Graduate Theses & Dissertations

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Purification and Identification of Selenium-containing C-phycocyanin from Spirulina
Selenium is an essential trace nutrient to many organisms, yet in high concentrations it is toxic. Organic selenium is more bioavailable to aquatic biota than inorganic selenium, but is usually found in much lower concentrations. Algae are known to biotransform inorganic selenium into several organo-selenium compounds, but it is unknown whether any of these bioaccumulate in the food chain. In this study, selenium was incorporated into the methionine residues of an algal photosynthetic protein, c-phycocyanin from Spirulina spp. The extent of selenium incorporation was quantified by inductively coupled plasma-mass spectrometry (ICP-MS), and the protein was identified using electrospray mass spectrometry (ES-MS). C-phycocyanin was isolated and purified from Spirulina with a final recovery of 20-30 % of the total c-phycocyanin present. Selenomethionine replaced 92.8% ± 1.22 of the methionine residues in c-phycocyanin when grown in 2.5 ppm sodium selenite. ES-MS was used to obtain protein spectra, and pure c-phycocyanin was identified. Data of full scans provided estimated masses of both protein subunits--α-chain measured at 18,036 Da; β-chain measured at 19,250 Da--close to the theoretical masses. Protein fragmentation by collision-induced dissociation and electron capture dissociation provided approximately 52 % amino acid sequence match with c-phycocyanin from Spirulina platensis. This study demonstrates the incorporation of selenium into an algal protein, and the identification of c-phycocyanin using electrospray ionization-mass spectrometry. Author Keywords:
Widespread changes in growth, diet and depth distribution of lake whitefish (Coregonus clupeaformis) in the Great Lakes are linked to invasive dreissenid mussels
Recent declines in growth and condition of Great Lakes' lake whitefish (Coregonus clupeaformis) have been linked to ecosystem-wide changes stemming from the invasion of dreissenid mussels. To test the influence of invasive mussels on this commercially important coregonid species, we collected archived scale samples from ten Great Lake locations and analyzed long-term changes in growth rates, delta 13C and delta 15N stable isotope ratios before and after mussel establishment. There was a decrease in pre-maturation growth after establishment in all four locations where we examined back-calculated growths. In six of the seven locations with dreissenid populations, a significant increase in delta 13C and a significant decrease in delta 15N was found. In dreissenid-absent locations of Lake Superior, we did not see changes in growth or isotope ratios indicative of a major regime shift. Observed shifts in isotopic signatures provide evidence for an increased reliance on nearshore food sources and shallower depth distribution as a result of dreissenids, which likely contributed to lowered growth of lake whitefish. Author Keywords: Diporeia, Dreissenids, food web, Great Lakes, invasive species, lake whitefish
Development and Use of Passive Samplers for Monitoring Dissolved and Nanoparticulate Silver in the Aquatic Environment
Silver nanoparticles (nAg) are the largest and fastest growing class of nanomaterials, and are a concern when released into aquatic environments even at low μg L-1+). Diffusive gradient in thin films (DGT) with a thiol-modified resin were used to detect labile silver and carbon nanotubes (CNT-sampler) were used to measure nAg. Laboratory uptake experiments in lake water provided an Ag+ DGT diffusion coefficient of 3.09 x 10 -7 cm2s-1 and CNT sampling rates of 24.73, 5.63, 7.31 mL day-1, for Ag+, citrate-nAg and PVP-nAg, respectively. The optimized passive samplers were deployed in mesocosms dosed with nAg. DGT samplers provided estimated Ag+ concentrations ranging from 0.15 to 0.98 μg L-1 and CNT-samplers provided nAg concentrations that closely matched measured concentrations in water filtered at 0.22 μm. Author Keywords: ICP-MS, mesocosms, nanoparticles, nanosilver, passive sampling
Fate of Silver Nanoparticles in Lake Mesocosms
The fate of silver nanoparticles (AgNPs) in surface waters determines the ecological risk of this emerging contaminant. In this research, the fate of AgNPs in lake mesocosms was studied using both a continuous (i.e. drip) and one-time (i.e. plug) dosing regime. AgNPs were persistent in the tested lake environment as there was accumulation in the water column over time in drip mesocosms and slow dissipation from the water column (half life of 20 days) in plug mesocosms. In drip mesocosms, AgNPs were found to accumulate in the water column, periphtyon, and sediment according to loading rate; and, AgNP coating (PVP vs. CT) had no effect on agglomeration and dissolution based on filtration analysis. In plug mesocosms, cloud point extraction (CPE), single-particle-inductively coupled mass spectroscopy (spICP-MS), and asymmetrical flow field-flow fractionation (AF4-ICP-MS) confirmed the temporal dissolution of AgNPs into Ag+ over time; however, complexation is expected to reduce the toxicity of Ag+ in natural waters. Author Keywords: AF4-ICP-MS, cloud point extraction, fate, mesocosms, silver nanoparticles, SP-ICP-MS
Seasonal variation in nutrient and particulate inputs and outputs at an urban stormwater pond in Peterborough, Ontario
Stormwater ponds (SWPs) are a common feature in new urban developments where they are designed to minimize runoff peaks from impervious surfaces and retain particulate matter. As a consequence, SWPs can be efficient at retaining particle-bound nutrients, but may be less efficient at retaining nutrients that are present primarily in the dissolved form, like nitrogen (N). However, the forms of nutrients (e.g. particulate vs. dissolved) likely differ with hydrologic and seasonal conditions and few studies have examined year-round differences in nutrient forms and concentrations at urban SWPs. In order to contrast total suspended solids (TSS), phosphorus (P) and nitrogen (N) levels between low and high flow conditions, sampling was conducted at an urban SWP in Peterborough, ON between November 2012 and October 2013. Only an increase in TSS levels at the outflow between low and high flow conditions was observed, as well as a decrease in TSS levels at the outflow compared to Inflow 1 under low flow conditions. Nitrate-N (NO3-N) was the dominant form of N entering the pond under all flow conditions, whereas the fraction of total-P (TP) that was particulate increased under high flow conditions. Nevertheless, the dissolved fraction of TP was consistently high in these urban inlets. Only NO3-N was significantly greater in the inflows than outflow and only under low flow conditions. Increases in the proportions of organic-N and ammonium-N in the outlet suggest that biological processing is important for N retention. Author Keywords: nitrogen, Ontario, phosphorus, stormwater ponds, total suspended solids
Carbon and Nitrogen Isotope Changes in Streams along an Agricultural Gradient
Nitrogen is a major constituent of agricultural fertilizers, and nitrogen inputs to stream water via runoff and groundwater lead to a variety of negative environmental impacts. In order to quantify the movement of nitrogen through aquatic food webs, fourteen streams with varying land uses across South-Central Ontario were sampled for two species of fish, freshwater mussels, and water for measurement of isotope ratios of δ15N and δ13C. I found that nitrogen isotopes in fish, water, and mussels were related to the percentage of riparian monoculture, and that carbon isotopes were unrelated to monoculture. Though all species were enriched as monoculture increased, the rate of δ15N enrichment as monoculture increased did not vary between species. This study has improved our understanding of how monoculture affects nutrient enrichment in stream food webs, and assesses the validity of using nitrogen isotopes to measure trophic positions of aquatic organisms across an environmental gradient. Author Keywords: agriculture, fish, food webs, nitrogen, stable isotopes, streams
Evaluating the Effects of Habitat Loss and Fragmentation on Canada Lynx
Current major issues in conservation biology include habitat loss, fragmentation and population over-exploitation. Animals can respond to landscape change through behavioural flexibility, allowing individuals to persist in disturbed landscapes. Individual behaviour has only recently been explicitly included in population models. Carnivores may be sensitive to changing landscapes due to their wide-ranging behaviour, low densities and reproductive rates. Canada lynx (Lynx canadensis) is a primary predator of snowshoe hares (Lepus americanus). Both species range throughout the boreal forests of North America, however lynx are declining in the southern range periphery. In this dissertation, I developed new insights into the effects of habitat loss and fragmentation on lynx. In Chapter 2, I created a habitat suitability model for lynx in Ontario and examined occurrence patterns across 2 regions to determine if habitat selection is flexible when different amounts of habitat are available. Although lynx avoided areas with <30% suitable habitat where suitable land cover is abundant, I found that they have flexible habitat selection patterns where suitable land cover is rare and occurred in low habitat areas. In Chapter 3, I investigated the effects of dispersal plasticity on occupancy patterns using a spatially explicit individual-based model. I showed that flexible dispersers, capable of crossing inhospitable matrix, had higher densities and a lower risk of patch extinction. In contrast, inflexible dispersers (unable to cross inhospitable matrix), were most limited by landscape connectivity, resulting in a high extinction risk in isolated patches. I developed three predictions to be explored with empirical data; (1) dispersal plasticity affects estimates of functional connectivity; (2) variation in dispersal behaviour increases the resilience of patchy populations; and (3) dispersal behaviour promotes non-random distribution of phenotypes. Finally, in Chapter 4, I examined the consequences of anthropogenic harvest on naturally cycling populations. I found that harvest mortality can exacerbate the effects of habitat fragmentation, especially when lynx densities are low. Dynamic harvest regimes maintained lynx densities and cycle dynamics while reducing the risk of population extinction. These results suggest that lynx display some flexibility to changing landscapes and that the metapopulation structure is more resilient to increasing habitat loss and fragmentation than previously understood. Future studies should focus on determining a threshold of connectivity necessary for population persistence and examining the effects of habitat loss on the fecundity of lynx. Author Keywords: Fluctuating Populations, Habitat Fragmentation, Landscape Ecology, Occupancy Dynamics, Population Ecology, Spatially Explicit Population Models
ASSESSING THE IMPACT OF ATMOSPHERIC DEPOSITION AND HARVEST INTENSITY ON SOIL ACIDITY AND NUTRIENT POOLS IN PLANTATION FORESTS
The objective of this thesis was to assess the influence of anthropogenic sulphur (S) and nitrogen (N) deposition, and harvesting on soil acidity and calcium (Ca2+), magnesium (Mg2+), potassium (K+) and N soil pools in plantation forest soils in Ireland. The response to reductions in anthropogenic S deposition was assessed using temporal trends in soil solution chemistry at two long-term monitoring plots--one on a blanket peat, the other on a peaty podzol. At the peat site, there was little evidence of a response to reductions in throughfall non marine sulphate (nmSO42-) and acidity; soil water acidity was determined by organic acids. In addition, temporal variation in soil water did not respond to that in throughfall. In the podzol, reductions in anthropogenic S and H+ deposition led to a significant improvement in soil water chemistry at 75 cm; pH increased and total aluminum (Altot) concentrations declined. The impact of harvest scenarios on exchangeable Ca2+, Mg2+ and K+ pools was assessed using input-output budgets at 40 sites (30 spruce, 10 pine). Harvest scenarios were stem-only harvest (SOH), stem plus branch harvest (SBH) and stem, branch and needle harvest (whole-tree harvesting; WTH). Average K+ and Mg2+ budgets were positive under these scenarios. However, exchangeable K+ pools were small and due to uncertainty in K+ budgets, could be depleted within one rotation. Average Ca2+ budgets for spruce were balanced under SOH, but negative under SBH and WTH. Nitrogen deposition was high, between 5 and 19 kg N ha-1 yr-1, but was balanced by N removal in SOH. However, N budgets were under SBH and WTH, indicating that these harvesting methods would lead to depletion of soil N over the long-term. Finally, monitoring of N cycling at a spruce plot indicated that N deposition was contributing to large NO3- leaching, and as such the site was N saturated. However, N cycling did not fit the criteria of the N saturation hypothesis; instead leaching was directly related to N deposition and supported the model of kinetic N saturation. Author Keywords: acidic deposition, base cations, input-output budgets, Ireland, nitrogen, whole-tree harvesting
Cytokinin Oxidase/Dehydrogenase (CKX) Gene Family in Soybeans (Glycine max)
Glycine max (soybean) is an economically important plant species that registers a relatively low yield/seed weight compared to other food and oil seed crops due to higher rates of flower and pod abortion. Alleviation of this abortion rate can be achieved by altering the sink strength of the reproductive organs of soybeans. Cytokinin (CK) plays a fundamental role in promoting growth of sink organ (flowers and seeds) by increasing the assimilate demand. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that catalyses the irreversible breakdown of active CKs and hence reduce the cytokinin content. The current thesis uncovers the members of CKX gene family in soybeans and the natural variations among CKX genes within soybean varieties with different yield characteristics. The identification of null variants of OsCKX2 that resulted in large yield increases by Ashikari et al. (2005) provided a rationale for current thesis. The soybean CKX genes along with the ones from Arabidopsis, Rice and Maize were used to construct a phylogenetic tree. Using comparative phylogeny, protein properties and bioinformatic programs, the potential effect of the identified natural variations on soybean yield was predicted. Five genes among the seventeen soybean CKXs identified, showed polymorphisms. One of the natural variations, A159G, in the gene GmCKX16 occurred close to the active site of the protein and was predicted to affect the activity of enzyme leading to higher accumulation of CKs and hence increased seed weight. Use of such natural variations in marker assisted breeding could lead to the development of higher yielding soybean varieties. Author Keywords: CKX, Cytokinins, Seed weight, Seed Yield, SNPs, Soybeans
Speciation of Aluminum and Zinc in Three Streams of a Forested Catchment of the Boreal Zone
This study presents a detailed assessment of the chemical speciation of aluminum and zinc in three streams of a small, acid-sensitive forested catchment on the southern edge of the Precambrian Shield. Speciation analysis was achieved using an in-situ analytical technique known as Diffusive Gradient in Thin film (DGT) which measures labile metals, and a predictive computer algorithm (WHAM VI) which calculates metal species concentrations. Three types of DGT with different metal scavenging capabilities were used and a total of 11 deployments performed across four seasons. WHAM VI predictions showed that the organic fraction of aluminum was the main contributor to the dissolved concentrations in the main inflow stream (PC1) (~ 80 %) and the lake's outflow (PCO) (~ 75%); in the upland stream (PC1-08) the inorganic fraction contributed ~ 75%. For zinc the free ion was the single most important contributor to the dissolved concentration (< 90%) in all three streams. A comparative study of the DGT and WHAM methods showed an agreement between their inorganic concentrations during the spring season. Both methods indicate the greatest environmental impact for Al takes place during snow melt period in PCO and PC1-08 and in the summer for PC1. The greatest environmental impact for Zn predicted with WHAM VI, occurs during the spring in all three streams. Author Keywords: Aluminum, DGT, Metal speciation, WHAM, Zinc
Ice age fish in a warming world
In the face of climate change, the persistence of cold-adapted species and populations will depend on their capacity for evolutionary adaptation of physiological traits. As a cold-adapted Ice Age relict species, lake trout (Salvelinus namaycush) are ideal for studying potential effects of climate change on coldwater fishes. I studied the thermal acclimation capacity and aerobic metabolism of age 2+ lake trout from four populations across four acclimation temperatures (8ºC, 11ºC, 15ºC, and 19ºC). One population had marginally significant higher active metabolic rate (AMR) and metabolic scope across all temperatures. There was no interpopulation variation for critical thermal maximum (CTM), standard metabolic rate (SMR), or thermal acclimation capacity. Acclimation resulted in a 3ºC increase in thermal tolerance and 3-fold increase in SMR for all populations. At 19ºC, SMR increased and AMR declined, resulting in sharply reduced metabolic scope for all populations. The limited intraspecific variation in thermal physiology suggests that climate change may threaten lake trout at the species rather than population level. Author Keywords: Climate Change, Lake Trout, Metabolic Rate, Salvelinus namaycush, Temperature, Thermal Acclimation
Evaluating the effects of landscape structure on genetic differentiation and diversity
The structure and composition of the landscape can facilitate or impede gene flow, which can have important consequences because genetically isolated groups of individuals may be prone to inbreeding depression and possible extinction. My dissertation examines how landscape structure influences spatial patterns of genetic differentiation and diversity of American marten (Martes americana) and Canada lynx (Lynx canadensis) in Ontario, Canada, and provides methodological advances useful for landscape geneticists. First, I identified the effects of map boundaries on estimates of landscape resistance, and proposed a solution to the bias: a buffer around the map boundary. Second, I assessed the sensitivity of a network-based estimate of genetic distance, conditional genetic distance, to incomplete sampling. I then used these landscape genetic tools in a pairwise, distance-based analysis of 653 martens genotyped at 12 microsatellite loci. I evaluated whether forest management in Ontario has influenced the genetic structure of martens. Although forest management practices had some impact, isolation by distance best described marten gene flow. Our results suggest that managed forests in Ontario are well connected for marten and do not impede marten gene flow. Finally, I used a site-based analysis of 702 lynx genotyped at 14 microsatellite loci to investigate spatial patterns of genetic diversity and differentiation at the trailing (contracting) edge of the lynx distribution in Ontario. I analyzed harvest records and found that the southern edge of lynx range has contracted by >175 km since the 1970s. I also found that neutral genetic diversity decreased towards the trailing edge, whereas genetic differentiation increased. Furthermore, I found strong correlations between gradients of lynx genetic structure and gradients of climate and land cover in Ontario. My findings suggest that increases in winter air temperature, decreases in snow depth, and loss of suitable habitat will result in further loss of genetic diversity in peripheral populations of lynx. Consequently, the adaptive potential of lynx populations on the southern range periphery could decline. In conclusion, my dissertation demonstrates the varying influences that contemporary landscape structure and climate gradients can have on genetic diversity and differentiation of different species. Author Keywords: Circuitscape, genetic network, landscape genetics, Lynx canadensis, Martes americana, range shift

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