in all bat species studied from the 20 sites in the Central Balkans. In our study, P. conspicua shows a high host specificity for its bat host, M. schreibersii, and is found almost exclusively on this bat host. In this study, P. conspicua was also found on two other bat species, M. blythii and M. capaccinii, which are not reported as main hosts in the literature. As the percentage is extremely low (< 1%), we assume that this is a random finding due to the high mobility of the flies within mixed colonies.

P. conspicua was previously identified as the main host for A. nycteribiae [11], and this is confirmed by the results of this study. Our results are somewhat similar to the results of Peter et al. [15], reporting their findings from the neighboring countries of Bulgaria and Romania. According to these authors, 33 of 142 (23.1%) of P. conspicua collected from M. schreibersii were infected by the same fungal species as in our study, A. nycteribiae. Peter et al. [15] discuss that P. dufourii is probably an accidental host for A. nycteribiae. Interestingly, in our study, we detected no infection of P. dufourii by A. nycteribiae, even though there was close body contact between the bat hosts of different species within the same colony at most of the investigated sites, at the time of sampling ectoparasites. Based on these results, we can conclude that the parasitic fungi, A. nycteribiae, show high host specificity to their primary host, P. conspicua, which in our study reaches an astonishing 100%.

Exploring the Parasite-Host Dynamics of Ectoparasitic Fungi Arthrorhynchus nycteribiae in the Central Balkans

A. nycteribiae is detected for the first time at all localities in Serbia, as previous recordings derive, based on the literature data, only from Popšička cave [11]. To the best of our knowledge, this is the first time the fungus is recorded in Bosnia and Herzegovina and Montenegro. It is interesting to note that, in some localities in Serbia, there was a very high prevalence of parasitic fungi, with its highest values in Sokolovica cave in eastern Serbia (60%). Besides this cave, a prevalence of 20% or more was registered at three other localities in eastern Serbia (Bogovinska cave 26.09%, Canetova cave 23.53%, and Dudićeva cave 20%). In comparison to caves of eastern Serbia, the western Serbia caves had 0% or less than 10% of prevalence of parasitic fungi. Although the number of investigated localities is relatively low, we can conclude that there is a difference in the prevalence of this fungal species between western and eastern Serbia, and more investigation should be done in the future in order to provide an explanation for these geographical differences in the parasite prevalence.

Results of our study show that the season has a significant effect on the presence and prevalence of A. nycteribiae on their primary host, P. conspicua. The highest prevalence was shown during the summer season. This can be explained by the fact that during this time of the year, the secondary host, M. schreibersii, forms maternity colonies made of females and their juveniles after weaning, and during this time of the secondary host life cycle, there is a high chance for transmission of the parasite. The prevalence drops during the autumn season when the colonies become smaller and less dense and when bats disperse to their winter roosts. The prevalence of infected bat flies is smallest during the spring season, which can be explained by the fact that after hibernation of individual bats, their low level of mobility, and low temperatures during the wintertime in their roost, the conditions for reproduction of the parasites are not convenient, and therefore, the prevalence of parasitic fungi is very low (2%) in comparison to the numbers during the summer (23%). Our results are in accordance with those already published from the neighboring countries of Bulgaria and Romania [15], where researchers found higher levels of infections in the season after weaning, in comparison to the season preceding birth and the nursing season. Their explanation for this phenomenon was also based on higher opportunities for ascospore transmission among flies when bat flies are much more abundant [40].

Host sex represents a significant factor in determining the prevalence of infestation by A. nycteribiae. Our results show that females of the primary host, P. conspicua, had significantly higher numbers of infestations in comparison to male bat flies. Our results are in accordance with the results of Haelewaters et al. [11] from their research in Romania and Hungary, where out of 38 infected P. conspicua flies (n = 152), 31 were female [11]. According to these authors, Laboulbeniales species seem to prefer female bat flies, likely because females have a longer lifespan, allowing more time for fungal spores to accumulate. Additionally, their larger size and the fat reserves they build up during pregnancy may make them more suitable hosts. Also, in the mentioned work of Peter et al. [15], which considers specimens from Bulgaria and Romania, the researchers found that bat fly sex had an impact on Laboulbeniales infection, where female bat flies were significantly more infected by fungi than males. More similar results were reported by Haelewaters et al. [11] and Szentiványi et al. [19] for specimens in Europe.

Finally, we found a slightly higher infestation of bat flies with fungi in female bat hosts than in male bats. This can be explained by the fact that the bat host—M. schreibersii—forms large and dense groups of female bats during pregnancy and when nursing their young, increasing the likelihood of ascospore transmission among the flies [3, 15].

In conclusion, in this work, we have confirmed the first record of A. nycteribiae for Bosnia and Herzegovina and Montenegro, and extended its distribution in Serbia from previously only one known location from the last century. Furthermore, we have provided more information on the molecular identification and phylogenetic analysis of this scarcely studied fungus, especially for this part of Europe. Our results show that this parasitic fungus species is highly species-specific, and parasitizes only one primary host, P. conspicua, and one secondary host, M. schreibersii. The prevalence of infections was shown to be the highest in the summer months when its secondary host forms dense and numerous colonies, and on female primary hosts, where the chances for new infections are the highest. This study represents a significant contribution to the body of knowledge on the distribution of A. nycteribiae and to the understanding of complex parasite-host relationship patterns existing in the poorly studied area of the Central Balkans.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

Jelena Burazerović and Miloš Stupar conceived the study and designed the experiments. Additionally, Jelena Burazerović collected material for analysis, developed the first draft of the manuscript and coordinated the process of manuscript development. Marija Jovanović performed laboratory processing and morphological identification. Miloš Stupar and Željko Savković supervised morphological identification and performed molecular identification and phylogenetic analysis. Katarina Breka performed the statistical analysis. All authors contributed to the final version of the manuscript.

Funding

The authors are grateful to the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, for the financial support according to the contract with the registration numbers (451–03 - 66/2024–03/200178 and 451–03 - 65/2024–03/200178).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval

All bat captures and handling were officially approved by the Serbian Ministry of Environmental Protection, the Montenegrin Agency for environmental protection, and the Republic of Srpska (Bosnia and Herzegovina) Republic Institute for protection of cultural-historical and natural heritage, in compliance with the Serbian, Montenegrin, and the Republic of Srpska laws and regulations.

Competing Interests

The authors declare no competing interests.