Predation of invasive raccoon (Procyon lotor) on hibernating bats in the Nietoperek reserve in Poland

The raccoon Procyon lotor is an invasive species inhabiting Europe and its impact on local populations, particularly on endangered species, is not well known. The aim of the study was to determine whether raccoons pose a threat to bats wintering in the Nietoperek nature reserve, one of the largest hibernation sites in Central Europe. In the winter of 2016–2017, 67 raccoon scats were collected in one of the main underground corridors in the reserve. It was confirmed, with the use of molecular methods, that the scats belonged to raccoons. Raccoon prey was identified based on the features of skulls, jaws or hair found in the scats. Analysis of the determinable remains indicates that bats (including Daubenton’s bat/Natterer’s bat/Brandt’s bat M. daubentonii/nattereri/brandtii, Natterer’s bat Myotis nattereri, greater mouse-eared bat Myotis myotis, brown long-eared bat Plecotus auritus) made up the largest percentage of raccoon diet (96%). Remains of other mammal species, lizards Lacerta spp., plant materials and anthropogenic food were also identified in raccoon scats. The results of the research indicate that predation by raccoons can constitute a significant factor in the increasing mortality of bats wintering in Nietoperek.

Information about the negative impact of the introduced species on European bats remains poorly described. Records of attacks on Leisler's bat Nyctalus leisleri by the rose-ringed parakeets Psittacula krameri come from Italy (Menchetti et al. 2014). In Spain, these parrots evicted bats from their hollows and attacked common noctules Nyctalus noctula (Hernández-Brito et al. 2018). Bats were also a prey of the domesticated cats (Ancillotto et al. 2013;Khayat et al. 2020).
The aim of the study was to determine the impact of the raccoon on bats wintering in the Nietoperek nature reserve. We propose the following hypotheses: (1) raccoons penetrate underground corridors, not only to find shelter, but also to feed on wintering bats which can constitute a significant contribution to the predator's food; (2) the presence of specific bat species in the raccoon diet is proportionate to the size of that particular bat species' population within the studied underground corridor.
The research was conducted in the Nietoperek nature reserve in Western Poland (N52.2343 E15.2920). The reserve is located in underground corridors ca. 30 km long, which are part of the fortifications from the Second World War period. The corridors consist of a series of shafts that lead from ground level battle shelters to interconnected underground tunnels, halls and train stations. The corridors are one of the largest bat wintering sites in Central Europe. During the hibernation period, over 38,000 bats, arriving from Poland and Germany, find shelter in the underground corridors. The following species were found in the area: greater mouse-eared bat Myotis myotis, pond bat Myotis dasycneme, Bechstein's bat Myotis bechsteinii, western barbastelle Barbastella barbastellus, Daubenton's bat Myotis daubentonii, Natterer's bat Myotis nattereri, Brandt's bat Myotis brandtii, brown long-eared bat Plecotus auritus and serotine bat Eptesicus serotinus (Cichocki et al. 2015).
The research was carried out in a single, 1500 m long corridor, which serves both as the main entrance to the underground corridors for visitors and one of the most important ways for bats to get into the hibernaculum. The entrance to the corridor starts on the ground level. Scattered light reaches about 500 m inwards, but beyond that the corridor stays in complete darkness (Fig. 1, Online Resource 1).
Raccoon predation on bats was investigated based on the analyses of the composition of predator scats (Online Resource 2). Single scats appeared underground in December 2016, and over time, their numbers increased, indicating a regular penetration of the underground corridor by the predator. We collected material in two rounds, i.e. at the beginning and in the middle of March 2017. A total number of 67 scats were collected and analysed for diet composition.
Predator species were initially identified as raccoon based on the size and shape of scats. At that time, raccoons had only been seen in the ground parts of the fortifications. We have made the DNA analysis to confirm the species. Species identification was performed based on sequencing of the mitochondrial COI (mini-barcode) gene fragment from 25 samples. The analysed COI gene segment was 187 bp long (239 bp including primers). The total number of 25 samples, frozen after collection, was subjected to the molecular tests. DNA was isolated with the QIAamp DNA Stool Mini kit (Qiagen) following the manufacturer's protocol. PCRs were performed in thermal cyclers (Sensquest) with the conditions and cycling conditions described by Chaves et al. (2012) as well as primers BC-F3 and BC-R2 (Chaves et al. 2012). Blank PCR controls were used in all reactions to monitor reagent contamination. Amplification products were run on 1.5% agarose/TBE gels stained with MidiGreen. PCR products were purified using EPPiC (A&A Biotechnology) following the manufacturer's protocol. Sequencing was performed in Genomed S.A. (Poland). The obtained sequences were analysed using the DNA Surveillance-Carnivora and a species assay by Chaves et al. (2012). According to the information from this database, the species present in ten samples was confirmed as raccoon. Raccoon prey was determined based on the analysis of skull features (Ruprecht 1987). Depending on the degree of material preservation, prey was identified in terms of their species, genus or order. In addition, the following features were taken into account: coat colour, length of metacarpal and phalange bones forming bat wings, undigested parts of bat ears. In the absence of bone elements, features of hair structure were analysed (Teerink 1991). Bats that were not identified to species level from the genus Myotis were described as: Daubenton's bat/Natterer's bat/Brandt's bat. Other unidentified small bats were assigned to Chiroptera indet., including small Myotis, Plecotus and Barbastella.
The occurrence of species in raccoon scats was calculated as a proportion of samples containing the remains of a given species or group of species to the total number of analyzed samples. The total number of bats included in the tests was determined on the basis of the maximum number of determinable skull elements: tooth series and right and left branches of the jaw. In the absence of skull elements, the sample was assumed to belong to a single individual. This assumption is justified by the composition of scats in which uniform fragments of fur and wing fragments were found. When feeding on greater mouse-eared bats, raccoons often left heads and forearms uneaten. However, this material was not taken into account in the analysis to avoid double-calculation of the same individual (Online Resource 3).
The proportions of scats containing bats (of respective species or group of species) and scats containing other components of raccoon diet, were compared using χ 2 test. The same test was used to assess the difference in the occurrence of two main groups of investigated bats in raccoon diet: the greater mouse-eared bat and Daubenton's bat/Natterer's bat/Brandt's bat.
The number of bats wintering in the underground section penetrated by raccoons was monitored every two weeks, from November 2016 to the end of March 2017. In total, 10 inspections were conducted. To demonstrate which of the species or group of species is most vulnerable to raccoon foraging, the number of individuals from a given group found in scats was compared to the average number of counted individuals hibernating along the investigated section of the underground corridor. The entrance corridor is used by bats dynamically. Therefore, the population inhabiting this section changes over the season. Due to the above reason, the average number of bats of a given species or a group of species in relation to the number of individuals from these groups found in scats was used in the analysis. For all the statistical tests, the significance level p = 0.05 was used. All statistical analyses were conducted using R program (R Core Team 2018).
The results of our research describe the first documented case of the negative impact of invasive raccoons on European bat species during their hibernation. Raccoons exploring the underground corridors of the Nietoperek nature reserve prey on hibernating bats during winter. We found 81 individuals in the predator scats out of which 32 were identified to species (20-Natterer's bat, 11-greater mouse-eared bat, 1-brown long-eared bat). Due to material fragmentation, other individuals were identified as Daubenton's bat/Natterer's bat/Brandt's bat (43 individuals) or were included in the group of unidentified small bats Chiroptera indet. (6 individuals).
Bats were present in 96% of the analysed scats. In 69% of these single individuals were found, in 28% two individuals were identified, and in 3% three individuals were recorded. Only three of the analysed samples did not contain any bats. Apart from bats, the total number of 15 samples also contained other diet components ( Table 1). The fraction test for two groups (χ 2 = 71.06, df = 1, p < 0.001) indicates a significantly higher contribution of bats in the raccoon diet compared to other food fractions (hypothesis # 1 confirmed).
The fraction test for two groups of bats found in raccoon scats, i.e. the greater mouse-eared bat and the Daubenton's bat/Natterer's bat/Brandt's bat (χ 2 = 37.45, df = 1, p < 0.001), indicates that the dominant group among the diet was Daubenton's bat/Natterer's bat/Brandt's bat (hypothesis #2 rejected). The percentage of individuals found in raccoon scats for the selected species or a group of species is inverse in relation to the average number of individuals hibernating in the examined underground section (Fig. 3).
Although the greater mouse-eared bat is the most abundant group of bats wintering in Nietoperek nature reserve, the main prey of raccoons included different species: Daubenton's bat/Natterer's bat/Brandt's bat. Furthermore, individuals of the greater mouse-eared bat species constitute the largest percentage of dead bats found in the reserve (J. Cichocki unpbl. data). The majority of greater mouseeared bats (99%) winter in places located more than 2 m above the bottom of the underground corridors. Due to this fact, they are inaccessible to raccoons. Based on that, we conclude that raccoons probably ate dead greater mouseeared bats that had fallen out of the clusters earlier. In contrast to the greater mouse-eared bats, Natterer's bats and Daubenton's bats often hibernate near the ground (even as low as 1 m), therefore, they stay within the feeding range of raccoons. It is possible that high winter activity and the close-to-the-ground hibernation were the reasons for the increased occurrence of this species in raccoon diet (Online Resource 4).
Raccoons are food opportunists (Rulison et al. 2012). In their natural range, raccoon diet is usually dominated by plants and invertebrates, with a significant presence of crustaceans, crabs and crayfish in the areas of their availability (Lotze and Anderson 1979). In Germany, the first European country where raccoons were introduced, the animal diet was dominated by plant food and various species of invertebrates (Engelmann et al. 2011). Consequently, vertebrates accounted only for 15% of biomass eaten by raccoons. However, the occurrence of birds and mammals in the predator diet was increasing in winter (Engelmann et al. 2011). In the floodplain areas of Western Poland, rodents represented the largest share in raccoon diet. However, the phenological period in which the material was obtained was not determined by the authors of the study (Bartoszewicz et al. 2008).  In our research, raccoons used the availability of bats opportunistically, treating them as the main component of their diet. Bats were present in the diet of raccoons inhabiting caves in North America (Munson and Keith 1984;McAlpine et al. 2011). Raccoons also fed on dead individuals with white nose syndrome (McAlpine et al. 2011). Until now, the only predator recorded to be preying on bats in the underground corridors of the Nietoperek nature reserve, was the stone marten Martes foina (Urbańczyk 1981;Lesiński and Romanowski 1988;Tryjanowski 1997). Research on the diet of the stone marten, conducted in the 1980s, revealed that its diet was dominated by Daubenton's bats and brown long-eared bats (Lesiński and Romanowski 1988). However, this fact does not seem to have a significant impact on the number of bats in the reserve. The emergence of a new effective and universal predator, such as raccoons, has increased predation pressure on bats. Consequently, this can lead to an increase of bat mortality, at least in the corridors accessible to raccoons, and become a factor of significant impact on the number of bats in wintering sites.
In North American caves, raccoons usually penetrated relatively small entrance sections but were also found in the deeper parts (Munson and Keith 1984;McAlpine et al. 2011). Some caves are used by raccoons as hideouts and resting place (Levis 2002;Moseley et al. 2013;Montalvo et al. 2017). In the Nietoperek nature reserve, raccoons penetrated the corridor section to a distance of about 900 m from the entrance. Scats were also found 1300 m from the entrance indicating that the predator was moving in complete darkness.
There are several dozen entrances in the reserve but only these accessible at ground level can be used by terrestrial animals. Therefore, only a few entrances are available to raccoons. The animal may probably use entrances to vertical shafts of staircases as a shelter, but this does not interfere with bats' wintering zone. However, the range of predation shown in this study indicates the need to monitor the situation and perhaps take more radical measures to protect bats.