My research investigated sialic acid metabolism in the opportunistic fungal pathogen, Aspergillus fumigatus. The sialic acid, N-acetylneuraminic acid (Neu5Ac), is a sugar found on fungal spore cell surface that mediates adhesion to host proteins and phagocytes. The aims of the thesis were to characterize a novel A. fumigatus exo-sialidase (AfS), and to clone and characterize putative A. fumigatus nucleotide sugar transporters (AfNSTs) to identify CMP-Neu5Ac or UDP-galactose transporters. The A. fumigatus sialidase gene was expressed in E. coli and crystallized; the crystal structure and Michaelis – Menten kinetic analysis revealed that the glycoside of another sialic acid, 2-keto-3-deoxynononic acid (KDN), was a better substrate for the enzyme than glycosides of Neu5Ac. This enzyme represents the first sialidase characterized from the Kingdom Fungi. To better understand why KDN is a better substrate for AfS than Neu5Ac, using the enzyme structure as a guide in conjunction with known sialidase structures, a point-mutation (R151L) was introduced in the substrate binding pocket to better accommodate glycans with terminal Neu5Ac. Activity of the R151L mutant was slightly enhanced toward Neu5Ac. Moreover, amino acid sequence comparisons revealed that this amino acid may be a hallmark of KDNases. In addition, I attempted to identify a CMP-sialic acid transporter in A. fumigatus, a type of nucleotide sugar transporter (NST). NSTs mediate nucleotide sugar transport into the endoplasmic reticulum and Golgi complex for subsequent addition to glycoproteins and glycolipids. STD-NMR analysis and 14C-transport assays were conducted to examine the substrate specificity of four putative A. fumigatus NSTs expressed in yeast. Two transporters (AfNST1 and AfNST5) bound UDP-glucose and UDP-galactose, and transported 14C-UDP-galactose. Epitope maps showed that the UDP-moiety anchored the nucleotide sugar and that sugar structure conferred specificity because not all UDP-sugars bound to the NSTs. No CMP-sialic acid transport was detected. Despite similarities in substrate preference between AfNST1 and AfNST5, growth and morphology of the corresponding knock-out mutants differed; only the Af∆NST5KO was compromised when grown on media containing cell wall stressors. Using lectins and flow cytometry, I found that the level of cell surface galactose was significantly reduced in both knockout strains as compared to the wild type; however, sialic acid density on conidia was significantly reduced only in the Af∆NST5KO mutant. This research demonstrates for the first time that NSTs are important for the integrity of the fungal cell and may represent novel targets for antifungal agents.
Many populations of forest Lepidoptera exhibit regular periodic cycles in abundance. Explicit mechanisms for such dynamics however, remain a subject of debate in Ecology. I used annual field data (1977-2015) from a cyclical species of forest Lepidoptera native to southwestern B.C., the western tent caterpillar (Malacosoma californicum pluviale), to elucidate how fecundity, viral disease and temperature contribute to its dynamics. Using time-series analysis and relationships between lagged population density, disease prevalence and annual population growth rate, I demonstrated that cyclical dynamics can be generated. I then used AIC model selection to show that fecundity and lagged population density had the greatest contributions to annual population rate of increase, followed by disease prevalence and warmer spring temperatures during larval development. Using these factors, I constructed a population model capable of generating population cycles similar to those observed in the field. These results indicate that fecundity, density-dependent disease prevalence and temperature contribute significantly to the cyclical dynamics of these populations.
Wildlife mass mortality events can have profound ecological consequences and may be becoming more frequent or severe due to climate change, anthropogenic factors or other stressors. Mortality events involving echinoderms are of particular concern because of the important role echinoderms play in structuring marine ecosystems. In this thesis I explore the local consequences of a widespread sea star mortality event, and investigate the global trends in echinoderm mass mortality events. I found that the mass mortality of the sunflower sea star Pycnopodia helianthoides, which began in the summer of 2013 as a result of a wasting syndrome, resulted in a trophic cascade involving urchins and kelp at the local scale (i.e., Howe Sound, BC). A global review of reports of echinoderm die-offs revealed that these events have not become more frequent or extensive since 1897. However, disease and climate change may be playing an increasing role. This study provides some of the first evidence of subtidal community shifts following sea star wasting syndrome, and highlights the need for consistent and comprehensive documentation of echinoderm population trends in the literature to increase our understanding of mass mortality events.
Spiders provide a fascinating opportunity for the study of animal communication. Web-building spiders build their own signalling environments - the web is the medium that transmits vibrations from prey, predators and potential mates. However, we know little about how information is conveyed through different types of webs, or how spiders distinguish between different types of vibrations. In this thesis, I studied elements of vibratory communication in two species of spiders with contrasting web architecture: the western black widow, Latrodectus hesperus, which builds a tangle-web, and the hobo spider, Eratigena agrestis, which builds a funnel-web. In chapter 2, I document formerly undescribed life history traits of E. agrestis, and conclude that life history traits are robust to differential predator and competitor densities across two study sites in British Columbia. In chapter 3, I present hitherto lacking quantitative descriptions of courtship behaviours in L. hesperus, revealing that web reduction by males correlates with reduced female aggression, and that it may improve mating success of courting males. In chapter 4, I describe how vibration frequencies are transmitted through the webs of L. hesperus and E. agrestis. I found little difference in propagation efficiency between longitudinal and transverse vibrations and that in both species vibration transmission is more variable within webs than between webs, suggesting that specific frequencies play a minor role in signalling. In chapter 5, I tested whether male courtship produces vibratory signals that differ from prey cues. I analysed vibrations produced by courting males and by two types of prey (flies and crickets) on the webs of L. hesperus and E. agrestis, and also played back male and prey vibrations through the webs of L. hesperus. Male vibrations differ more from those of prey in L. hesperus than in E. agrestis. This finding supports the hypothesis that L. hesperus males, faced with aggressive females, produce vibrations that prevent them from being mistaken for prey. The low-amplitude vibrations caused by abdominal tremulations of L. hesperus males may be linked with lowered female aggression.
Methylmercury is a widespread contaminant that has been shown in multiple studies to cause behavioural and reproductive effects on piscivorous birds. Previously, it was thought that non-aquatic birds (such as passerines) were not at risk for methylmercury toxicity. However, in recent years high blood mercury levels have been found in free-living passerines. In the current study, zebra finch (Taeniopygia guttata) chicks were treated with methylmercury during the nestling stage of early development to simulate exposure from food provisioning by the parents. Despite a dose response relationship shown in the blood mercury analyses, no effects of dose were found for growth, development, or behaviour of the chicks. No long-term effects were seen on male courtship and song or female reproductive success. The lack of treatment effects in these experiments indicates that the nesting stage may be less sensitive in passerines, possibly due the sequestration of mercury into growing feathers.
The lysosomal storage disease Mucopolysaccharidosis I, involving lysosomal deficiency of α-L-iduronidase (IDUA), is often characterized by extensive ER-associated degradation (ERAD) of missense mutant IDUAs, folding variants which often retain residual activity. Compound X-372, identified from a plant cell-based screen of 1,040 FDA-approved drugs, enhanced the post-ER transport of ERAD-prone P533R-IDUA. Here, dose response studies of compound treatment on Nicotiana tabacum BY2 protoplasts, expressing human wild-type and ERAD-prone R383H-IDUA, not only validated the rescuing effect of X-372, but also indicated this effect occurred via augmenting a general cellular process. Microarray analysis and subsequent quantitative PCR validation of candidate genes showed that the therapeutic effect of X-372 was linked to augmented sulfur assimilation. Reduced glutathione - a product of the sulfur assimilation pathway that is also a potent cellular antioxidant – is a target for future studies to identify the specific means by which X-372 enhances the post-ER transport of ERAD-prone folding mutants.
The microtubule-associated protein EB1b inhibits root responses to mechanical stimulation. The goal of this study was to understand more clearly how EB1b regulates these responses. Loss of EB1b did not alter root elongation rates in response to mechanical cues. However, overexpressing EB1b had an inhibitory effect on root elongation. Mutant eb1b-1 Arabidopsis plants expressing truncated EB1b proteins, with and without GFP fusions, were generated. Truncations included both N-terminal (microtubule-binding) and C-terminal (protein-interaction) domains. Transgenic mutants expressing a truncated version of EB1b missing part of the C-terminal domain were analyzed. The responses of these mutant roots to mechanical stimulation was similar to untransformed eb1b-1 mutants. Since previous analyses have shown that responses of mutants expressing full-length EB1b are equivalent to wild type, this result indicates that the EB1b C-terminus is required for normal regulation of root responses to mechanical cues and that interactions between EB1b and other, non-tubulin proteins is involved.
Based on sequence similarity to the well-studied Arabidopsis thaliana MP/ARF5 gene, we hypothesized that the Oryza sativa Auxin Response Factor 11 (OsARF11) gene is a prime candidate for auxin-signaling mediated development in rice. Here we describe characterizations of two independent insertion mutants in the OsARF11 gene. Our results reveal that homozygous plants of both allelic mutants have reduced shoot and root growth and produce fewer seeds compared to wild type plants grown under the same growth conditions. In addition, the number of leaf veins per leaf and per unit leaf width is reduced, as is the width of leaf mid veins. Taken together, the results demonstrate for the first time that OsARF11 contributes to plant growth, fecundity, and the regulation of leaf vein patterning in rice. The results also suggest that OsARF11 may be a suitable target for breeding on these traits.
Lewis’s Woodpeckers (Melanerpes lewis) are Threatened in Canada and rely on pre-existing cavities for nesting. I studied how cavity density, competition, and predators influence Lewis’s Woodpecker breeding performance across three habitats in British Columbia, and investigated the broad-scale patterns of nest tree persistence and reuse over time. I found that Lewis’s Woodpecker breeding performance was high in riparian cottonwood habitat, moderate in live pine, and lowest in crown-burned pine habitat. Cavity density explained habitat-based breeding performance. Nest tree persistence was generally high, but declined over time, and while nest tree reuse varied dramatically across years, there was no consistent temporal pattern. Our results suggest that 1) resource managers should use regionally-specific data for managing Lewis’s Woodpecker populations, and 2) cavities may be a limiting factor for the recovery of Lewis’s Woodpecker populations in Canada, particularly in regions where nest tree persistence is lower and may not support recruitment.
Attract-and-kill tactics for control of house flies (Musca domestica) often use foraging cues as attractants. To investigate foraging resources for indoor attraction of house flies, I tested the response of flies to various human foods and a floral resource. In two-choice laboratory bioassays, only dandelion flowers and dandelion honey attracted flies. Analytical attempts to capture the essential semiochemicals from these resources failed, highlighting the need to develop alternative approaches. Another potentially effective foraging cue is the “fly factor”, the phenomenon that food currently or previously fed on by flies attracts more flies than the same type of food kept inaccessible to flies. In two-choice laboratory bioassays, I demonstrate that the fly factor exists in house flies. Of the mechanisms tested potentially causing the fly factor, only fly feces and regurgitate attract flies. Attraction of flies to fly feces and regurgitate indicates that flies sense airborne semiochemicals emanating from these sources.