Donaldson, Michael

White-nose syndrome (WNS) is a skin disease of bats caused by the fungus Pseudogymnoascus destructans (Pd) that damages flight membranes during hibernation and can lead to death. The disease causes mortality of multiple bat species in eastern North America and is spreading into western North America. Future impacts of WNS on naïve bat populations are unknown. Variation in host susceptibility occurs among and within species, but mechanisms driving this variation are unclear. Multiple studies have characterized immunological responses to WNS, but skin physiology as a barrier to pathogens is understudied. The unique ability of Pd to actively penetrate the normal, intact skin of its mammalian host makes WNS an interesting study system to understand skin defenses. Aspects of the mammalian skin environment that can influence disease susceptibility include pH, sebaceous lipids, and microbiomes. I found skin mycobiomes of WNS-susceptible species had significantly lower alpha diversity and abundance compared to bat species resistant to Pd infection. Using these data, I predicted that most naïve bat species in western North America will be susceptible to WNS based on the low diversity of their skin mycobiomes. Some fungi isolated from bat wings inhibited Pd growth in vitro, but only under specific salinity and pH conditions, suggesting the microenvironment on wings can influence microbial interactions and potentially WNS-susceptibility. I measured the wing-skin pH of bats in eastern Canada and found that Eptesicus fuscus (WNS-tolerant) had more acidic skin than M. lucifugus (WNS-susceptible). Differences in sebum quantity and composition among and within mammalian species may help explain variation in skin disease susceptibility and the composition of skin microbiomes. This is due to the antimicrobial properties of sebum and the use of sebum as a nutrition source by microbes. Outcomes of this work further our understanding of inter- and intra-specific differences among bat species and individuals in skin mycobiomes and physiology, which may contribute to variation in WNS-susceptibility. Future research should focus on characterizing the physical and chemical landscape of skin as this is essential for understanding mechanisms structuring skin microbial assemblages and skin disease susceptibility in wildlife.

Author Keywords: bat, fungi, microbiome, mycology, physiology, white-nose syndrome

Fungal pathogens adapt to environmental changes faster than their hosts, due in part to their adaptive mechanisms exhibited in response to stress. Ustilago maydis was used to investigate potential natural antisense transcript (NAT) RNA-mediated mechanisms that enhance fungal adaptation to stress. Of the 349 NATs conserved amongst U. maydis and two related smut fungi, five NATs were identified as having altered transcript levels in response to multiple stress conditions. Subsequently, antisense transcript expression vectors were created for select NATs and transformed into U. maydis haploid cells. When exposed to stress conditions, two antisense expressing mutant strains exhibited alterations in growth. RT-qPCR analysis of mRNA complementary to expressed NATs revealed no significant change in mRNA levels, which suggests NAT expression may influence stress response through dsRNA formation or other RNA mediated mechanisms. These results establish a basis for further investigations into the connection between NATs and the stress response of fungi.

Author Keywords: natural antisense transcripts, non-coding RNAs, stress response, Ustilago maydis

Heavy metal pollution threatens human and ecosystem health. E. gracilis was investigated for its potential use in bioremediation due to its tolerance for heavy metals and ability to sequester them from the environment. E. gracilis can remove metals by producing metal binding compounds enriched in sulfur and nitrogen. In this thesis, E. gracilis cultures that were pretreated with elevated levels of sulfur or nitrogen had increased tolerance to CdCl2 compared to non-pretreated cultures. RNA-sequencing revealed that both pretreatments led to transcript level changes and that exposure to CdCl2 led to further transcript level changes. Gene ontology (GO) enrichment analysis reflected changes in nitrogen and sulfur metabolism as well as physiological processes related to metal binding. The data from this thesis revealed important transcription level changes that occur when E. gracilis is challenged with CdCl2 and helps us understand how organisms adapt to heavy metal pollution in the environment.

Author Keywords: bioremediation, Cadmium, Euglena gracilis, GO-enrichment, metal-binding, RNA-Sequencing

The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they infect a common host (Zea mays), can hybridize, and tools are available for their analysis. Hybrid dikaryons exhibited filamentous growth on plates but reduced virulence and limited colonization in Z. mays. Select virulence genes in the hybrid had similar transcript levels on plates and altered levels during infection of Z. mays relative to each parental dikaryon. Virulence genes were constitutively expressed in the hybrid to determine if its pathogenic development could be influenced. Little impact was observed in hybrids with increased expression of effectors known to modify host response and metabolism. However, increased expression of transcriptional regulators of stage specific pathogenic development increased the hybrid's capacity to induce symptoms. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence.

Author Keywords: effectors, hybrid pathogenesis assays, Sporisorium reilianum, transcription factors, Ustilago maydis, virulence factors

Ophidiomycosis (snake fungal disease) is caused by the pathogen Ophidiomyces ophiodiicola. Infected snakes exhibit dermal lesions, occasional systemic infections, and, in some cases, mortality. We studied snakes at Rondeau Provincial Park, Ontario, Canada, to explore whether ophidiomycosis develops during brumation or year-round. Throughout their active season, we quantified the prevalence of clinical signs of the disease on snakes and conducted qPCR of skin swabs to determine the prevalence of O. ophiodiicola on snakes. Prevalence of O. ophiodiicola and disease symptoms were highest on eastern foxsnakes (Pantherophis vulpinus) and very rare on other snake species. In P. vulpinus, pathogen and clinical sign prevalence was highest, directly after emergence from overwintering, with the majority of P. vulpinus being able to resolve clinical signs of ophidiomycosis by the return of winter. When we analyzed the survivorship of P. vulpinus we determined that the likelihood of a snake dying with ophidiomycosis is similar to a snake dying without ophidiomycosis. Given that P. vulpinus were the most affected species at our study site, ophidiomycosis does not appear to pose an imminent threat to our study population of P. vulpinus under current conditions.

Author Keywords: Eastern Foxsnake, Fitness, Ophidiomycosis, Pantherophis vulpinus, Seasonal trends, Snake fungal disease