Donaldson, Michael

Molecular and genomic tools provide a deeper understanding of the ecology and evolution of species and their capacity to adapt to changing selective pressures, where diversity is presumedly linked to higher fitness and evolutionary potential. Molecular tools can also illustrate how historical processes affect contemporary genetic variation to predict how current population trends may influence future genetic diversity. Genomic investigations increasingly extend beyond variation within host genomes to include diversity of their associated microbiomes, recognized to influence host/environment interactions and adaptation. Muskoxen (Ovibos moschatus) are iconic, Arctic herbivores of ecological, economic, and cultural significance. Demographically, most mainland muskox populations have remained stable or grown over the last century, yet the biggest herds, found on Victoria and Banks Islands (Nunavut and the Northwest Territories, Canada) have experienced recent and drastic population declines. These Arctic island population declines have been associated with warming trends leading to shifting ranges of forage biodiversity, and pathogen expansions directly associated with increased mortality. Genomic investigations have the potential to enhance understanding of these contrasting trends and the adaptive capacities of muskox to cope with rapid ecological change. In this thesis, I assess genetic, genomic, microbiome and diet diversity to better understand the ecology, and evolution of muskoxen. I found extremely low levels of genetic variation associated with population bottlenecks coinciding with major glaciation events and contemporarily low levels of gene flow among populations. Whole genome analyses identified signatures of selection between muskox populations, providing a genetic basis for the divergence of two previously proposed muskox subspecies. Significant differences in diversity, effective population size and inbreeding among subspecies suggests animals from Arctic islands and Greenland are more vulnerable to environmental change. Molecular investigations of diet and microbiome diversity reflected unique capacities of muskoxen to survive on high-fiber forage and exploit shifts in Arctic vegetation that may include continued shrubification. Overall, these data provide insight into the complex relationship between genetic diversity and changing environments, setting a foundation for expanded future investigations of muskox seeking to promote the future viability of this species.

Author Keywords: Genetic Diversity, Genome Assembly, Metabarcoding, Microsatellites, Muskox, Persistence

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Heterogeneous environments impose discordant selective pressures on natural populations, where disparate biotic/abiotic factors and variable population connectivity, yield mosaic patterns of genetic variation on the landscape. The ability to maintain or change genetic mosaics of populations becomes key to persistence, as species increasingly need to adapt to rapidly changing environmental and human-associated selective pressures. Specifically, infectious diseases can impose strong and rapid selective pressures on populations, where anthropogenic disruptions of co-evolutionary patterns and altered distributions of hosts and pathogens exacerbate disease risk. Genomic tools provide means to evaluate disease-associated impacts on the genetic landscape of host populations and facilitate implementation of informed conservation efforts. In this thesis, I evaluate disease dynamics in: 1) a long-standing arctic rabies/arctic fox (Vulpes lagopus) system affected by influxes of red fox (V. vulpes), and 2) an invasive bat pathogen system, where the North American introduction of Pseudogymnoascus destructans (Pd) has had variable impacts on bat species and populations. In these systems, signatures of host selection were estimated from temporal and spatial shifts in allelic diversity within genomic regions associated with immune response, highlighting different host mechanisms to enzootic and invasive diseases. In the arctic rabies/fox system, pathogen variants did not influence red fox local disease responses, reflecting more recent expansions of this host to Arctic regions. In contrast, arctic fox revealed genomic patterns consistent with long-term, co-evolutionary processes. In Pd/bat systems, genomic evidence supported the hypothesis that eastern small-footed bats (Myotis leibii) were inherently resistant or tolerant to Pd, the causative agent of white-nose syndrome (WNS). In contrast, WNS-impacted little brown bat (M. lucifugus) populations had varied genomic impacts subsequent to strong selective sweeps from disease. My research illustrates how immunogenetic profiling, in context of demographic processes inferred from neutral genetics, enhances understanding of the varied impacts of changing disease landscapes on host populations/species; insights relevant to other host-pathogen systems. Building on this thesis, future explorations of low coverage genomes, host-imposed reciprocal selection, and impacts on methylation, transcriptomic and proteomic patterns associated with shifts in genetic diversity, would enable more holistic understanding of the geographic mosaics within these disease systems.

Author Keywords: Disease Dynamics, High-throughput Sequencing, Immune System, Natural Selection, Population Genetics, Reduced Representation Sequencing

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