First report of the bat fly species Basilia italica in Romania

Abstract Bat flies are haematophagous ectoparasites, highly specialised to bats and are also considered to have vectorial potential for several pathogens like Bartonella spp. or Polychromophilus spp. In Romania, past studies mostly focused on the ectoparasitic fauna of cave-dwelling bats, listing the occurrence of 10 bat fly species in the country, with only scarce information on bat flies infesting crevice-roosting bat species. Here we report the occurrence of Basilia italica, a rare nycteribiid species infesting primarily forest-dwelling bats. This is the first country-record for Romania and also represents the easternmost occurrence of this species. Further studies are needed to evaluate the vectorial potential of B. italica.


Introduction
Bat flies (Diptera, Hippoboscoidea) are highly specialised ectoparasites of bats representing two families in the superfamily Hippoboscoidea (Streblidae and Nycteribiidae; Reeves and Lloyd 2018). Species belonging to Nycteribiidae are wingless, while most streblids have functional wings (with some exceptions). These dipterans are exclusive parasites of bats and are highly adapted to a parasitic life style. Bat flies are haematophagous and have highly specialised reproductive organs. These parasites reproduce by viviparous puparity and only females leave the host, when larvideposit on the roost-substrate (Dick and Patterson 2006). Most bat flies show high host-specificity (monoxenous), being exclusive parasites of one or a few morphologically similar and phylogenetically closely related bat species (Dick 2007). Bat flies are increasingly recognised as vectors for several pathogen groups like bacteria of the genus Bartonella spp. or parasitic protozoa, like Polychromophilus spp. (Megali et al. 2010, Dick and Dittmar 2014, Sándor et al. 2018. Although the research interest is growing worldwide, most papers focus on parasites of cave-roosting bats in Romania, listing 10 species (Burghele-Balacescu 1966), namely: Basilia nana, B. nattereri, Nycteribia latreillii, N. kolenatii, N. pedicularia, N. schmidlii, N. vexata, Penicillidia duforii, P. conspicua and Phthiridium biarticulatum). Gheorghiu (2006) reported the presence of several species at the Piatra Craiului forest and Sándor et al.

Material and methods
(Methods used for this study were published at protocols.io with the following DOI: dx.doi.o rg/10.17504/protocols.io.bi2ykgfw).
Bats were trapped using mist nets erected close to the roosting site of an all-male colony of multiple species in an abandoned building close to Ic Ponor (Munții Apuseni, 46.629842N, 22.806450E, Fig. 1), at an altitude of 1044 m on 17.06.2020 and 01.07.2020. Upon capture, bats were examined, species were identified, based on morphological characters (Dietz et al. 2009) and all external parasites were collected using tweezers and were stored in 70% ethanol (single tube/host). In the laboratory, bat flies were identified under stereomicroscope, using morphological keys (Theodor 1967).

Results
On both trapping occasions, a single bat fly individual was collected from a single adult male Myotis mystacinus. None of the other examined individuals (all males, Vespertilio murinus -106, Pipistrellus pipistrellus -17, M. mystacinus -16 and M. brandtii -2) hosted bat flies (Suppl. material 1). Bat flies were identified as a male and a female Basilia italica, based on the following morphological characteristics: a small-sized fly with visible eyes, but a body shape similar to the genus of Nycteribiae, a group of spines at the end of sternite 5. The surface of tergites are more or less bare, but on the marginal ends, there are some longer hairs. Lastly, there are long setae on the tibiae and the overall form of sternite 5 is unique ( Fig. 2; see Theodor 1967, page 202).
The finds reported here are the first records of B. italica in Romania, hence increasing to 11 the number of known bat fly species in this country. This is a novel and a geographically-distant record, therefore considerably extending the range towards the East. (Fig. 3). The geographically closest occurrence of this fly species is in Hungary, where it was reported from six different species (Czuppon andMolnár 2001, Haelewaters et al. 2017).   Although bat flies are usually abundant on cave dwelling species (like Miniopterus schreibersii or M. myotis) and their flies are relatively well-known (Zahn and Rupp 2004);, however, the host species of B. italica tend to roost in crevices (primarily tree-holes, but also abandoned buildings) and they are captured in smaller numbers (Dietz et al. 2009). After examining almost 500 individuals from the potential host species of B. italica in the past five years, these are the first observed individuals of this species in Romania.

Acknowledgements
Hereby, we would like to acknowledge the help provided by numerous volunteers in the fieldwork throughout the last five years, with special thanks for the assistance of B. Sándor and E. Sándor in data collection in 2020. ÁP was supported by the Collegium Talentum programme of Hungary. ADS was the beneficiary of the János Bolyai Research Scholarship of Hungarian Academy of Science and the ÚNKP 19-4-ÁTE-10 New National Excellence Program of the MIT.

Funding program
The NKFIH 132794 (National Research, Development and Innovation Office of Hungary) provided financial resources to ADS.

Hosting institution
University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Ethics and security
Bat capture permits were issued by the Underground Heritage Commission (Romania).

Author contributions
ÁP contribuited to the bat capture and sample collection, did the identification and wrote the early version of the manuscript.
ADM made the photos and corrected the manuscript.
SDA organised the field trip, revised and corrected the manuscript.

Conflicts of interest
We (all authors) declare that there are no confllict of interest.
First report of the bat fly species Basilia italica in Romania