Fungal sampling of a maternity roost of Big Brown Bats (Eptesicus fuscus) on the Baca National Wildlife Refuge.

Erin M Lehmer, Stephen Fenster & Kirk Navo Background

The initial research was focused on sampling fungal community diversity on the migratory Mexican free-tailed bat (Tadarida brasiliensis) population from the Orient Mine upon arrival and prior to departure from Colorado. However, in June 2015 because of cold spring temperatures and higher than average precipitation, arrival of the free-tailed population was delayed, and we were unable to capture bats after repeated sampling efforts. Because of these failed efforts, it was decided to move to the nearby Baca National Wildlife Refuge in an attempt to capture resident (i.e. non-migratory) bats, using a stacked mist net system. During the single night of sampling at the Baca NWR, we captured 32 adult female big brown bats (Eptesicus fuscus) from a single maternity roost located in the attic of an abandoned outbuilding on the refuge property. These bats were processed in the same manner that we had processed the free-tailed bats in previous seasons; after capture, they were weighed, sex and reproductive condition were determined, and forearm lengths were measured. Fungal spores were collected by swabbing the wing membranes and dorsal and ventral fur with sterile cotton swabs dipped in sterile water. During routine processing of the fungal spores (i.e. culturing, PCR and DNA sequence barcoding analysis), we determined that 2 of the samples were a very close genetic match to P. destructans based on sequence alignment data of the internal transcribed spacer (ITS) region of the genome. A sequence alignment for 2 of the initial samples is shown in Figure 1. An additional 8 samples collected from the big browns were identified as belonging to either the genus Psuedogymnoascus or the genus Geomyces during initial genetic analysis (i.e. sequencing of the ITS region; Table 1). These results were noteworthy, as they demonstrated for the first time that fungi belonging to the genus Pseudogymnoascus were present on bats in Colorado, including fungi that were a very close match (i.e. 99% genetic similarity) to P. destructans. However, because these findings were based on only a single region of the genome, sequencing of additional regions was needed to confirm the identity of the Pseudogymnoascus at the species level. As such, these 10 samples were selected for further analyses.

Table 1. All potential samples identified as Pseudogmnoascus or Geomyces based on sequencing of the ITS region from big brown bats (Eptesicus fuscus) captured in the San Luis Valley, CO, June 2015.

Sample Bat Wing or Body Temperature Growth (°C) Genus Species Percent Overlap 5 73 Wing 8 Pseudogymnoascus destructans 99.80% 9 66 Body 8 Pseudogymnoascus sp J AM 2013 99.61% 12 79 Body 8 Geomyces sp F12 99.57% 13 77 Wing 8 Geomyces pullulans 100.00% 14 76 Wing 8 Geomyces sp 04NY10 99.80% 46 82 Wing 8 Geomyces pullulans 100.00% 95 77 Wing 8 Geomyces pullulans 100.00% 99 65 Body 8 Geomyces pannorum 100.00% 135 65 Body 8 Pseudogymnoascus pannorum 100.00% 138 79 Body 8 Pseudogymnoascus destructans 99.80%

Methods Culture and Morphological Analysis The 10 fungal isolates initially determined to be a close genetic match to Pseudogymnoascus were re-cultured in duplicate in 100 mL of sabouraud broth and then transferred to sabouraud dextrose agar plates containing streptomycin and tetracycline for further analysis; plates were incubated at 8˚C and 20˚C. The remaining inoculated broth was frozen at -80˚ C for future use. Photos of the gross morphology were taken every 2 - 3 days to monitor growth, using a standard digital camera. For the first two weeks, stains (i.e. microscope slides) of reproductive structures were done every time a digital picture was taken, using methylene blue. After this, stains were only done when a significant morphological change occurred. Photos of the stains were taken using a fluorescent light microscope and the program Cell Sense.

DNA Purification, PCR and Sequence Barcoding Analysis Following incubation (14-40 days) fungal samples were suspended in 293 µL of EDTA then homogenized using a Q55 Sonicator at 40 amps. Genomic DNA was extracted from samples using the Wizard© Genomic DNA Purification Kit yeast protocol (Promega Corp., Madison, Wisconsin). The manufacturer’s instructions were followed explicitly excluding the addition of lyticase to the samples.

To more precisely identify the genetic identity of the 10 “suspect” samples, PCR was used to amplify the large subunit (LSU) and intergenic spacer (IGS) regions. Primers (Invitrogen©, Waltham, Massachusetts) and thermocycler conditions were modified from Shoch et al. (2012) and Muller et al. (2012) to promote a better polymerization reaction. For IGS Long fragments, initial denaturation occurred at 98°C for 2 min followed by 34 cycles of 98°C for 10s, 50°C for 50s, 72°C for 2 min 40s, 72°C for 4 min 20s, and final extension at 12°C. For IGS Short, ITS, and LSU fragments initial denaturation occurred at 98°C for 30s followed by 29 cycles of 98°C for ten seconds, 55°C for 30s, 72°C for 30s, 72°C for 3 min, and final extension at 12°C. The master mix for all reactions contained 2.5 µL DNA with 1 µL of each forward and reverse primer, 12.5 µL of 2x Blue Taq Master Mix (New England Biolab©, Ipswich, Massachusetts), and 8 µL of deionized water for a total volume of 25 µL. Products were cleaned using ExoSAP-IT using the manufacturer’s protocol (Affymetrix©, Santa Clara, California) and sent to Functional Biosciences (Madison, Wisconsin) to obtain DNA sequence data. The results were processed using CLC Main workbench 7 (Qiagen) and sequences were aligned using FungalBarcoding.org and NCBI nucleotide blast. DNA sequence samples were identified to the species level if there was a minimum of a 99% overlap between sequences, genus level if there was a 95% overlap, family level if there was a 90% overlap, class level if there was an 85% overlap, and order level if there was less than an 80% overlap.

Results Growth of fungal samples on the sabouraud agar plates occurred at both 20° C (Figure 2) and 8° C (Figure 3), with the 8° C growing slower as seen in Figures 1 and 2. Individual colonies were white for a majority of their growth period up until 15 days, when they started to turn grey. Their edges had a fuzzy appearance, initially with convex surfaces. Ten days after inoculation, colonies began to form ring-like structures around their edges. Fifteen days later, the ring structures became thicker and the colonies began to produce secretions.

Figure 3. Morphological characteristics of Pseudogymnoascus isolates Figure 2. Morphological characteristics of grown at 8 °C. A) Sample #12 three days AI, B) Sample #12b three days Pseudogymnoascus isolates grown at 20 °C. A) Sample #12 AI, C) Sample #138 three days AI, D) Sample #12 five days AI, E) Sample three days after inoculation (AI), B) Sample #138 three days #12 seven days AI, F) Sample #138 seven days AI, G) Sample #12 ten AI, C) Sample #12 five days AI, D) Sample #138 five days AI, days AI, H) Sample #12b ten days AI, I) Sample #138 ten days AI. E) Sample #12 seven days AI, F) Sample #138 seven days AI, G) Sample #12 ten day AI, H) Sample #138 ten days AI.

Stains using methylene blue allowed us to visualize fungal spores. On samples grown at 8 °C, the hyphae were filamentous (Figure 4A) with branching conidiophores (Figure 4B, C). Extending from the conidiophores were groups of spherical conidia occurring as a single unit or as a small chain of conidia (Figure 4C). Samples cultured at 20 °C had hyphae that were larger in diameter and started to become septate after 5 days in incubation (Figure 5 G, H). The conidia of the 20 °C samples had a similar spherical shape (Figure 5 A, B, C).

Figure 4. Methylene blue stains of fungal spores of potential Pseudogymnoascus isolates 10 days after inoculation. A) Sample #138 grown at 8°C, B) Sample #12 grown at 8°C, C) #138 grown at 20°C, D) Sample #12 grown at 20°C.

Figure 5. Methylene blue stains of fungal spores of potential Pseudogymnoascus isolates. A) Sample #138 3 days after inoculation, grown at 20°C, B) Sample #138 3 days after inoculation, grown at 20°C, C) Sample #12 3 days after inoculation, grown at 20°C, D) Sample #138 5 days after inoculation, grown at 8°C, E) Sample #12 5 days after incubation, grown at 20°C, F) Sample #12 5 days after incubation, grown at 20°C, G) Sample #12 5 days after incubation, grown at 20°C, H) Sample #138 5 days after incubation, grown at 20°C.

Amplification of IGS and LSU regions of the “suspect” fungal isolates was successful. DNA sequence barcoding analysis for the IGS region identified all (100%) samples as matches to unknown Pseudogymnoascus species, whereas the LSU sequences were a mixture of matches to P. pullulans (33.3%) and P. pannorum (77.8%; Table 2). Sequence alignments comparing the IGS regions of the 10 “suspect” samples to Pd are shown in Figure 6; sequence alignments comparing the LSU regions of the suspect samples are shown in Figure 7.

Table 2. DNA sequence results of the IGS and LSU genes of our fungus sampled from big brown bats (Eptesicus fuscus) in the San Luis Valley, CO, June 2015.

Gene Sample Bat Genus Species IGS 5 73 Pseudogymnoascus unknown 9 66 Pseudogymnoascus unknown 14 76 Pseudogymnoascus unknown 46 82 Pseudogymnoascus unknown 99 65 Pseudogymnoascus unknown 135 65 Pseudogymnoascus unknown 138 79 Pseudogymnoascus unknown LSU 5 73 Pseudogymnoascus pannorum 9 66 Pseudogymnoascus pannorum 13 77 Pseudogymnoascus pullulans 14 76 Pseudogymnoascus pannorum 46 82 Pseudogymnoascus pullulans 95 77 Pseudogymnoascus pullulans 99 65 Pseudogymnoascus pannorum 135 65 Pseudogymnoascus pannorum 138 79 Pseudogymnoascus pannorum

Discussion Based on both morphological characteristics and sequence analysis of the IGS and LSU regions, it is not likely that the 10 “suspect” fungal samples collected from big brown bats at the Baca NWR were P. destructans. However, because of inconsistencies in growth patterns of this species of fungus, as well as limited information in fungal barcoding databases, these results should be interpreted with caution.

Gross morphology of the psychrophilic fungi belonging to the genus Pseudogymnoascus is still largely unexplored. Previous studies have attempted to characterize growth of P. destructans under a range of environmental conditions with varying results. In our study, we found that the morphology of “suspect” fungal samples did not vary greatly when cultured at different temperatures. Isolates grown at 8° C were slow growing compared to those grown at 20° C, but they displayed similar growth characteristics. Near the end of 40 days of growth, the 20°C samples began to show changes in morphology, indicating that perhaps after a longer period of growth differences in the phenotype would be observed. It was surprising that immediate phenotypic differences were not observed, as several previous studies have indicated that P. destructans not only displays differences in phenotype according to the environment in which it is grown but also displays atypical morphology at any temperature above 12° C (Khanket et al., 2014; Verant et al., 2012). The fact that our 20°C samples did not display morphology of tangible variance from those grown in 8°C casts doubts on these unknown species being P. destructans.

Optimal growth temperatures for P. destructans have been recorded between 4oC and 12°C, however there is some discrepancy in what the actual upper critical growth temperature is, with some researchers reporting no growth above 19.8oC (Verant et al. 2012) and others reporting no growth above 25°C (Chaturvedi et al., 2010). While we observed growth at 8° C, our samples (genetically identified as Pseudogymnoascus) most readily grew at 20°C, a characteristic uncommon for psychrophilic fungi, including those belonging to the genus Psedugymnoascus. Because of such inconsistencies in the data regarding the range of growth temperatures for Pseudogymnoascus, future studies should continue to grow an assortment of fungi belonging to this genus (including Pd) at a range of temperatures to more conclusively determine whether certain temperature ranges truly inhibit growth.

Our “suspect” fungal species shared a number of morphological resemblances with P. destructans. Morphology of P. destructans spores have been previously characterized as having smooth conidiophores 1.5 – 2 µm wide, frequently having verticils with 2 - 4 branches featuring 5 - 12 asymmetrically curved conidia (Gargas et al., 2009). Morphological changes have been observed in the hyphae and conidia at temperatures above the optimal growth range. At 12° C and above, hyphae are reportedly thicker and diffusely septate and conidia reportedly change from a curved shaped to pyriform or globoid and are produced in short chains. As temperatures increase, these changes become more prominent, eventually leading to the total loss of typical characteristics in the hyphae and conidia at above 18° C (Verant et al., 2012). We observed similar morphology in the hyphae of our “suspect” samples to those described by Gargas et al. (2009) and Verant et al. (2012); however, we did not observe the characteristically curved conidia, but rather conidia were more spherical in shape. Rather than seeing the significant level of change with increasing temperature, our samples only showed a slight change in morphology. The 20°C samples were morphologically similar to the 8°C samples for the first three days after inoculation (Figure 5A, B). Soon after, the structures began to degrade (Figure 5 C, F). After being incubated for five days at 20°C, the hyphae began to appear septate and had a wider diameter than the samples grown at 8°C. Again, these results demonstrate the difficulty in using morphological characteristics to definitively identify fungi, and illustrate the variability in growth patterns that exist, even when 2 cultures of very closely related species are grown under identical conditions.

Although initial DNA sequence analysis identified 2 of our “suspect” samples as P. destructans based on the ITS region of the genome, subsequent sequence analysis of the IGS and LSU regions of the genome did not confirm these findings. However, these subsequent analyses did verify that all 10 “suspect” samples belonged to the genus Pseudogymnoascus. Because of variability in the ITS region among species, it has long been used to identify fungi to the species level (Shoch et al. 2012) and, as such, the database for the ITS region of the fungal genome is extensive. For these regions, the ITS region is typically the first region that is sequenced. However, additional regions with high variability among species, like the IGS and LSU, are often sequenced in order to confirm an initial identification (Shoch et al. 2012; Muller et al., 2013). Unfortunately, database information for the LSU and IGS regions are not nearly as extensive as that for the ITS region. This means that a large number of unknown samples cannot be matched to a reference sample, and therefore cannot be identified. In other words, even though the LSU and ITS genes have clearly defined barcoding gaps and significant variability between species, in order to determine the identity of an unknown, one needs a standard to compare it to and these standards are somewhat lacking for the LSU and IGS regions. This was particularly evident in our DNA barcoding analysis of the IGS region, as all 10 samples could be identified to the genus level, but could not be identified to the level of species. Furthermore, each region of the genome that we sequenced (ITS, LSU, IGS) returned different species identification, illustrating the inconsistencies that exist in databases which house genetic sequences for a fungal species. Because amplification of the DNA was successful (confirmed by electrophoresis) and sequence analysis returned clean chromatographs (indicating sequencing was successful) for all 10 “suspect” samples, we are confident that discrepancies in species identification between the three regions of the genome were not the result of our methodology. Rather, these discrepancies were likely the result of fungal databases containing limited information, particularly for the IGS region. Furthermore, because of a lack of consistency in identifying our 10 “suspect” samples between the three regions of the genome, it is possible that some of the samples we analyzed are a novel fungal species that require further morphological and genetic characterization.

The recent identification of Pd in Nebraska illustrates the need for increased monitoring in Colorado, including direct fungal sampling of bats and bat roosting sites. The fact that Pd was identified on big brown bats in Nebraska, and that we identified Pseudogymnoascus in a number of samples collected from big brown bats in Colorado is significant. Although big brown bats may have a natural resistance to WNS (Frank et al. 2014), they are in frequent contact with other susceptible bat species during seasonal roosting, and thus may have opportunities to transfer fungal spores to these individuals. Extending direct monitoring of fungal communities to include a larger number of bat species from a number of locations across the state would help to determine the extent to which fungal spores are shared among different species of bats and whether the composition of the fungal community differs among bats from different regions of the state. In addition to increasing monitoring efforts in Colorado, comparison of sequence data from the Pseudogymnoascus obtained from bats in Nebraska with the data we obtained in Colorado would allow us to determine the degree of variability that exists in the ITS, LSU and IGS regions across different geographic locations. The Baca NWR provides an important location for continued research into the fungal community of Eptesicus fuscus, and insights into the relationship of species of bats and the potential role in transmission of Pd within the State of Colorado. The abandoned house on the refuge, as well as other possible out buildings and structures, provide roosting habitat for bats and conservation of these species. The riparian and wetland habitats found on the Baca NWR provide ideal foraging opportunities for both resident population of bats, and the significant colony of free-tailed bats at the Orient Mine. We strongly recommend the preservation of known roosting habitat, and continued support for WNS research and acoustic monitoring efforts in partnership with the State on the Baca NWR.

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