**4. White‐nose syndrome: a threat to bat populations hibernating in caves**

Bats are threatened by a range of both natural and anthropogenic stressors, including preda‐ tion, lack of food, pathogenic agents, climate change, habitat loss, ecological disasters, illegal trade, chemical and light pollution, roosts and hibernacula disturbance, and wind turbine construction. Considering their economic importance to agriculture, the general decline in bat populations documented around the world is of some importance [70]. Recently, a novel threat to insectivorous bat species hibernating in caves and mines has been recognized in North America [71, 72]. White‐nose syndrome (WNS), a fungal infection characterized by fungal growth on the bat's muzzle (**Figure 12A**), has caused a dramatic decline in American bat populations. *Pseudogymnoascus destructans*, the causative agent, is a psychrophilic fungus [73] that thrives at the body temperatures displayed by bats in torpor [74].

Despite intensive research, the origin of the pathogenic agent associated with this disease remains unknown and it is still not known why the disease appeared so suddenly [75]. The disease was first registered as a point‐source epidemic at Howe's Cave, Albany, New York, in 2006 [71], since when it has spread westward at approx. 200–900 km annually [76]. Based on the 'novel pathogen hypothesis', Europe was initially thought to be the source of the agent which the following findings tending to suggest that WNS did indeed originate in Europe: (1) a single *P. destructans* genotype was identified in North American hibernating bats [77]; (2) the WNS fungal agent was also found in bats in European bats [78]; (3) no mass mortality events

**Figure 12.** Skin infection with white‐nose syndrome fungus. Greater mouse‐eared bat (*Myotis myotis*) showing fungal growth on the muzzle, ears and forearm photographed in April 2016 (A). Transillumination of the wing membrane by ultraviolet light: a technique to visualize and detect wing membrane lesions based on yellow fluorescence of riboflavin produced by the white‐nose syndrome agent *Pseudogymnoascus destructans* in the skin of affected bats (B). The scale is the same for both pictures. Photos by Jiri Pikula.

were reported in European hibernating bats harbouring the causative agent [79–81]; (4) inoculation with European fungus isolates induced WNS in the North American the little brown bat *Myotis lucifugus* [82]; and (5) European *P. destructans* isolates exhibited higher genetic diversity [83, 84]. However, recent findings of both the causative agent and WNS infection in Asia have refuted most of the hypothesis arguments and pushed the search for the pathogen source to non‐European hibernacula [85, 86]. Likewise, the detection of bat WNS in western North America in March of 2016 does not fit the previously documented pattern of *P. destructans* geographic spread [76].

Diagnosis of WNS is based on identification of the fungal agent growing on bats using cul‐ tivation, morphological characteristics (e.g. crescent shaped conidia), and molecular assays [73, 87]. One of the most useful diagnostic methods is wing membrane transillumination with ultraviolet (UV) light, which reveals fluorescent lesions produced by the infection [88]. The method is non‐lethal, can be used under field conditions and, in combination with photogra‐ phy, can be used to estimate infection intensity (**Figure 12B**) as it is highly sensitive and spe‐ cific for WNS. Histopathology findings of typical cupping erosions (**Figure 13**) are the gold standard of WNS diagnosis [89, 90].

**Figure 13.** Microscopic appearance of white‐nose syndrome infection. Thick‐type skin of the muzzle extensively infected with *Pseudogymnoascus destructans* fungus (black arrow). Greater mouse‐eared bat (*Myotis myotis*) sampled in a hibernaculum (Czech Republic) in May 2016 (A). Thin‐type skin of the wing membrane with a white‐nose syndrome pathognomonic cupping erosion that contains densely packed fungal hyphae (black arrows, B). The same bat specimen as in (A). Both skin sections stained with periodic acid‐Schiff stain.

Surprisingly, the WNS fungal infection is restricted to the skin only, with no evidence of systemic fungal invasion in infected bats [71, 89]. Hence, bat mortality is thought to follow complex pathophysiological mechanisms, and a multi‐stage WNS model has recently been proposed to explain the disease's progression [91]. Hibernating bats positive for WNS have been reported as displaying abnormal behaviour, higher arousal frequency from torpor, ema‐ ciation and fat depletion, dehydration, acidosis and electrolyte disbalance [82, 92–94]. The extent of wing pathology in infected bats appears to be directly related to mortality [95]. In general, Palearctic bats tend to have a lower disease intensity (measured as the percentage of wing membrane area affected by WNS lesions) than Nearctic bat species [96], possibly explaining the intercontinental differences in bat mortality.

Riboflavin or vitamin B2 is the main compound responsible for the distinctive orange‐yellow fluorescence observed under UV light (**Figure 12B**) after invasive *P. destructans* growth has replaced living tissues. Pathogenic *P. destructans* strains produce considerably more ribofla‐ vin than non‐pathogenic *Pseudogymnoascus* spp. strains. Importantly, high riboflavin concen‐ trations accumulated in WNS skin lesions are toxic to cells under conditions typical for bat hibernation and euthermia. As such, riboflavin may act as a key virulence factor for WNS [96].

were reported in European hibernating bats harbouring the causative agent [79–81]; (4) inoculation with European fungus isolates induced WNS in the North American the little brown bat *Myotis lucifugus* [82]; and (5) European *P. destructans* isolates exhibited higher genetic diversity [83, 84]. However, recent findings of both the causative agent and WNS infection in Asia have refuted most of the hypothesis arguments and pushed the search for the pathogen source to non‐European hibernacula [85, 86]. Likewise, the detection of bat WNS in western North America in March of 2016 does not fit the previously documented pattern of

**Figure 12.** Skin infection with white‐nose syndrome fungus. Greater mouse‐eared bat (*Myotis myotis*) showing fungal growth on the muzzle, ears and forearm photographed in April 2016 (A). Transillumination of the wing membrane by ultraviolet light: a technique to visualize and detect wing membrane lesions based on yellow fluorescence of riboflavin produced by the white‐nose syndrome agent *Pseudogymnoascus destructans* in the skin of affected bats (B). The scale is the

Diagnosis of WNS is based on identification of the fungal agent growing on bats using cul‐ tivation, morphological characteristics (e.g. crescent shaped conidia), and molecular assays [73, 87]. One of the most useful diagnostic methods is wing membrane transillumination with ultraviolet (UV) light, which reveals fluorescent lesions produced by the infection [88]. The method is non‐lethal, can be used under field conditions and, in combination with photogra‐ phy, can be used to estimate infection intensity (**Figure 12B**) as it is highly sensitive and spe‐ cific for WNS. Histopathology findings of typical cupping erosions (**Figure 13**) are the gold

*P. destructans* geographic spread [76].

same for both pictures. Photos by Jiri Pikula.

66 Cave Investigation

standard of WNS diagnosis [89, 90].

As *P. destructans* is a generalist pathogen, all bat species hibernating within contaminated caves may be at risk of infection [97]. However, adverse population‐level effects depend on the species and appear to differ considerably between North America and Eurasia. Hibernating Palearctic bats appear to have evolved infection tolerance mechanisms to cope with the ende‐ micity and extensive spatial distribution of virulent *P. destructans* in the Palearctic region [86]. These mechanisms include behavioural adaptations, such as specific patterns of hibernation and shelter selection [29] that ensure low pathogen impact. While our knowledge of this threat is growing, there are still numerous unanswered questions that require study, at the local and global levels.
