Peromyscus Maniculatus ) in a White-Nose Syndrome Positive Bat Hibernaculum in Eastern Canada KAREN J

Psychrotolerant Microfungi Associated with Deer Mice ( Peromyscus maniculatus ) in a White-nose Syndrome Positive Bat Hibernaculum in Eastern Canada KAREN J. V ANDERWOLF 1, 2, 3, 4 , D AVID MALLOCH 1, and DONALD F. M CALPINE 1 1Department of Natural Science, New Brunswick Museum, 277 Douglas Avenue, Saint John, New Brunswick E2K 1E5 Canada 2Canadian Wildlife Federation, 350 Michael Cowpland Drive, Kanata, Ontario K2M 2W1 Canada 3Current address: Department of Pathobiological Sciences, University of Wisconsin, 1656 Linden Drive, Madison, Wisconsin 53706 USA 4Corresponding author: [email protected] Vanderwolf, Karen J., David Malloch, and Donald F. McAlpine. 2017. Psychrotolerant microfungi associated with Deer Mice (Peromyscus maniculatus ) in a white-nose syndrome positive bat hibernaculum in eastern Canada. Canadian Field- Naturalist 131(3): 238 –245. https://doi.org/10.22621/cfn.v131i3.1906 With the exception of recent work on bats, no reports on the fungi present on live mammals in underground habitats have been published. We cultured psychrotolerant fungi from the external surface and faeces of live Deer Mice ( Peromyscus man - iculatus ), and from the intestinal contents of a single freshly killed P. maniculatus , overwintering in a white-nose syndrome positive bat hibernaculum and from adjacent summer forest in eastern Canada. A low diversity of psychrotolerant fungi was cultured from P. maniculatus compared with that found in previous studies of the mycoflora of bats and arthropods occupy - ing bat hibernacula in the region. Although the grooming habits of P. maniculatus may reduce the accumulation of a diverse psychrotolerant fungal assemblage on their external surface, we demonstrate that active euthermic mammals in underground habitats can carry viable spores of psychrotolerant fungi, both externally and internally. Small rodents using cave habitats may also play a role in dispersing psychrotolerant fungi between caves and suitable low-temperature habitats (i.e., burrows) in adjacent forest. Key Words: Pseudogymnoascus destructans ; Deer Mouse; Peromyscus maniculatus ; cave fungi; cave mycota; cold-tolerant fungi; fungal dispersal; white-nose syndrome Introduction afield in late summer (Fenton 1970; Trevor-Deutsch Mammals introduce organic matter, including fungal 1973). Here we report on psychrotolerant fungi asso - spores, into underground habitats, where nesting mate - ciated with overwintering P. maniculatus using cave rial, food caches, scat, carcasses, and shed hair and skin habitat in eastern Canada, where overwintering bat pop - serve as substrates for various fungi (Nelson and Smith ulations were severely reduced after the 2011 arrival 1976; Jurado et al. 2010). The introduction to North of the fungus Pseudogymnoascus destructans to the area American caves and cave-like habitats (i.e., mines; here - (McAlpine et al. 2011). As a comparison, during the after we include such habitats under the generic term summer months, we also sampled fungi on mice from “caves”) of the psychrotolerant (cold-tolerant) fungus forest adjacent to this cave habitat. Pseudogymnoascus destructans , causative agent of the lethal bat disease, white-nose syndrome (WNS; Lorch Methods et al. 2011), has prompted increased interest in the my - Winter Sampling cology of caves. However, with the exception of recent Peromyscus maniculatus were live-trapped in Dorch - work focusing on bats (Johnson et al. 2013; Vander - ester Mine, an abandoned copper mine and bat hiber - wolf et al. 2013, 2016a; Lorch et al. 2015), no reports naculum near Sackville, New Brunswick, 11–14 March on the fungi present on live mammals in caves have 2014 (42 trap nights). Two trap sizes were used: 5.1 × been published. In addition, the literature on how psy - 6.4 × 16.5 cm and 7.7 × 8.9 × 22.9 cm (H. B. Sherman chrotolerant fungi might be dispersed from cave habi - Traps, Inc., Tallahassee, Florida, USA). All traps were tats is limited (Stephenson et al. 2007; Vanderwolf et al. soaked in fungicide and rinsed prior to sampling. Traps 2016a,b). were baited with a mixture of peanut butter and oats, Deer Mice ( Peromyscus maniculatus Wagner, 1845) furnished with cotton nesting material, checked daily, are a common and widespread North American small and re-baited as required. Traps were placed on the floor rodent that may reside in small numbers in caves, where and on ledges along the walls adjacent to a mouse nest available, during the winter (Trevor-Deutsch 1973). Dur - (1–2 m above the floor; Figure 1A), approximately 45– ing the warmer months, this species disperses into sur - 80 m from the mine entrance. The temperature in Dor - rounding woodland, staying relatively close to cave en - chester Mine was measured using ibuttons (model trances in spring and early summer and ranging farther DS1920-F5, Maxim Integrated Products Inc., Sunny - 238 ©The Ottawa Field-Naturalists’ Club (2017) 2017 VANDERWOLF ET AL .: MICROFUNGI ASSOCIATED WITH DEER MICE 239 vale, California, USA) in the manner of Vanderwolf were spread on separate petri plates containing DPYA et al. (2012). medium with no dilution. Each trapped mouse was transferred to a fresh plastic Faeces produced by mice held in bags were trans - bag for swabbing by inverting the trap over the bag. ported to the lab for processing. Faeces (~3 pellets per Two swabs per mouse were taken using a new, sterile, mouse) were suspended in 100 mL of autoclaved water dry, cotton-tipped applicator for each swab. The swabs and vigorously shaken for 10 minutes. The sample was were rubbed over the fur both dorsally and ventrally. then serially diluted five times, with 10 mL of each suc - Af ter swabbing, the applicator was immediately streaked cessive solution mixed with 90 mL of water. For each across the medium surface in a petri plate. Three dilut - of the five dilutions, plus the undiluted sample, 10 mL ing streaks were completed in the mine within 1 h of the were spread over the surface of separate petri plates initial streak, after which plates were sealed in situ with containing hardened DPYA medium. parafilm (Pechiney Plastic Packaging, Chicago, Illi - Summer Sampling nois, USA). Two media types were inoculated for each On the night of 27 August 2014, mice were trapped mouse: dextrose–peptone–yeast extract agar (DPYA) with live traps (100 trap nights) baited with bird seed and Sabouraud–dextrose agar (SDA), both of which adjacent to the entrance of Dorchester Mine. All traps were infused with the antibiotics chlortetracycline (30 were soaked in fungicide and rinsed the day before use. mg /L) and streptomycin (30 mg/L). Mice were not Mice were processed using the methods described directly handled or marked during any part of the pro - above, except that faeces and intestinal contents were cedure and were immediately released after sampling. not collected. Three mice were swabbed twice, with one One mouse was removed from the mine after swabbing swab inoculated on DPYA medium and the other on of its external surface (New Brunswick Museum spec - SDA medium. For all other mice, only one swab was imen 12946), euthanized using isoflurane, and the con - taken and inoculated on DPYA medium because of con - tents of its stomach, small intestine, and large intestine tamination issues with the SDA medium. FIGURE 1. A. Deer Mouse ( Peromyscus maniculatus ) nesting material (a source and substrate for fungal spores) and a live trap in place on a wall ledge in Dorchester Mine near Sackville, New Brunswick. B. In winter, P. maniculatus were active in the dark zone on wall ledges and the floor of the mine. Photos: K. J. Vanderwolf. 240 THE CANADIAN FIELD -N ATURALIST Vol. 131 Fungal Culturing and Data Analysis (Table 1). Most fungal taxa were obtained from the un - In the laboratory, samples were incubated, inverted, diluted sample and the first dilution; the fifth dilution in the dark at 7°C in a low-temperature incubator (Mod - produced no cultures. Seven fungal taxa plus one sterile el 2015, VWR International, Mississauga, Ontario, morph were isolated from mouse gut contents. Can ada) to approximate the subterranean environment The mean temperature in Dorchester Mine during the and target psychrotolerant fungi. Samples were mon - sampling period, March 2014, was −0.98°C (SD 1.24) itored over four months until either no new cultures had in the twilight zone and 6.63°C (SD 0.00) in the dark appeared for three weeks, or the plate had become over - zone. The twilight zone ibutton was located 3 m inside grown with hyphae. Once fungi began growing on the the entrance. The mean temperature outside the mine, plates, each distinct colony was subcultured to a new approximately 20 m from the entrance and above snow plate. DPYA without oxgall and sodium propionate was cover, was −2.49°C (SD 5.31). Mice were generally ob - used for maintaining pure cultures (Figure 2). Identi - served on wall ledges and the floor deeper in the mine fications were carried out by comparing the micro- and (Figure 1B) where air temperatures were warmer, and macro-morphological characteristics of the microfungi where they were subsequently captured. to those traits appearing in the taxonomic literature and Summer Sampling compendia (Domsch et al. 2007; Seifert et al. 2011) and Twenty-two fungal taxa plus five sterile morphs were by comparing isolates to a reference collection of fungi cultured from 15 mice, with a mean of 4.87 fungal taxa assembled from previous studies in underground habi - per mouse (SD 2.13, range 2–8). Female mice ( n = 8) tats in the region, which were identified using a mix of carried a mean of 4.75 (SD 2.31) fungal taxa and males morphological and molecular methods (Vanderwolf et (n = 5) carried 4.6 (SD 2.30). Two mice escaped before al. 2013, 2016a,b). Permanent desiccant-dried vouchers sex was determined. The second swab contributed two of the collected fungi are deposited in the New Bruns - fungal taxa (SD 2, range 0–4, n = 3 mice) that were wick Museum mycological collection (NBM numbers not detected with the first swab.

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