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.