Mycobiome of the Bat White Nose Syndrome Affected Caves and Mines Reveals Diversity of Fungi and Local Adaptation by the Fungal Pathogen Pseudogymnoascus (Geomyces) destructans

Abstract

Current investigations of bat White Nose Syndrome (WNS) and the causative fungus Pseudogymnoascus (Geomyces) destructans (Pd) are intensely focused on the reasons for the appearance of the disease in the Northeast and its rapid spread in the US and Canada. Urgent steps are still needed for the mitigation or control of Pd to save bats. We hypothesized that a focus on fungal community would advance the understanding of ecology and ecosystem processes that are crucial in the disease transmission cycle. This study was conducted in 2010–2011 in New York and Vermont using 90 samples from four mines and two caves situated within the epicenter of WNS. We used culture-dependent (CD) and culture-independent (CI) methods to catalogue all fungi (‘mycobiome’). CD methods included fungal isolations followed by phenotypic and molecular identifications. CI methods included amplification of DNA extracted from environmental samples with universal fungal primers followed by cloning and sequencing. CD methods yielded 675 fungal isolates and CI method yielded 594 fungal environmental nucleic acid sequences (FENAS). The core mycobiome of WNS comprised of 136 operational taxonomic units (OTUs) recovered in culture and 248 OTUs recovered in clone libraries. The fungal community was diverse across the sites, although a subgroup of dominant cosmopolitan fungi was present. The frequent recovery of Pd (18% of samples positive by culture) even in the presence of dominant, cosmopolitan fungal genera suggests some level of local adaptation in WNS-afflicted habitats, while the extensive distribution of Pd (48% of samples positive by real-time PCR) suggests an active reservoir of the pathogen at these sites. These findings underscore the need for integrated disease control measures that target both bats and Pd in the hibernacula for the control of WNS.

Detecting viable Pseudogymnoascus destructans (Ascomycota: Pseudeurotiaceae) from walls

“…We swabbed cave walls more than 1 m from roosting bats, if any were present, and obtained results with DPYA that generally match or exceed those obtained with culture-independent methods, while also allowing us to confirm the viability of Pd. Our yield of Pd from wall swabs using SAB is similar to Lorch et al (2013) and Zhang et al (2014).…”

“…These inconsistencies may reflect differences in the detection of Pd DNA using cultureindependent approaches compared to culture-dependent methods, which reveal only viable spores, or possibly site differences. Lorch et al (2013) and Zhang et al (2014) found that the probability of detecting Pd was higher using molecular methods compared to culture techniques. Viable Pd was cultured from only 13.8 % (n ¼ 195) of soil samples in Pdpositive hibernacula using SAB (Lorch et al, 2013).…”

“…Viable Pd was cultured from only 13.8 % (n ¼ 195) of soil samples in Pdpositive hibernacula using SAB (Lorch et al, 2013). Zhang et al (2014) obtained viable Pd from 18 % of sediment samples from Pd-positive hibernacula in New York using SAB and Rose Bengal medium. In Estonia, 100 % of wall swabs (n ¼ 4) on SAB yielded Pd, but these swabs were taken within a few centimeters of where one bat with visible white fungal growth had been roosting a few days earlier (Puechmaille et al, 2011).…”

“…Previous studies have produced low yields of viable Pd from Pd-positive hibernacula, with only 13 to 33 % positive samples using culture-dependent methods (Lorch et al, 2013;Martinkova et al, 2010;Zhang et al, 2014), as compared to 44 to 91 % with culture-independent methods (Zhang et al, 2014;Langwig et al, 2015). Lorch et al (2013) found that the probability of detecting Pd in a Pd-positive hibernaculum was 56 % for each sample using real-time PCR, compared to 14% using culture-dependent methods.…”

Diverse Applications of Environmental DNA Methods in Parasitology

“…Sampling of individual hosts to monitor such potential spread is often not feasible (particularly in the cases of migratory birds, mammals, and insects), but eDNA analyses of fecal material has much potential as a host-independent and non-invasive method of parasite monitoring; for example, helminths in rat feces [66], fungal community characteristics associated with the spread of Bat White Nose Syndrome [67], Blastocystis in dog feces [68], and Cryptosporidium species in cattle feces [69]. Hitch-hiking is a particular concern with micro-eukaryotic parasites, which are at least in some cases more easily dispersed than larger metazoans [70,71] and more difficult to find and/or identify in pathology screens of hosts.…”