Recent Submissions

  • Flood Survival Strategies of Overwintering Snakes

    Yagi, Anne .R.; Department of Biological Sciences
    This thesis investigates snake flood survival during hibernation and how anthropogenic habitat alteration and climate variability may affect habitat quality and overwintering survival. Chapter one reviews the current understanding of ecophysiology of hibernation in snakes. In chapter two, I introduce a winter habitat model of a subterranean space that remains flood and frost-free, referred to as the “life zone,” where snakes survive winter. I analyzed 11- winters of hibernation habitat data and 18-yrs of population mark-recapture data to assess the effects of the first flood event on an endangered Massasauga population. Following the flood event, snake observations declined despite hundreds of hours of search-effort. At the population level this was evidence of poor winter survival and recruitment post flood. The direct cause of mortality was not determined but poor winter survival in areas with a depleted life zone was statistically supported. In the third chapter, I measured the metabolic rate (M ̇_(O_2 )) at 5°C for three snake species that inhabit my study area. I varied water level conditions and measured activity and dive behaviours continuously during experiments. I found differences between species in their resting metabolic rate, which I attributed to body size differences. I confirmed, cutaneous respiration occurs at a low rate and was significantly upregulated during a forced dive (flood event). Therefore, there is an intrinsic physiological response to a flood event in neonatal snakes. However, post-flood recovery indicated a greater oxygen demand after the short-forced dive. An oxygen debt was incurred during a short-forced dive under normoxic conditions. My conclusions are, 1) hibernation habitat (i.e., life zone) must include a non-freezing, non-flooding aerobic space throughout winter to maintain snake survival. 2) cutaneous respiration is a short-term flood survival strategy. I found no support for a complete aquatic hibernation strategy 3) the energy costs of a full-dive is additive to the recovery energetic costs of a flood event. A neonatal snake wintering energy budget is proposed, and winter mortality conservation issues are discussed in chapter 4.
  • Characterization of plant, leafhopper, and spider communities in perimeter plantings and vineyards in the Niagara region

    Hughes, Margaret Moira; Department of Biological Sciences
    Vineyards are large agroecosystems associated with high external inputs and intervention leading to local decreases in biodiversity. With trends towards sustainable agriculture, there is a push to maximize natural ecosystem functions through methods of on-farm diversification, such as perimeter plantings. Increased plant diversity has been found to increase the ability to exploit natural ecosystem functions such as pest management, through the bottom-up control of species richness displayed by increased plant species richness. The aim of this thesis was to investigate the effect of perimeter plantings on vineyard plant and invertebrate communities. I hypothesized that perimeter plantings would have greater plant diversity and habitat complexity than vineyard interiors. Perimeter plantings would also support increased assemblages of natural enemies with decreased pest populations when compared to the vineyards. Plant communities in the perimeter plantings and the vineyards were first surveyed using transects within the perimeters and perpendicular transects from the perimeters towards the interior of the vineyards. Invertebrate communities were also surveyed within the perimeter plantings and adjacent vineyards, focusing on leafhoppers and spiders. Seven commercially operating vineyards throughout the Niagara region were surveyed both within the perimeter planting and adjacent vineyard during the 2018-growing season. It was found that perimeter plantings not only had increased plant species richness and functional diversity, but the species and functional composition within the perimeters differed from vineyard interiors. This indicated that perimeter plantings did not increase weed pressure but allowed for increased habitat complexity adjacent to the vineyards. Leafhoppers showed significantly higher abundance in vineyard interiors than perimeter plantings, and as distance from perimeter planting increased, leafhopper abundances also increased. Spiders were more abundant in perimeter plantings, decreasing in abundance with distance from perimeter. Overall, the results suggest that perimeter plantings have the ability to support biological pest control, while not increasing both weed or pest pressure observed within vineyards.
  • The Physiological and Behavioural Consequences of Reduced Scalation in Captive-bred Phenotypes of the Bearded Dragon (Pogona vitticeps Ahl 1926)

    Sakich, Nicholas; Department of Biological Sciences
    Lepidosaurs as a group are known for their tough, scaled integument and low rates of evaporative water loss. Whether or not there is a causal relationship between the two has been a contentious issue. There also remains the question of whether the lepidosaur scale forms a barrier to ultraviolet (UV) light. Thirdly, there is evidence to suggest that rate of evaporative water loss influences behavioural thermoregulation in lepidosaurs. Lepidosaurs with higher rates of evaporative water loss should be expected to choose cooler temperatures than lepidosaurs with lower rates of evaporative water loss in order to reduce water loss. To investigate these ideas, I used three captive-occurring phenotypes of the bearded dragon (Pogona vitticeps Ahl 1926): Wild Type, animals exhibiting scales of reduced prominence (“Leatherback”), and scaleless animals (“Silkback”). I a priori expected that Silkbacks would have the highest rates of evaporative water loss, the lowest thermal preferences, and the lowest UV light intensity preferences. By the same token, I expected Wild Types to be at the opposite end of the spectrum from Silkbacks for each of these measurements, and I expected Leatherbacks to be intermediate between the two. I used respirometry to measure the animals’ rates of evaporative water loss, a thermal gradient to measure their thermal preferences, and a UV light intensity gradient to measure their UV light intensity preferences. Silkbacks on average lost water at about twice the rate that Wild Types did, with Leatherbacks being intermediate in their water loss rates. The three phenotypes did not visibly differ in their thermal preference. Silkbacks had lower UV light intensity preferences than either Leatherbacks or Wild Types. These results suggest that the lepidosaur scale is indeed a barrier to evaporative water loss and suggest that it is also a barrier to UV light. However, the lack of obvious difference in thermal preference suggests that thermal preference in bearded dragons is not plastic enough to respond to a phenotype that increases the animal’s rate of evaporative water loss. In addition to answering basic questions about lepidosaur biology, my data have relevance to the fields of animal welfare and conservation.
  • Examining the Roles of Octopamine and Proctolin as Co-Transmitters in Drosophila melanogaster

    Kornel, Amanda; Department of Biological Sciences
    The nervous system is a highly complex and intricate system that interacts with and controls nearly all the other body systems. The basic functions of nerve cells are conserved across most species and are very similar between vertebrates and invertebrates. Chemical transmitters (neurotransmitters) facilitate communication between nerve cells and their targets. The effects of these signals can be modified by co-transmitters that are released from neurons in conjunction with neurotransmitters, and by neuromodulators that are released as hormones. This thesis examines the effect of two neuromodulators on neuromuscular junctions of the fruit fly, Drosophila melanogaster. Two modulators, proctolin and octopamine, have been identified in motor nerve terminals and are thought to be released as co-transmitters to modify the effects of glutamate, the neurotransmitter that depolarizes muscle cells and triggers contraction. The neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) was found to increase the amplitude of body wall muscle contractions elicited by glutamate in the absence of nerve stimulation. Thus, proctolin appears to enhance contractions by acting postsynaptically. Previous work reported that increasing neural activity lowers the threshold and EC50 for proctolin’s ability to enhance nerve-evoked contractions by two orders of magnitude. To determine whether such activity-dependence is caused by increased release of glutamate, effects of varying glutamate concentrations on the effectiveness of proctolin are examined here. The threshold for proctolin to increase body wall contractions decreased from 100 nM to 10 nM when glutamate concentration increased from 5 mM to 7 mM, but the threshold increased again to 100 nM for glutamate concentrations of 10-20 nM. Thus, although the effectiveness of proctolin shows some dependence on glutamate concentration, alterations in glutamate levels do not appear to account entirely for the more substantial and more consistent changes in proctolin threshold that occur with increasing neural activity, reported elsewhere. Since octopamine in known to be present in motor neurons innervating most of the body wall muscles of 3rd instar larvae, it was hypothesized that stimulating the motor neurons should release octopamine together with glutamate, and that increasing motor neuron activity should increase the release of both octopamine and glutamate. This hypothesis led to the prediction that an octopamine antagonist, phentolamine, should reduce the amplitude of nerve-evoked contractions, and that the antagonist should be more effective when the motor neurons are stimulated at higher frequencies. Phentolamine, however, did not alter the amplitude of body wall muscle contractions elicited by stimulating the motor axons using impulse bursts with intraburst stimulus frequencies of 5, 32 and 50 Hz. Surprisingly, exogenously applied octopamine did enhance the amplitude of nerve-evoked contractions, and, this effect was antagonized by phentolamine when contractions were elicited by impulse bursts with frequencies of 5 and 50 Hz. At a concentration of 1x10-6 M, octopamine did not induce contractions or alter the amplitude of glutamate-evoked contractions. These results do not support the hypothesis that endogenous octopamine is released onto muscle fibers as a co- transmitter to augment contraction amplitude. One possible explanation for these findings is the octopamine may be released at higher concentrations at neuromuscular synapses, and the effects of octopamine on nerve-evoked contractions are mediated presynaptically, by increasing transmitter release. Overall, the results of this thesis indicate that both octopamine and proctolin modulate muscle contractions in an activity-dependent manner; the level of external nerve-stimulus or exogenous glutamate concentration alter the effectiveness of the contransmitters. However, further work is needed to elucidate the mechanisms of such activity-dependence.
  • Behavioural Thermoregulation and Escape Behaviour: Investigating the Thermal Biology of Round Gobies

    Bridgeman, Justin; Department of Biological Sciences
    The invasive round goby (Neogobius melanostomus) has successfully colonized all of the Great Lakes since its discovery in the region in 1991, yet little is known about its thermal biology. The focus of this thesis was to examine the effect of acclimation to unseasonably warm temperatures on round goby behavioural thermoregulation, as well as behavioural and physiological performance during escapes with warm acute temperatures. Juvenile gobies were acclimated to either 21°C or 24°C for each set of experiments. I first examined goby thermal preference in a shuttlebox through their ability to escape from unfavourable temperatures. I found that escape temperatures were plastic following acclimation to a rise in 3°C rise in temperature (+3°C) and associated positively with acclimation temperature, even though gobies showed slightly lower-than-expected average escape temperatures in each acclimation treatment. Interestingly, acclimation to +3°C leads to lower exploratory behaviour in warm waters and lower overall activity levels during behavioural thermoregulation. In risky situations involving threat of predation, exploratory behaviour is often linked to boldness. Next I investigated exploratory swimming through two behavioural traits: ability to voluntarily enter a tunnel and subsequent swimming activity while being chased in a detour task. Detour tasks require a fish to swim down a narrow space and then detour to the left or right as they approach a barrier. A strong correlation between behavioural traits suggests the two behavioural traits are a good measure of risky swimming behaviour. With respect to the impact of warm acute temperatures, I observed an increase in strength of risky swimming behaviour following a rise in acute temperature of 6°C (+6°C). Finally, I examined burst swimming performance (C- and S-starts) during the detour task. While there was no observed effect of warm acute temperatures on most C- and S-start performance measures, there was a significant increase in S-start time to maximum velocity at +6°C. Along with the observed plasticity in strength of risky swimming behaviour, my findings showed that acclimation to +3°C shifted physiological performance of escapes. My findings provide a greater understanding of round goby physiological and behavioural functioning during short- and long-term rises in temperature.
  • Quantification of gamma-secretase activity in an endogenous context reveals biphasic GSI-mediated Notch/APP selectivity switch and the novel detection of potential proteolytic cleavage fragments of the Notch Intracellular Domain

    Mueller, Matthew Craig; Department of Biological Sciences
    Gamma-secretase is a promiscuous intra-membrane protease implicated in the proteolytic processing of two notable substrates: amyloid-precursor-protein (APP), in which gamma-secretase will irreversibly cleave to produce Amyloid-beta (Aβ) in Alzheimer’s disease; and the Notch receptor, where gamma-secretase is essential for liberating the Notch intracellular domain (NICD) to activate Notch-mediated transcriptional regulation in the nucleus. Gamma-secretase inhibitors such as DAPT and Avagacestat have been tested as therapies to prevent the formation of amyloid plaques, however, off-target interference with the Notch signalling pathway leading to Notch-related malignant side effects in clinical trials makes these pharmaceuticals unsuitable. The high-throughput search for selective drugs that block the production of Aβ but don’t interfere with the Notch signalling pathway has been hindered by a lack of reliability in detecting Notch signal inhibition in pre-clinical, cell-based assays with ectopic substrate expression. Therefore, the development of a highly-sensitive, high-throughput cell-based assay to quantify the level of proteolytic processing of APP and Notch by gamma-secretase is a promising addition to the gamma-secretase inhibitor drug discovery pipeline. This thesis presents the combination of immunofluorescence staining, western blotting, and the bromo-deoxyuridine (BrdU) cell proliferation assay as three orthogonal methods to sensitively quantify the gamma-secretase cleavage of Notch and APP in SH-SY5Y human neuroblastoma cells, which rely on active Notch signalling to maintain proliferation. Using our assay, we found that the selectivity for gamma-secretase to cleave APP versus Notch was dependent on the time the GSI was replenished before harvesting, which may directly reflect a GSI concentration-dependent selective potency. Using this high-throughput cell-based assay, cleavage of APP and Notch activation by gamma-secretase was sensitively quantified; while a novel profile of cell-type specific proteolytic fragments of the Notch ICD have been identified that may have biological implications in normal development and the pathological proliferation and metastasis of some cancers.
  • Role of Exopolysaccharide in Pantoea agglomerans Interactions with Bacteriophages

    Zaprzala, Patrick; Department of Biological Sciences
    Fire blight is the common name given to a disease caused by phytopathogen, Erwinia amylovora. This pathogen infects plants belonging to the rosaceous family, particularly the commercially grown apple and pear cultivars in North America. Agriculture and Agri-Food Canada Research team has focused on the use of bacteriophages as biological control agents for the control of fire blight. Bacteriophage persistence in the orchard environment can be affected by various environmental factors such as exposure to ultraviolet light and dry conditions. In order to improve phage viability during application, a carrier-phage system was developed that uses a non-pathogenic epiphytic bacterium, named “the carrier”, that protects the bacteriophages during processing and field applications. The bacterium used is Pantoea agglomerans, which delivers and propagates the phages to the open blossoms, prior to the arrival of E. amylovora. In addition, the carrier alone may act as a biological control agent on the blossom surface, competing for space and producing antibiotics targeting E. amylovora. The aim of this study was study the interaction between the phage and the carrier bacterium, P. agglomerans. The exopolysaccharide (EPS) layer, is the first cellular component which bacteriophages encounter during the infection of P. agglomerans. PCR was conducted and it confirmed that 6 genes involved in EPS biosynthesis were present within the four different isolates of P. agglomerans. A CRISPR/Cas9 knockout and lambda red recombineering protocol was used to construct EPS deficient P. agglomerans mutants. The deficient populations were confirmed using colony appearance, PCR and Sangar sequencing. A quantitative PCR was conducted to quantify and compare the different bacteriophage populations between the wild-type and deficient EPS mutants. The EPS mutants were found to have shown a significantly decreased or complete lack of growth in both Myoviridae and Podoviridae infections.
  • Engineering Optogenetic Control of Endocytic Recycling: Controlling Rab11 Function in Drosophila melanogaster using Engineered Light-Responsive Nanobodies

    Ward, Devin; Department of Biological Sciences
    The regulated transport of materials in cells is an essential function of all living organisms. In eukaryotes, one main family of transport regulators is the Rab GTPases. Rab GTPases utilize GTP to move materials throughout the cell by binding to the membrane of vesicles or endosomes, and trafficking distinct, membrane-associated components throughout the cell. One member of this large family of proteins is Rab11. Rab11 is responsible for endosome recycling: returning membrane proteins and receptors from intracellular recycling endosomes to the cell membrane, where these membrane proteins and receptors may be reused. Although the exact mechanism of Rab11 trafficking is not known, Rab11 appears to be critical for the development and survival of many organisms. Drosophila mutants for the Rab11 gene are not viable, where lethality manifests during embryonic development. This early lethality has imposed significant limitations on elucidating the immediate effects of Rab11 inhibition. Thus, the goal was to engineer a novel method of inhibiting Rab11 in vivo in Drosophila melanogaster. Specifically, the goal was to generate a genomically non-invasive construct (Opto-Nanobody) utilizing an optogenetic, light-sensitive Cryptochrome 2 (Cry2) fused to YFP-targeting nanobodies to bind functional, endogenous, YFP-tagged Rab11. This system promises to provide precise light-responsive spatio-temporal control of Rab11 function in response to blue-light exposure through homo-oligomeric clustering, which has been shown to inhibit Rab-dependent trafficking. Using the Drosophila embryo as a model system, these tools were applied to directly determine the effects of Rab11 inhibition on Notch signaling, and to determine the mechanisms that govern Rab11 trafficking. The Opto-Nanobody was tested in vitro in S2 cells, and was shown to form homo-oligomeric clusters in the presence of blue light and demonstrated the ability to bind to YFP-Rab11. This Opto-Nanobody construct has been inserted into a D. melanogaster injection vector, so that the Opto-Nanobody may be inserted into the D. melanogaster genome, and used to control YFP::Rab11 activity in vivo to elucidate the role of Rab11 in Notch signalling.
  • Exploring Diversified Vineyard Ecological Soil Management Strategies: Impacts of cover cropping, Spanish River Carbonatite, and smooth pigweed (Amaranthus hybridus) interactions on an agroecosystem

    VanVolkenburg, Heather; Department of Biological Sciences
    Agroecosystems are one of the most heavily managed ecosystems that provide essential services for human well being. Intense management of agroecosystems has led to global degradation of soil and reduced biodiversity, two of the major challenges faced by agriculture today. Soil, the foundation of an agroecosystem, forms the base for cropping systems and how it is managed matters. My research focused on investigating how two ecologically-based soil management techniques, cover cropping (using a species mixture of Cichorium intybus, Raphanus sativus, Medicago sativa, Trifolium pratense, and a monoculture of Lolium multiflorum) and agromineral amendment application (using Spanish River Carbonatite – SRC), affect both biotic and abiotic variables such as soil nutrients, plant growth, and plant and soil invertebrate community composition over time. My study included control greenhouse trials and trials in an operational vineyard. In greenhouse trials, SRC was found to be especially effective for growth of leguminous species, M. sativa and T. pratense while forb species, R. sativus and C. intybus, tended to grow better in synthetic fertilizer. Residual effects on a second period of growth were minimal. This suggests that, depending on species used, cover crops when combined with SRC may be able to support an operational system in a similar way to when synthetic inputs are used. Trials were also conducted in an operational vineyard. While the added complexity of a field setting did not significantly affect any of the measured variables between amendment treatments, cover crop type mattered with significantly higher vegetation-index diversity and total abundance values found in cover crop mixture plots than in monoculture. Annual weather and farm management practises were most likely the main driver of the variation found in soil invertebrate community diversity. An additional experiment aimed to determine whether Amaranthus hybridus, a known allelopathic plant species present in the vineyard, affects the same cover crop species germination and growth using an A. hybridus tea extract treatment in controlled conditions. Here, A. hybridus was found to inhibit germination and growth of M. sativa and T. pratense yet stimulate growth in R. sativus and L. multiflorum. Careful consideration should be given to which weeds can be found in an agroecosystem and how they may influence management outcomes. Long-term investigations are needed to truly understand how to best manage the various components found in an agroecosystem.
  • Utilization of unmanned aerial vehicles and proximal sensing to detect Riesling vineyard variability

    Dorin, Briann; Department of Biological Sciences
    A single vineyard block can consist of significant spatial variability for several grape-growing attributes. The ability to detect and subsequently respond to this variation can lead to improved vineyard management, a growing practice termed precision viticulture. The overall goal of this research study was to determine if remote-sensing technologies could be used to detect Riesling vineyard variability, thus enhancing precision viticulture implementation. Approximately 80 grapevines in a grid pattern were geo-located within each of six commercial Riesling vineyards across the Niagara Peninsula in Ontario. From these grapevines the following variables were measured to determine their vineyard variation: soil and vine water status, vine size/vigor, winter hardiness, virus titer, yield components, and berry composition. Subsequently, remote-sensing technologies collected thermal [by unmanned aerial vehicle (UAV)] and multispectral (by UAV and ground-based proximal sensing technology GreenSeeker™) data from each block. Multispectral data were transformed into the Normalized Difference Vegetation Index (NDVI). Vineyard UAV NDVI maps were further used for selective harvesting of areas corresponding to low vs. high NDVI and wines made from these two zones were compared chemically and sensorially. The hypothesis was that remote and proximal sensing technologies could accurately detect vineyard variation for manually collected variables and further implicate differences in wine attributes upon zonal harvesting. Direct positive correlations were observed between remotely and proximally sensed NDVI vs. vine size, leaf stomatal conductance, leaf water potential, virus infection, yield, berry weight, and titratable acidity and inverse correlations with Brix and potentially-volatile terpene concentration. Maps created from remotely and proximally sensed data demonstrated similar spatial configurations to interpolated maps of these variables. In general, GreenSeeker NDVI demonstrated the most significant relationships with measured variables compared to UAV NDVI and UAV thermal data. Wines created from areas of low vs high NDVI differed inconsistently in their wine pH. Sensorially, in certain sites and vintages, panelists were able to distinguish between wines made from low vs high NDVI zones. Overall, remote sensing demonstrates the ability to detect vineyard areas differing in measures of vine health, size, yield, berry composition, and wine attributes, though more research is needed to understand the inconsistent results observed between vineyard sites and vintages.
  • Identifying the effect of clone and rootstock on viticultural performance, fruit composition and winemaking potential for Pinot Noir, Chardonnay and Riesling, in Niagara, Ontario.

    Barker, Andrea; Department of Biological Sciences
    Previous research globally has demonstrated that the performance of grapevine clone and rootstock combinations on vine performance and fruit quality are region-specific. Consequently, it is imperative for the Ontario wine industry to have locally-relevant information on the performance of vine combinations before these selections are purchased and established in vineyards. The objective of this research is to identify the influence of clone and rootstock combination on vine performance, fruit composition and oenological potential for core Ontario varieties: Pinot noir (clones: 113, 114, 115 and 777 on rootstock Riparia Gloire), Chardonnay (clones: 548, 96, 95 and 76 on rootstock Selektion Oppenheim 4), and Riesling (clones 9, 12 and 21 on rootstock SO4; clones 239, 49 and 21 on rootstock SO4; rootstocks 101-14, SO4 and 5C on clone 9; and rootstocks 3309C and SO4 on clone 21) over the course of multiple growing seasons and vintages, 2017 and 2018. Replicated blocks for each treatment were established in one of three commercial vineyards within the Niagara region. Vine performance was measured by timing of phenological stages, yield components, vine balance, disease and winter injury. Fruit composition was determined by measuring acid and soluble solid content of 100-berry samples taken from sentinel vines. Fruit was harvested from select research blocks and fermented into wine to evaluate oenological potential through must composition analysis, fermentation kinetics and finished wine composition analysis. Results indicate significant (p≤0.05) differences in all cultivars in both years, for the effects of clone and rootstock on vine performance, fruit composition and oenological potential in Niagara. However, trends were generally not consistent across years, indicating further vintages will be required to eliminate the weather-related differences between years. This project is limited by its duration of two years. Research from additional vintages is required in order to further understand the effects of clone and rootstock selection under Niagara vineyard conditions.
  • Cellular Mechanisms Underlying Retinoic Acid-Induced Growth Cone Guidance During Neuronal Regeneration

    Johnson, Alysha; Department of Biological Sciences
    During the period of neuronal development, neurons must make correct synaptic connections with their appropriate targets. The intricate connections of the nervous system are established in part by growth cones, located at the tips of extending neurites. These unique structures are essential for axon pathfinding and target cell selection by sensing and integrating numerous guidance cues from their environment. Retinoic acid, the active metabolite of vitamin A, is an important regulator of neurite outgrowth during vertebrate development, but there is substantial evidence that it also plays a role in axon guidance. Previous studies have provided preliminary evidence of a potential role of retinoid receptors in mediating the chemotropic effects of retinoic acid. In this study, I demonstrated that a synthetic retinoic acid receptor agonist was able to mimic the effects of retinoic acid in inducing growth cone turning. I also examined the intracellular pathways activated by retinoic acid that induce changes in growth cone behaviour. Previously it has been shown that retinoic acid-induced growth cone turning of invertebrate motorneurons requires local protein synthesis and calcium influx, similar to other known guidance cues in the central nervous system. However, the signalling pathways that link calcium influx to the regulation of cytoskeletal dynamics involved in growth cone turning are not currently known. Here, I examined potential effectors downstream of retinoic acid and have provided evidence that the intracellular pathways likely involve the Rho GTPases, Rac and Cdc42. I demonstrated that the inhibition of Rac or Cdc42 prevented growth cone turning towards retinoic acid. However, it was shown that the involvement of Rac differed depending on whether the growth cones maintained communication with the cell body or not. Moreover, the inhibition of Cdc42 not only blocked growth cone turning towards retinoic acid, but also induced a switch in growth cone responsiveness from attraction to repulsion. Overall, these studies provide new knowledge of the mechanisms underlying growth cone pathfinding by retinoids during nervous system development and regeneration.
  • Identification and characterization of polymorphic mobile elements (MEs) in humans

    Dahi, Zakia; Department of Biological Sciences
    Retrotransposons are mobile elements (MEs) that propagate in a “copy and paste” fashion in the genomes via RNA intermediates. In the human genome, retrotransposons consist of long terminal repeats (LTRs), long interspersed elements (LINEs), short interspersed elements (SINEs), SINE-VNTR- Alus (SVAs), and processed pseudogenes (PPSGs), and they collectively contribute close to 50% of the genome. Some members of these MEs continue to undergo retrotransposition, thereby generating a type of structural variations (SVs) within and between human populations by the presence and absence of ME insertions at specific genomic locations. A large number of such polymorphic MEs have been previously reported and documented, including cases associated with diseases, but with limited sequence characterization and genotype analysis. In this study, we performed extensive computational analysis and compilation of polymorphic MEs from multiple sources. We focused on characterization of complete sequences representing the insertion alleles and pre-integration alleles of ME polymorphic loci, using methods including local sequence assembly based on rich personal genome sequence data for many entries. Further, we performed in silico genotyping and population distribution for these polymorphic MEs for 2600 human subjects representing 28 well recognized populations around the world, as well as phylogenetic analysis of these human subjects using these polymorphic MEs as markers. We identified a total of 4400 polymorphic MEs with full sequence characterization for both the pre-integration and insertion alleles. Among these, 1267 entries represent new insertions not previously documented in the Database of Retrotransposon Insertion Polymorphisms in humans (dbRIP), and 1777 entries represent ME insertions outside the current human reference genome. By individual populations and all samples as whole, all 5 ME types displayed a similar allele distribution pattern with the majority having an allele frequency at 0.5, while differences across ME types are also seen at the very low frequency range. Nevertheless, polymorphic MEs do show substantial geographic differentiation, with numerous continent-specific loci identified. Polymorphic ME-based clustering of human subjects seems to correlate well with what we know about the history and relationship of human populations, indicating the usefulness of polymorphic MEs as markers for studying human evolution. Furthermore, polymorphic MEs were found to participate in both coding and regulatory sequences, signifying their potential contribution to the phenotypic diversity present among human populations and individuals. In conclusion, polymorphic MEs represent a significant source of human genetic diversity with potentials on impacting the structure, function, and evolution of the human genome.
  • Thermal Preference and Habitat Use of Eastern Garter Snakes (Thamnophis sirtalis sirtalis) in a Southern Ontario Peatland

    Abney, Curtis; Department of Biological Sciences
    Garter snakes are the most widespread reptile in North America. Despite occupying vastly different biogeoclimatic zones across their range, evidence suggests that the thermal preference (Tsel) of garter snakes has not significantly diverged among populations or different Thamnophis species. The reason for garter snake’s wide thermal tolerance could lie in their flexible thermoregulatory behaviours and habitat use. I aimed to investigate this relationship by first identifying the Tsel of a common garter snake species (Thamnophis sirtalis sirtalis) via a thermal gradient in the lab. I used this Tsel parameter (27.82 – 32.15 ºC) as a benchmark for calculating the thermal quality of open, mixed, and forested habitats all used by the species. I measured the thermal profiles of these habitats by installing a series of operative temperature models that mimicked the thermal properties of living garter snakes and recorded environmental temperatures as living snakes experience them. Lastly, I used coverboards to survey the habitat usage of T. s. sirtalis. Of the three habitats, I found that the open habitat offered the highest thermal quality throughout the snake’s active season. In contrast, I recorded the greatest number of snakes using the mixed habitat which had considerably lower thermal quality. Although the open habitat offered the greatest thermal quality on average, environmental temperatures regularly exceeded the upper limits of the animals’ thermal tolerance, greatly restricting the activity window for Eastern garter snakes. Therefore, the open habitat may be less thermally attractive to T. s. sirtalis. My data show that not all habitat types used by Eastern garter snakes share the same quality and the relationship is not as simple as warm, open habitats are better. Rather, these animals may preferentially seek out areas that offer a mix of both open and closed-canopy spaces to suit their thermoregulatory needs. In conclusion, T. s. sirtalis may select more thermally stable habitats over those that present them with thermal extremes and greater constraints to their surface activity.
  • Taste responsiveness and beer behaviour

    Small-Kelly, Stephanie; Department of Biological Sciences
    Orosensory perception strongly influences food and beverage liking and consumption. Variation in the perception of these oral sensations presents an opportunity to conceptualise and commercialise products based on consumers’ taste responsiveness. This thesis investigates the role of orosensory responsiveness in alcoholic beverage behaviour. Specifically, examining the role of thermal tasting in beer and cider liking and consumption. 60 participants (31 thermal tasters (TTs) and 29 thermal non-tasters (TnTs) rated the intensity of aqueous solutions of beer- and cider- relevant tastants: iso-α-acid (bitterness), ethanol (irritation, bitterness, sweetness), dextrose (sweetness) and citric acid (sourness) at concentrations typically found in commercial beers on generalised labelled magnitude scales (gLMS). Taste intensities (gLMS) and liking (9-point hedonic scale) of eight beer and cider samples differing in iso-α-acid and ethanol content were also rated. Participants self-reported on their beer and cider consumption. They also rated the importance of select factors when purchasing beer. TTs experienced the bitterness of ethanol more intensely than did TnTs (p(t)<0.05), they also rated the bitterness, sourness, astringency, and overall taste intensity of sampled beers and ciders higher than TnTs (p(F)<0.05). Agglomerative hierarchical and k-means clustering of liking scores revealed 3 clusters of consumers, characterized as ‘bitter dislikers’, ‘beer likers’, and ‘alcohol lovers’. ‘Taste’ was the most important factor consumers used when purchasing beer. It can be concluded that thermal taster status is an important determinant in the perception of beer and cider flavour. These results should assist product developers in designing beers and ciders targeted for specific segments of the population.
  • Testosterone’s regulation of the HPA axis differs for adolescent and adult male rats

    Zeidan, Mostafa; Department of Biological Sciences
    Stress hormones such as corticosterone (CORT), enables rodents to cope and meet the demands of their environment. In adult male rats, CORT release in response to stressors is dampened by the gonadal hormone testosterone. Adolescent male rats secrete more CORT in response to stressors than do adults and our lab has previously reported that gonadectomised pre-pubertal adolescent male rats (postnatal day [P] 35) do not show the dampening effect of testosterone replacement on CORT release in response to restraint stress found in adult rats (postnatal day [P] 75); whereas, post-pubertal adolescent rats (postnatal day [P] 45) show heightened CORT release when given testosterone replacement. Therefore, the main question would be what is the basis of the age difference in response to testosterone? And so, my mechanistic hypothesis that is tested in the thesis is that the greater stress response of adolescents than adults is because of greater conversion of testosterone to estradiol and/or less conversion of testosterone to DHT in adolescents than in adults. In Experiment 1, I replicated the results for P45 and P75 male rats. In Experiment 2, rats were gonadectomised (GDX) and given implants of testosterone, dihydrotestosterone (DHT), or empty, control implants (CTL) and plasma was obtained after 30 minutes of restraint. Although no significant differences were obtained for CORT levels, the pattern of means was consistent with our previous findings. Further, DHT and testosterone-treated rats had lower vasopressin (AVP) and corticotrophin releasing hormone (CRH), and a trend toward lower aromatase-immunoreactive cell counts in the parvocellular paraventricular nucleus (PVN) than did CTL rats, irrespective of age, and there were no group differences in the magnocellular PVN. In Experiment 3, gonadally-intact P45 and P75 males were treated with fadrozole (aromatase inhibitor), finasteride (5a-reductase inhibitor), flutamide (androgen receptor antagonist), or vehicle (VEH). Higher CORT concentrations after restraint were found in P45 than in P75 only among VEH rats. Among P45 rats, CORT levels were higher in VEH than in fadrozole-treated rats only. Among P75 rats, VEH rats had lower CORT levels than did finasteride-treated rats. These results suggest that the higher stress release of CORT in P45 may involve greater conversion of testosterone to estradiol at the level of the adrenal cortex.
  • Reproductive behaviour of male Xylocopa virginica and the influence of body size, nestmates, and siblings on territory defence

    Duff, Lyndon; Department of Biological Sciences
    Males of Xylocopa virginica are territorial like many other bee species. Males interact aggressively to displace other males from territories. Body size is known to influence resource holding potential in many other taxa, and studies of bees suggest that body size is important for territorial males. Familiarity and the avoidance of kin competition are also known to influence territorial behaviours in other taxa but has not been studied in male bees. Recent evidence suggests that nestmate recognition occurs in X. virginica and there is also evidence for the avoidance of kin competition. This thesis tests whether body size, familiarity, and kinship influence territorial interactions using social networking tools. Around half of all males attempted to establish or defend a territory. Males that established or defended territories are larger than males that did not. Male body size has a weak positive influence on hover rates, related to holding territories, the number of hovering neighbours each male had, and the number of males each male chased or fled in defence of territories. I found no evidence to support that familiarity influences aggressive behaviours, but there is a strong correlation with the number of neighbours a male had and the number of males it chased or fled. Brothers estimated from microsatellite genotypes are no more aggressive to each other than to non-siblings. However, the results indicate that several sets of brothers overwintered in different nests, which does not coincide with the behavioural patterns described in the literature. This study is the first step towards understanding the influence of familiarity and kin competition on male behaviour in a taxonomic group with a wide array of mate-locating strategies. Discussed herein are the importance of continued research on mating systems and mate-locating strategies of bees, as well as outlining future projects to address several gaps in knowledge that remain after this study.
  • The effect of Resveratrol on the Upregulation of Ngb

    Rezk, Mohamed; Department of Biological Sciences
    Apoptosis involves a series of biochemical events that leads to the eventual death of the cell. One pathway – intrinsic pathway, involves the fragmentation of mitochondria and the release of pro-apoptotic proteins, such as cytochrome c. A certain globin protein has been shown to be able to protect cells from apoptosis, called Neuroglobin (Ngb). Ngb is a globin haem protein that has been shown to reduce the ferric form of cytochrome c to inhibit apoptosis. In addition, Ngb has been shown to translocate into the mitochondria under stress, where it reacts with cytochrome c. Estradiol (E2) has been shown to greatly upregulate the levels of Ngb and also stimulates the translocation of Ngb into the mitochondria. The upregulation of Ngb has been shown to be mediated via the ER subtype, ERβ. Even though literature covers the effects of E2 and the ERβ agonist DPN (Diartylpropiolnitrile), there is a lack of evidence on the ER agonist, Resveratrol (RES); RES is a phytoestrogen that has been shown to induce mitochondrial biogenesis and abrogate mitochondrial fragmentation, ameliorating apoptosis. The hypothesis of this study is that RES will upregulate Ngb levels as E2 does, and will translocate Ngb into the mitochondria as E2 does. The results of this study showed that Ngb bands could not be detected via western blots, and the mRNA transcript levels in MCF-7 and DLD-1 could not be quantified. The Ngb-GFP fusion protein did not fluoresce and Ngb’s translocation into the mitochondria could not be determined. Ngb overexpression did not inhibit mitochondrial fragmentation and did not induce mitochondrial fusion.
  • Retinoic Acid and the Underlying Cellular Mechanisms involved in Neurite Outgrowth and Growth Cone Turning during Regeneration

    Nasser, Tamara Israa-Nadine; Department of Biological Sciences
    The involvement of retinoic acid (RA) in nervous system regeneration has been well documented, though the precise cellular and molecular mechanisms have not yet been fully determined. During regeneration, RA can exert trophic support for cells, as well as tropic effects to guide neurite outgrowth. Cultured neurons of the Lymnaea central nervous system (CNS) have previously been used to investigate the role of RA in neurite outgrowth and growth cone guidance. Recently however, a novel phenomenon has been identified, in which neurite outgrowth occurs from cut nerves of Lymnaea CNS “floating” on the surface of cultured medium. In this study, I examined whether RA could induce or guide neurite outgrowth from this novel preparation. Unlike previous findings with cultured neurons, there was a lack of consistent effects of 9-cis and all-trans RA in promoting neurite outgrowth from the floating CNS. However, the growth cones of these floating neurites were found to turn toward a local source of RA, indicating for the first time, that they are capable of responding to guidance cues, even in the absence of adhesion to a solid substrate. The cellular mechanisms of RA-induced growth cone turning were then conducted on cultured neurons. I demonstrated that various retinoid receptor agonists could mimic the chemotropic effects of RA, providing further evidence for a potential role of retinoid X receptor and retinoic acid receptors in growth cone turning. I also examined potential downstream effectors involved in this chemotropic response to RA and provided the first evidence that the intracellular signaling pathway likely involves the Rho GTPase, Rac. However, it was also shown that the involvement of Rac in mediating the RA-induced growth cone turning differed depending on whether the turning was induced by endogenous or synthetic retinoids, and also whether the growth cones were still attached to the cell body or not. Overall, these data provide new insights into the mechanisms by which retinoids affect neurite outgrowth and growth cone behaviour during regeneration of the nervous system.
  • Behavioural Thermoregulation and Energetics in Two Intermediate Hosts of Trematode Parasites

    Wang, Susan Yao Shan; Department of Biological Sciences
    Infection by macroparasites, such as trematodes (flatworms), can negatively impact survival of hosts such as larval amphibians, potentially altering host energy use in response to infection, and also through alterations of host behaviour that may increase infection tolerance or instead benefit the parasite. However, physiological consequences of macroparasite infections are not well studied, despite heavy parasite burdens in the field. The purpose of this work was to examine altered thermoregulatory behaviours in two taxa (snails and larval amphibians) used as intermediate hosts by trematodes, as well as to study the metabolism of naturally-infected tadpoles. Both infected and uninfected tadpoles (Lithobates sylvaticus and L. pipiens) and snails (Helisoma trivolvis) were placed in thermal gradients to observe thermal preferences in hosts. Oxygen consumption in naturally-infected bullfrog tadpoles (L. catesbeiana) was measured to determine whether macroparasites could impact host metabolism. The trematode-infected, L. sylvaticus tadpoles exhibited “behavioural fever” by choosing warmer temperatures by the end of the experiment compared to uninfected tadpoles, but this did not occur in L. pipiens. Active, infected snails also selected warmer temperatures relative to inactive snails and active uninfected snails. Trematode infection intensity did not affect respiration in L. catesbeiana tadpoles, but those with higher metabolic rates and larger fat bodies had lower parasite counts. These results suggest that behavioural fever may occur in ectotherms infected with macroparasites, but may be more important for species which are relatively intolerant of infection given that fever was not seen in L. pipiens. As infected snails selected warmer temperatures, this may be a case of parasite manipulation to increase production and emergence of infectious stages in warm microhabitats to facilitate transmission. Metabolic rate increased with fat body content, and larger fat bodies were observed in tadpoles with lower parasite intensity, suggesting more heavily parasitized animals had lower energy stores. Globally, infectious diseases are known to contribute to amphibian declines, thus more research is needed to understand the possible consequences of parasitism and mechanisms by which hosts to may defend themselves.

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