Cent. Eur. J. Biol. • 9(4) • 2014 • 437-443 DOI: 10.2478/s11535-013-0280-z

Central European Journal of Biology

Prevalence and phenology of white-nose syndrome Pseudogymnoascus destructans in bats from Poland

Research Article Konrad Sachanowicz1*, Arkadiusz Stępień2, Mateusz Ciechanowski3

1Museum and Institute of Zoology PAS, 00-679 Warszawa, Poland

241-933 Piekary Śląskie, Poland

3Department of Vertebrate Ecology and Zoology, University of Gdańsk, 80-308 Gdańsk, Poland Received 21 May 2013; Accepted 23 October 2013

Abstract: Pseudogymnoascus destructans (Pd), a parasitic fungus (being responsible for a disease known as white-nose syndrome, WNS) that caused mass mortality of cave-dwelling, hibernating bats in North America, appears to be native of Europe, where it also occurs on wintering bats, but no similar outbreaks of WNS have been recorded. Herein, we provide the first account on prevalence and phenology of P. destructans in Poland. Bats were counted once per month, from October or January to May (2010-2013), in an abandoned ore mine in southern Poland. Presence of P. destructans in two samples was confirmed by sequencing of isolated fungal DNA. Observations of phenotypically identical mycosis on bats hibernating at this site in March 2006 are likely to be the first known records of P. destructans from Poland. All Pd-suspected individuals were Myotis myotis with an exception of one Myotis daubentonii. The first Pd-suspected bats were noted in mid-February, but their number was the highest in March, what overlapped with maximum numbers of hibernating M. myotis. The prevalence in March was 7%-27% of M. myotis individuals. No mass mortality of bats was observed in the mine, with only three dead individuals found in the hibernaculum which hosted up to 130 bats, representing 6-7 species.

Keywords: White-nose syndrome • Geomycosis • Chiroptera • Hibernation • Myotis myotis • Mortality © Versita Sp. z o.o.

1. Introduction caused mass mortality of cave-dwelling, vespertilionid bats in North America, being responsible for, so-called, A number of emerging fungal pathogens appear to be white-nose syndrome (WNS) [2,3]. increasing their frequency and abundance worldwide, The syndrome itself, first noticed in 2006, can be leading to severe, often continental or global, crises recognized by white hyphae and conidia growing on in biodiversity and ecosystem functioning [1]. The the muzzle, ears, wing membranes and even invading mechanisms hidden behind such an increase remain hair follicles of infected bats. WNS appeared only largely unknown, although environmental pollution, during hibernation, thus has been restricted to the climate change and intercontinental transport are often temperate zone, and affected only the species wintering blamed. Some of the mentioned fungal pathogens in underground roosts (hibernacula). The fungus was gained international interest, e.g. the chytrid fungus described as new for science in 2009, as Batrachochytrium dendrobatidis, responsible for series destructans [5]. Later, phylogenetic analysis of Nearctic of extinctions or declines of more than 500 amphibian Geomyces and its allies placed the new pathogen in a species worldwide, or aquatic oomycete Aphanomyces separate Pseudogymnoascus [6]. It appeared astaci that caused a devastating decline in native to be psychrophilous, with no growth above 19.8°C European freshwater crayfish [1]. Recently, another and optimum at 12.5-15.8°C, but with production parasitic fungus, Pseudogymnoascus destructans, of abundant microconidia only below 12°C, a clear

* E-mail: [email protected] 437 White-nose syndrome fungus in bats from Poland

adaptation for propagation in microclimate typical for Although it became clear that the visual bat hibernacula [7]. Experimental infections of captive manifestation of P. destructans infections on the bats confirmed that P. destructans is responsible for exterior of bats in Europe developed mostly before the WNS pathogenesis [8], most often causing death of end of the hibernation period, i.e. March [18], the only infected individuals in case of North American species. detailed investigation of seasonal dynamics has been Infection causes ulcers, lesions, skin irritation and conducted at a single northwest German locality during severe damage to the invaded tissue [9,10]. Immune two consecutive seasons [20]. No such data is available reconstitution inflammatory syndrome (IRIS) probably for central Europe. The only published record of the triggers or contributes to that damage, as hibernation fungus from Poland concerns a studied sample from is associated with immune suppression, while return a M. myotis individual, collected in March 2010 at an to euthermia results in its rapid reversal [11]. Bats unknown locality in the southwest of the country [20]. In infected with P. destructans probably face severe this paper we provide the first account of the temporal evaporative water loss, thus increased frequency of occurrence of P. destructans infection in vespertilionid arousal from hibernation, with the animals often flying bats hibernating in an underground roost in south out of their roost, rapidly depleting their subcutaneous Poland. fat reserves and consequently dying of starvation [12,13]. WNS spreads rapidly during the first years after its appearance, leading to population collapse, even in 2. Experimental Procedures the common and widespread species Myotis lucifugus, that lost 30-99% of individuals spending winter in large We monitored a number of bats in corridors of a bat hibernacula in the eastern part of USA [14]. Also disused ore mine in the Blachówka dolomite quarry rare and endangered species, like Myotis sodalis, suffer (50°24.292´´N, 18°51.239´´E) in Bytom-Sucha Góra from the disease [15], and strong decrease in activity of (Śląsk Upland, south Poland). The mine is the most Myotis spp. during summer was recorded in the areas important part of Tarnowskie Góry-Bytom Undergrounds surrounding underground roosts affected by WNS [16]. designated in 2004 as Special Protected Area (Nature The pathogen being a causative agent of WNS, 2000) for bats and their habitats. The mine maintains P. destructans, has been recorded on bats hibernating the largest winter bat aggregation (up to 130 individuals, underground in several European countries (Austria, representing 6-7 species, recorded in the winter season Belgium, Czech Republic, Denmark, Estonia, France, 2012/2013) in the Silesian Upland (K. Sachanowicz, The Netherlands, Poland, Romania, Slovakia, A. Stępień, unpubl. data). In the mine, the average Switzerland, Turkey, Ukraine) [17-20]. Records of annual temperature reached 5.8°C measured ca 80 m P. destructans confirmed by fungal culture and genetic from its entrance. In the winter months, from January to analyses, were obtained from at least eight bat species: March, mean monthly temperature ranged from 0.5 to Myotis myotis, M. blythii oxygnathus, M. bechsteinii, 4.0°C, while during the period from April to November M. nattereri, M. mystacinus, M. brandtii, M. dasycneme – from 5.0 to 8.0°C; humidity was close to 100% [23]. and M. daubentonii [18,20] and WNS itself was confirmed During three winter seasons, 2010-2013, two in Europe by histopathological evidence [21]. However, authors (A. Stępień, K. Sachanowicz) counted bats it did not lead to mass mortality in any European country on a fixed transect ca 2500 m length, starting from the [20]. Most of bats with visible fungal growth probably grilled entrance of the mine. In the 2010-2012 seasons recovered after the end of hibernation and the most we counted bats once per a month, from October to affected species, M. myotis, reveals constant population May, while in 2013 – from January to May, including growth during the last 20 years with no signs of decline an additional count in March. All visible bats hanging [18]. The WNS-suspected bats have been recorded on on walls, ceiling and hidden in crevices of low (ca 1 to photographs since 1995 [18], ten years before the first 4 m high) walls of corridors were counted and identified American case, leading to the suspicion that the fungus based on external features, without handling. Thus, is native for Europe, transferred to America by humans, several individuals representing sibling species, either presumably by cave explorers. Further experiments M. brandtii or M. mystacinus, were left unidentified to with inoculation of American bats with European and species. American isolates of P. destructans provided direct After observing individuals of M. myotis with evidence that the fungus is most likely a recent invader white fungal growth (suspected P. destructans – later in America, while its high pathogenicity is associated described as ‘Pd-suspected’ [20]) in February 2011, we with lack of resistance to that pathogen in Myotis bats of collected samples of the fungus for genetic analysis North America [22]. in March 2011. Using cotton swabs we collected the

438 K. Sachanowicz et al.

fungus from the muzzles of two hibernating M. myotis March 2011. The sequences obtained were submitted and placed them into 1.5 ml plastic tubes. The fungal to the European Nucleotide Archive with accession material was inoculated onto Sabouraud dextrose agar numbers HF911370-HF911371. plates and incubated in the dark at two temperatures (ca. In three consecutive seasons of bat monitoring, we 7°C and 15°C). After 14 days, the outgrowing colonies recorded the first individuals of Pd-suspected M. myotis of the fungus were isolated. The isolates were identified (Figure 2) in February, with the earliest observation based on their phenotypic characteristics, i.e. shape on 16th February 2013. The numbers or Pd-suspected and size of conidia and conidiophores. Fungal DNA bats were highest in March, when we observed also was also isolated from samples to confirm the species maximum numbers of hibernating M. myotis. In the identity. For details of DNA isolation, gene amplification, consecutive years, the prevalence in March was 13%, PCR reactions, products purification, sequencing and 27% and from 11 to 7% of individuals, respectively. We analysis protocols, see [18]. The other bat individuals observed the last Pd-suspected bats on 10th April 2011, were considered Pd-suspected in every case when while on 21 April 2012 no bats remained in the mine white fungal colonies were seen growing on muzzle, (Table 1). An additional count on 30 March 2013 showed ears or membranes. that increasing number of M. myotis was not associated with an increase in prevalence of infection towards the 3. Results end of the hibernation period. Although individuals of M. myotis with visible white fungal With the exception of one M. daubentonii, we did growth (suspected P. destructans) on their muzzles and not observe P. destructans in bats of other species ears had been observed (Figure 1) in the Blachówka hibernating in the Blachówka mine, where all species, mine on 4 March 2006 (K. Sachanowicz, Ł. Polonius), except Plecotus auritus, occurred in low numbers we confirmed this fungus in two samples collected in (Table 2). The only Pd-suspected M. daubentonii (among five individuals recorded on 17th March 2013), hibernated in contact with an infected individual of M. myotis (Figure 3). During and after hibernation (in April-May), in the period of bat monitoring, we did not find any dead Pd- suspected bat. We found only single dead individuals: P. auritus (in February 2012), M. daubentonii (in April 2012) and M. myotis (in February 2013); there was, however, no evidence that fungal infection was responsible for their death and other fungal species could be involved with the decomposition of dead bats.

Figure 1. Myotis myotis with white fungal growth of suspected Figure 2. Myotis myotis with white fungal growth of suspected P. P. destructans on a muzzle and ears, the Blachówka destructans on a muzzle, ears and wing membranes, mine, 4 March 2006 (Ł. Polonius). hibernating in the Blachówka mine, 16 February 2013 (A. Stępień). 439 White-nose syndrome fungus in bats from Poland

2010/2011 2011/2012 2012/2013 Date Date Date n prevalence n prevalence n prevalence

October- October- October- 8 0 8 0 - - December December December 24 January 14 0 24 January 12 0 26 January 9 0

25 February 46 2 27 February 29 4 16 February 35 3 17 March 70 8 25 March 62 8 23 March 40 11 30 March 102 7 10 April 34 3 21 April 0 0 19 April 4 0

17 May 0 0 14 May 0 0 4 May 0 0

Table 1. The abundance (n) of M. myotis hibernating in the Blachówka ore mine and numbers of individuals with visible white fungal growth of suspected P. destructans on muzzles (prevalence), during three winter seasons 2010-2013.

Maximum number of bats Number of dead bats Maximum number of Pd- Species recorded during one count found suspected individuals

Myotis bechsteinii 1 0 0

Myotis brandtii/M. mystacinus 3 0 0

Myotis daubentonii 16 1 1

Myotis myotis 102 1 11

Myotis nattereri 1 0 0

Plecotus auritus 37 1 0

Table 2. Species composition and the abundance of bats hibernating in the Blachówka ore mine during three winter seasons 2010-2013.

Figure 3. Myotis myotis (left) and M. daubentonii with white fungal Figure 4. Group of hibernating M. myotis including one bat with growth (suspected P. destructans) on a muzzle, ears and typical symptoms of Pd infection, Nietoperek bat reserve, wing membranes, hibernating in the Blachówka mine, 20 February 2010 (K. Kaliński) 17 March 2013 (A. Stępień).

440 K. Sachanowicz et al.

4. Discussion 5%) or xeric areas dominated by limestone karst (2-3%) [18]. The presence of P. destructans in the Blachówka Thermal conditions of winter roosts may support or mine has been confirmed by genetic analysis. The prevent development of psychrophilous P. destructans. observations of Pd-suspected M. myotis in March 2006 Low and rather stable temperatures of winter months are apparently the first known records of this fungus (from 0.5 to 4.0°C) and high humidity in the Blachówka from Poland [20]. mine [23] seem to facilitate the presence and Although M. myotis is the species most frequently development of the fungus. In southern and central infected by P. destructans, in Europe the fungus has Poland (mountain and upland areas), its presence may been reported on all species of Myotis bats which be expected in other underground roosts (mainly caves hibernated in the Blachówka mine [18,20]. The apparent and mines with similar microclimate) used by M. myotis, absence of the fungus in these species and all but one a widespread species in these parts of the country. M. daubentonii individuals, in the Blachówka mine, Therefore, these combined factors seem to be the could also be a result of the behavioural differences most important for predicting P. destructans distribution. among species. There, only individuals of M. myotis Recently, observations of the fungus presence have formed clusters, but these aggregations were not mixed become available for several sites. Single hibernating with bats of other species which wintered singly or rarely individuals of M. myotis with an apparent P. destructans in groups of two-three individuals. Myotis myotis formed on their muzzles were recorded and photographed clusters from February onward, with the largest ones on 28 March 2013 in an artificial cave in Bochotnica, (up to 45 bats) at the end of hibernation – in March and central-eastern Poland (M. Piskorski, pers. comm.) and April, what coincided with the highest prevalence of at three localities in the western Carpathians: Oblica P. destructans. The only M. daubentonii infected with the cave (since 2006), Bania w Jarmucie mine (2012) and fungus was in body contact with infected M. myotis. This Dymiąca Piwnica cave (2013), (W. J. Gubała, pers. may support the hypothesis that clustering behaviour, comm.). The fungus likely occurs also in other parts higher abundance and sociality in general facilitate of central and northern Poland within the geographic the spread of the fungus among different species and range of M. myotis [25], e.g. in the largest bat individual bats [24]. The lack of a continual increase hibernaculum in Poland and one of the most important of prevalence in late March seems to be related to European bat sites – Nietoperek Bat Reserve, used by increasing arousals and activity of bats in the mine. On thousands of M. myotis in winter [26]. Two individuals 30 March 2013, numerous individuals of M. myotis were of M. myotis with typical symptoms of Pd infection already awake, including a copulating pair. Although were photographed there in February and March 2010 detectability of Pd-suspected bats was relatively good (Figure 4, J. S. Boratyński, pers. comm.). Microclimate in the Blachówka mine, particularly in M. myotis which of the Nietoperek Bat Reserve seemed suitable for the all hibernated on low ceilings and walls and were not fungus development and the same may be true of other hidden in crevices, the numbers of bats infected with the important Polish bat hibernacula, which are mainly fungus should be regarded as minimal. The absence of abandoned military objects. Occasional bat counts in visible white fungal growth on bats does not mean the March 2009-2013 in three larger bat hibernacula on the lack of P. destructans infection [20]. Polish Baltic Sea Coast (Gdańsk-Orunia, Gdańsk-Oliwa Our results agree with published data on the and Elbląg, inhabited by 78-579 bats, predominantly temporal occurrence of P. destructans on bat hosts. M. daubentonii, M. nattereri and M. myotis), revealed The fungus usually appeared on bats in February, but not a single individual with fungal cover resembling it was observed from the middle of January till late geomycosis (M. Ciechanowski, A. Przesmycka, June, and the number of infected bats peaked in March M. Więckowska, unpubl. data). [20]. The highest prevalence in our observations was Our data indicate that bat mortality was very low and generally low (from 11% to 27%) and varied similarly no increased bat mortality has been related to infection in the same months in consecutive years. During one by P. destructans in the Blachówka mine. Thus it is season at a German site, the prevalence was similar probable that all of the individuals with visible fungal (18-25%), but a year later it reached 28-55% [20]. In the hyphae recovered from infection or did not suffer from Czech Republic and Slovakia, where 98 hibernacula it. We cannot diagnose their cases as WNS, as this were inspected between late February and March, refers to a certain pathological state, confirmed either the highest percentages of Pd-suspected M. myotis by histological examination [9,21] or by bat mortality, were recorded in submountain humid or mesic regions preceeded by a sequence of arousals, visible loss of (11-100%), while being much lower in mountains (0- condition and damage of tissue. Only single dead bats

441 White-nose syndrome fungus in bats from Poland

of the two species infected by P. destructans were found: fat reserves. More frequent arousals are suggested to be M. myotis, which died presumably in the late autumn or associated with increased evaporative water loss [12]. early winter (before February) and M. daubentonii, found However, it seems doubtful that this may be a reason in April, after hibernation. A dead P. auritus was found in in hibernacula with extremely high relative air humidity February, when the fungus just begins to become visible (e.g. close to 100% as it was in some parts of the on hibernating European bats. Pseudogymnoascus Blachówka mine). For the majority of bats hibernating in destructans has never been confirmed in either European subterranean roosts, P. destructans appears P. auritus or other non-Myotis bats of the western to act as a commensal, rather than a typical parasite, Palearctic [18,20], but in the Nearctic, members of other as mild fungal growth may not decrease the bats fitness vespertilionid genera, also hibernating in subterranean and disappears before the end of the hibernation period, sites (Perimyotis subflavus and Eptesicus fuscus), can probably groomed off by arousing individuals. be affected by typical WNS [2]. Problems with discriminating live and freshly dead bats hanging in clusters on ceilings in March-April (due Acknowledgements to a lack of the possibility to check all individuals), can lead to underestimation of real mortality in most We thank Natalia Martínková, who confirmed the bat hibernacula. That bias can be partially reduced fungus species based on genetic analysis, Łukasz by additional counts to search for dead bats after Polonius for providing the photo from the Blachówka hibernation in April-May. Even without causing mass mine (March 2006), Jan S. Boratyński and Krzysztof mortality of bats in Europe, it is likely that P. destructans Kaliński for providing the photo of Pd-suspected may increase the probability of death of infected bats, M. myotis from the Nietoperek bat reserve. Monitoring mainly injured or the most exhausted individuals at the of bats was conducted under an authorization from end of hibernation, possibly as a result of an increased the General Directorate for Environmental Protection frequency of arousals and the resulting depletion of body (DOPozgiz-4200/IV.D-7/1930/10/km).

References

[1] Fisher M.C., Henk D.A., Briggs C. J., Brownstein [7] Verant M. L., Boyles J. G., Waldrep W. Jr., Wibbelt J.S., Madoff L.C., McCraw S.L., et al., Emerging G., Blehert D. S., Temperature-dependent growth fungal threats to animal, plant and ecosystem of Geomyces destructans, the fungus that causes health, Nature, 2012, 484 (7393), 186-194 bat white-nose syndrome. PLoS One, 2012, 7, [2] Foley J., Clifford D., Castle K., Cryan P., Ostfeld e46280, doi: 10.1371/journal.pone.0046280 R.S., Investigating and managing the rapid [8] Lorch J.M., Meteyer C.U., Behr M.J., Boyles emergence of White-Nose Syndrome, a novel, J.G., Cryan P.M., Hicks A.C., et al., Experimental fatal, infectious disease of hibernating bats, infection of bats with Geomyces destructans Conserv. Biol., 2011, 25(2), 223-231 causes white-nose syndrome, Nature, 2011, 480 [3] Blehert D.S., Fungal Disease and the Developing (7377), 376–378 Story of Bat White-nose Syndrome, PLoS [9] Meteyer C.U., Buckles E.L., Blehert D.S., Pathogens 8(7), 2012, e1002779. doi:10.1371/ Hicks A.C., Green D.E., Shearn-Bochsler V., journal.ppat.1002779 Histopathologic criteria to confirm white-nose [4] Blehert D.S., Hicks A.C., Behr M., Meteyer C.U., syndrome in bats, J. Vet. Diagn. Invest., 2009, 21, Berlowski-Zier B.M., Buckles E.L., et al., Bat white- 411–414 nose syndrome: An emerging fungal pathogen? [10] Reichard J.D., Kunz T.H., White-nose syndrome Science, 2009, 323, 227 inflicts lasting injuries to the wings of little brown [5] Gargas A., Trest M.T., Christensen M., Volk myotis (Myotis lucifugus), Acta Chiropterol., 2009, T.J., Blehert D.S., Geomyces destructans sp. 11, 457–464 nov. associated with bat white-nose syndrome, [11] Meteyer C., Barber D., Mandl J., Pathology in Mycotaxon, 2009, 108, 147–154 euthermic bats with white nose syndrome suggests [6] Minnis A. M., Linder D. L., Phylogenetic evaluation a natural manifestation of immune reconstitution of Geomyces and allies reveals no close relatives inflammatory syndrome, Virulence, 2012, 3 (7), of Pseudogymnoascus destructans, comb. nov., 1-6, http://dx.doi.org/10.4161/viru.22330 in bat hibernacula of eastern North America, [12] Willis C.K.R., Menzies A.K., Boyles J.G., Fungal Biol., 2013, 117, 638-649 Wojciechowski M.S., Evaporative water loss is

442 K. Sachanowicz et al.

a plausible explanation for mortality of bats from fungus (Geomyces destructans) in bats, Europe, White-nose syndrome, Integr. Comp. Biol., 2011, Emerg. Infect. Dis., 2010, 16, 1237-1243 51(3), 364-373 [20] Puechmaille S., Wibbelt G., Korn V., Fuller H., [13] Reeder D.M., Frank C.L., Turner G.G., Meteyer Forget F., Mühldorfer K., et al., Pan-European C.U., Kurta A., Britzke E.R., et al., Frequent Arousal distribution of white-nose syndrome fungus from Hibernation Linked to Severity of Infection and (Geomyces destructans) not associated with Mortality in Bats with White-Nose Syndrome, PLoS mass mortality, PLoS ONE, 2011, 6(4), e19167, ONE, 2012, 7(6), e38920, doi:10.1371/journal. doi:10.1371/journal.pone.0019167 pone.0038920 [21] Pikula J., Bandouchova H., Novotný L., Meteyer [14] Frick W. F., Pollock J. F., Hicks A., Langwig K., C.U., Zukal J., Irwin N.R., et al., Histopathology Reynolds D. S., Turner G., Butchowski C., Kunz confirms white-nose syndrome in bats in Europe, T. H. 2010. An emerging disease causes regional J. Wildlife Dis., 2012, 48, 207-211 population collapse of a common North American [22] Warnecke L., Turner J.M., Bollinger T.K., Lorch bat species. Science 329: 679-682 J.M., Misra V., Cryan P.M., et al., Inoculation [15] Thogmartin W.E., McKann P., King R.A., Szymanski of bats with European Geomyces destructans J.A., Pruitt L., Population-level impact of white- supports the novel pathogen hypothesis for the nose syndrome on the endangered Indiana bat, J. origin of white-nose syndrome, P. Natl. Acad. Sci. Mammal., 2012, 93(4), 1086–1098 USA, 2012, 109, 6999–7003 [16] Ford W. M., Britzke E. R., Dobony C. A., Rodrigue [23] Caputa Z., Influence of external conditions on the J. L., Johnson J. B., Patterns of acoustical activity Microclimate in a Portion of the Tarnowskie Góry- of bats prior to and following white-nose syndrome Bytom Underground System, In: The influence of occurrence, J. Fish Wildl. Manage., 2011, 2(2): environmental conditions on the bat hibernaculum 125–134 choice, Association of Public Ecological – [17] Puechmaille S.J, Verdeyroux P., Fuller H., Ar Recreational – Sports Movement in Bytom-Sucha Gouilh M., Bekaert M., Teeling E.C., White-nose Góra, 2008 syndrome fungus (Geomyces destructans) in [24] Langwig K. E., Frick W. F., Bried J. T., Hicks A. bat, France, Emerging Infect. Dis., 2010, 16, C., Kunz T. H., Kilpatrick A. M., Sociality, density- 290–293 dependence and microclimates determine the [18] Martínková N., Bačkor P., Bartonička T., Blažková persistence of populations suffering from a novel P., Červený J., Falteisek L., et al., Increasing fungal disease, white-nose syndrome, Ecol. Lett., Incidence of Geomyces destructans Fungus in 2012, 15, 1050–1057 Bats from the Czech Republic and Slovakia, PLoS [25] Sachanowicz K., Ciechanowski M., Piksa K., ONE 5(11), 2010, e13853, doi:10.1371/journal. Distribution patterns, species richness and status pone.0013853 of bats in Poland, Vespertilio, 2006, 9–10:151–173 [19] Wibbelt G., Kurth A., Hellmann D., Weishaar M., [26] Urbańczyk Z., Northern Europe’s most important Barlow A., Veith M., et al., White-nose syndrome bat hibernation site, Oryx, 1990, 24, 30-34

443