Distribution of the soft tick Carios vespertilionis in lowlands and low mountain regions of Germany

In Germany, the knowledge about ticks infesting bats is limited, and is restricted only to a few studies, most of them dating back decades. To further improve our knowledge on ticks parasitising bats, healthy and sick bats in central Germany were examined for ticks. In total 519 larvae and one nymph of Carios vespertilionis were collected from nine bat species: Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii, Myotis myotis, Nyctalus leisleri, Pipistrellus nathusii, Pipistrellus pygmaeus, Pipistrellus pipistrellus, and Vespertilio murinus. Either the presence of C. vespertilionis was new for some areas or it was confirmed in some federal states in central Germany. The infestation rate was mostly low (n = 1–5 larvae/bat). However, in two cases a high number of ticks was observed. The highest infestation of 97 C. vespertilionis larvae was recorded on one Parti-coloured bat (V. murinus).

Carios vespertilionis (short-legged bat tick) and Argas reflexus (pigeon tick) are the only soft tick species in Germany and have been reported in many German Federal States. Carios vespertilionis has been reported for the first time in 1906 in Bremen-Vegesack, northern Germany, and is the only bat-associated soft tick (Voigt and Oudemanns 1906). There are only a few reports about its distribution in Walter (1992) summarised C. vespertilionis hosts (totally 12 bat species) in nine Federal States. In Bavaria (southern Germany), 11 bat species were reported as hosts for C. vespertilionis, with P. pipistrellus being the primary host (Rupp et al. 2004).
In this study, we describe the distribution of C. vespertilionis in central Germany collected from bats caught with mist nets and sick or debilitated bats found on the ground.

Materials and methods
Ticks were collected from captured bats or bats delivered to animal rescue stations across six German federal states: Lower Saxony, Brandenburg, Saxony, North Rhine-Westphalia, Hesse, and Rhineland-Palatinate (Fig. 1). Ethical approval to catch bats had been issued by local Nature Conservation Authorities.
Bat ticks were collected from June to September 2021 and in August 2022, in six federal states in central Germany. Ticks were collected from bats caught with mist nets, and sick or debilitated bats coddled up in two volunteer bat rescue stations in Fulda city, eastern Hesse from 2002 to 2009 and in the urban region of Hanover, Lower Saxony, during summer 2022 and early 2023.
The bats were searched for ticks by brushing their fur with a small soft brush (to reduce the risk of possible infections of the handling person). The ticks were removed using finetipped tweezers and preserved in microtubes containing 70% ethanol, labelled according to each host. The ticks were identified using morphological characters according to Feider (1965).

Results
In total 519 C. vespertilionis larvae and one nymph were collected from 47 bats (Tables 1  and 2). Those bats belonged to nine species: Eptesicus nilssonii, E. serotinus, Myotis daubentonii, M. myotis, Nyctalus leisleri, Pipistrellus nathusii, P. pygmaeus, P. pipistrellus, and Vespertilio murinus. In the current study, P. pipistrellus was the dominant bat species from which C. vespertilionis larvae were collected. Generally, a few C. vespertilionis larvae were collected from bats, except for two cases: a P. nathusii was infested with 72 larvae and a V. murinus was infested with 97 larvae (Tables 1 and 2).

Discussion
In the Palearctic Region, bats are generally infested with a low number of ticks (Rupp et al. 2004;Hornok et al. 2017). In the UK, only 26 bats from the 7,606 submitted bats were infested with C. vespertilionis larvae (Lv et al. 2018). The distribution of C. vespertilionis in central Germany is known (Hutter et al. 2005) and results show that most of the examined bats did not carry any ticks or were only infested with a few ticks (n = 1-5 C. vespertilionis/bat). However, a few bats carried > 10 ticks. Nine P. pipistrellus were infested with 11, 13, 16, 28, 29 (2x), 32, 37, and 38 C. vespertilionis larvae, respectively. One P. nathusii bat was infested with 72 C. vespertilionis larvae. The highest number of larvae (n = 97) was collected from one V. murinus.
A study carried out in Bavaria (southern Germany) showed that generally, 1-2% of the bats is infested with ticks and the number of C. vespertilionis larvae found on 11 bat species averaged between 2 and 10 ticks (Rupp et al. 2004). There are few reports about bats infested with an uncommonly high number of ticks. Among 51 P. pipistrellus examined in Thuringia (Germany) in 2001/2002, three carried 12, 13, and 27 larvae of C. vespertilionis, respectively (Heddergott 2004). In total 231 C. vespertilionis larvae were collected from a young E. nilssonii in Lower Saxony (Germany), which was severely debilitated and died not long after. This was the highest number of tick larvae found on a bat in Germany (Walter and Rackow 2007).
It is unclear if sick bats are more likely to be infested by ticks or if high tick infestation causes the bats to become unwell. In the present study, the bat infested with 32 ticks   (Socolovschi et al. 2012). Tompa et al. (2023) identified Rickettsia species, genetically closely related to Rickettsia parkeri, Rickettsia conorii, Rickettsia slovaca, Rickettsia sibirica subsp. mongolotimonae, Rickettsia rickettsii, and an uncultured Rickettsia sp. Several of these species are considered pathogenic to humans, nevertheless most of them are not usually circulating in European tick species. The Sweden studies from Jaenson and Wilhelmsson (2021) and Tompa et al. (2023) (using the same set of samples) concluded that further investigations are needed to confirm C. vespertilionis' vector and/or reservoir capacity for the various pathogens. The aim of the current study was to gather more information about the distribution of C. vespertilionis on bats in Germany. Therefore, no studies on pathogens were planned and conducted. In the light of the Swedish results, future studies also have to focus on bacterial pathogens in bat soft tick to elucidate their potential to support the circulation of zoonotic pathogens.
During the present study, only larvae of C. vespertilionis were collected, except one nymph. Carios vespertilionis is frequently found in caves and its larvae are found on bats, with few exceptions. This can be explained by the long feeding time of larvae from a few days up to 14 or even 31 days. Nymphs and adult ticks, on the other hand, feed very quickly for only a short period of < 1 h (Walter and Kock 1985;Hoogstraal 1956;Walter 1992).
In a recent review (Sándor et al. 2021), 44 bat species were listed as hosts of soft ticks. Among them, C. vespertilionis had the most diverse host spectrum with 42 bat species in total. In the present study, C. vespertilionis larvae were found on a wide range of bat species, mainly on P. pipistrellus (common pipistrelle). These results support findings from previous studies where this bat species was identified as the main host in Germany. However, C. vespertilionis was also found on eight other bat species: N. leisleri (Leisler's bat), E. nilssonii (northern bat), E. serotinus (serotine bat), M. daubentonii (Daubenton's bat), M. myotis (greater mouse-eared bat), P. nathusii (Nathusius's pipistrellus), P. pygmaeus (soprano pipistrelle) and V. murinus (parti-coloured bat). In Lv et al. (2018), of the nine bat species included in the study three species -P. pipistrellus, Plecotus auritus, and M. daubentoniid -carried C. vespertilionis larvae, with P. pipistrellus being the most infested. In Sweden, C. vespertilionis have been recorded from two bat species -E. nilssoni and P. pipistrellus -and from dog (Canis familiaris) (Jaenson et al. 1994), whereas in Norway, four bat species -M. mystacinus, M. daubentoni, P. pipistrellus, and E. nilssoni -are known as hosts of this tick (Mehl 1983).
In the latest overview about the distribution of C. vespertilionis in Germany (Rubel et al. 2023), no georeferenced data points were reported in Saxony and Saxony-Anhalt federal states, but one in Thuringia and several in Brandenburg and Mecklenburg-West Pomerania. Here, the presence of C. vespertilionis in eastern Saxony was documented, which fills the gap between central German Federal States and adjacent Poland, where this tick species has been reported (Siuda et al. 2009;Mierzyński et al. 2018).
The currently known distribution of C. vespertilionis in Germany has been confirmed for the following federal states: Lower Saxony (Scheffler and Hiller 2010), Brandenburg (Cornely and Schultz 1992), North Rhine-Westphalia, Hesse, and Rhineland-Palatinate (Walter 1992). When reporting C. vespertilionis for a specific location, the collection time should be considered, as some bats have a wide distribution area. Pipistrellus nathusii, for example, migrates long distances of up to 2,486 km in one direction (Vasenkov et al. 2022). Migrating bat species (e.g., N. leisleri, N. noctula, P. nathusii, V. murinus) can travel > 4000 km in total each year during their flights between their summer and winter habitats (Hutterer et al. 2005).
Regarding the winter activity of C. vespertilionis, there are only two reports on ticks found on bats. One was in January 1985 and the other in February 1987, both in Baden-Wuerttemberg, southern Germany (Walter 1992). On top of those reports a few specimens of C. vespertilionis were found in bat faeces. In the present study C. vespertilionis larvae were collected during the winter months (December, January, and February) in the federal states Hesse and Lower Saxony. Larvae, nymphs and adults were collected in summer (June, July, and August 2015 and 2018) using a water-filled tray placed bellow a wood bat box harbouring an estimated number of 250-500 adult females and young P. pygmaeus (Jaenson and Wilhelmsson 2021). In the UK, larvae and nymphs where collected from P. pipistrellus in May and July and in material from roost in house in July (Arthur 1963).

Conclusions
There are still considerable knowledge gaps regarding the presence of several tick species in Germany, especially concerning rare species, such as C. vespertilionis. This study supplies more detailed information for some federal states in central Germany, but additional studies are warranted on C. vespertilionis presence, density, and evaluation of public and veterinary risks.