Now showing items 1-20 of 311

    • Investigating the Modulation of Drosophila melanogaster Body-wall Muscle Contraction by the Neuropeptide DPKQDFMRFamide

      Wasilewicz, Luc; Department of Biological Sciences
      The chemical synapse is the site of communication between a neuron and its target cell, where an electrical impulse depolarizes the presynaptic cell causing chemical release. The chemicals released at the synapse are signaling molecules referred to as transmitters and co-transmitters that exert effects on the target cell and can sometimes modulate the effects of each other. A class of signaling molecules, known as neuropeptides, can act as transmitters or can be released as hormones that can modulate chemical synapses and ultimately affect many physiological functions. The neuropeptide, DPKQDFMRFamide, is an important neuromodulator of neuromuscular junctions in the fruit fly, Drosophila melanogaster. DPKQDFMRFamide has previously been shown to enhance excitatory junctional potentials (EJPs) elicited by specific neurons, to enhance nerve-evoked contractions, and to induce contractions directly in Drosophila 3rd instar larval body-wall muscles. This thesis investigated how the DPKQDFMRFamide peptide modulates muscle contractions elicited by the excitatory transmitter of the neuromuscular junction, L-glutamate, in D.melanogaster 3rd instar larvae. Effects were assessed by co-applying peptide with L-glutamate after removing the central nervous system. The results indicate that DPKQDFMRFamide enhances glutamate-evoked contractions in a dose-dependent manner, and there was synergy between the effects of L-glutamate and DPKQDFMRFamide on muscle contraction. DPKQDFMRFamide increased membrane depolarization in muscle when co-applied with glutamate, and it enhanced contractions induced by caffeine in the absence of extracellular calcium. Thus, the peptide appears to act at the cell membrane to increase depolarization and at, or downstream of the sarcoplasmic reticulum (SR) to enhance caffeine-induced contractions. However, the effects of DPKQDFMRFamide do not appear to involve the 2nd messenger nitric oxide or the calcium/calmodulin activated protein kinase, CaMKII.
    • Selecting cover crop species for vineyards of the Niagara region

      Ben kalifa, mohamed lahbib; Department of Biological Sciences
      Organic viticulture challenges growers to think and act sustainably when managing variables such as weeds, pests, and overall crop production. Ongoing climate change is adding to this challenge with projected increases in extreme weather events such as heavy rainfall and drought. Cover crops can be considered as an ecosystem-based adaptation measure when chosen carefully. They can help growers mitigate effects of climate change as well as increase vineyards biodiversity. Despite their common use, local knowledge of which species work best in what conditions is lacking. Furthermore, species are seldom tested for response to drought and flood conditions in both controlled and operational settings. The first objective of this project was to evaluate the responses of nine different cover crop species to simulated drought and flood conditions under greenhouse-controlled conditions. Of the nine species, Pennisetum glaucum (pearl millet) and Melilotus officinalis (yellow clover) were the only two species to withstand both extreme conditions without being significantly affected. Trifolium alexandrinum (berseem clover), Vicia villosa (hairy vetch), and Trifolium incarnatum (crimson clover) produced higher biomass in saturated condition, while Festuca rubra (red fescue), and Thinopyrum intermedium (pubescent wheatgrass) survived the drought without visual clear symptoms except for puny plants. The second objective was to screen 13 cover crop species in two vineyards under operational settings, where weed pressure, local weather and management may influence species establishment. After the two screening years, Pennisetum glaucum, Trifolium incarnatum, Trifolium repens, Trifolium pratense, Vicia villosa, and Medicago sativa showed promising results in terms of establishment despite facing weather challenges.
    • The development of a new genetic test for grapevine cultivars using a computational genomics approach

      Martin, Robert; Department of Biological Sciences
      Due to the sale and consumption of wine and table grapes, the grapevine is an important crop for many countries, including Canada. One of the main issues in viticulture is the identification of cultivars. Many of the over 6000 different types of grape cultivars look similar in colour or shape but may have very different taste profiles and require different growing conditions, while some have the same name but are genetically different (homonym) or having different names but are genetically identical (synonyms). Genetic tests based on the use of simple single repeat (SSR), or short tandem repeats (STR) markers have been developed to determine the genetic identity of different grapevine cultivars. However, the markers used in existing tests were identified more than 2 decades ago without optimization, and with the service limited to a few places around the world imposing many hurdles for international users. This research aims to develop a new grapevine genetic test by selecting the best STR markers in taking advantage of recently available rich grapevine genomic resources. Using a computational genomics approach, a total of 13 top performing STR markers were selected based on their discrimination power for 304 grapevine cultivars. A Polymerase Chain Reaction (PCR) based test was designed to group these 13 STR markers into 5 multiplex PCR groups for assaying using the QIAGEN QIAxcel Advanced System™ for its speedy and cost-efficient DNA fragment analysis. As a way of evaluating the performance of the designed test, in silico genotyping was performed for 304 grapevine cultivars and 37 Chardonnay clones based on available whole genome sequencing data. The results showed that the test was able to distinguish all these grape cultivars and Chardonnay clones, and furthermore, the number of STR markers used in the test can be reduced to a minimum of 6 for distinguishing these cultivars and clones. Genotype-based phylogeny analysis of these cultivars and clones showed meaningful clustering patterns matching their known or assumed relationships, indicating the validity of the test. In conclusion, despite not being able to perform evaluations of the STR markers in the laboratory, the preliminary in silico results demonstrate the high efficiency of the computation genomic approach in finding top performing STR markers and predicts an excellent performance of the designed grapevine genetic test.
    • The ecological and physiological consequences of sun vs. shade nesting for the small carpenter bee, Ceratina calcarata, and their offspring

      de Haan, Jessie; Department of Biological Sciences
      Ceratina calcarata mothers choose their nesting location and that choice can have developmental consequences for their offspring. Nests in the sun experience higher developmental temperatures, reducing self-feeding insect body size through a phenomenon called the Temperature-Size Rule. How does developmental temperature affect body size in insects whose mothers’ feed them; for example, in mass-provisioning bees where all the food an offspring needs to complete development is provided by the mother upfront? What are the physiological advantages or disadvantages conveyed to offspring in sunny nests? In this thesis I used C. calcarata to investigate the ecological and behavioural consequences of nest location choice (sun or shade) on mothers, as well as the physiological consequences of developmental temperature on their offspring. Nests randomly allocated to the shade treatment were more likely to be empty when opened, indicating that shaded nesting locations were not preferred. Mothers nesting in the sun foraged more often for nectar than shade mothers, but provisioned similar sized pollen masses. Sunny nests were hotter than shaded nests, even more so if they were oriented on an angle. Offspring from sunny nests were smaller than shade bees in agreement with the Temperature Size Rule. Sunny offspring also had higher thermal tolerance than offspring from the shade but less frequent DGE bursts at 25°C compared to shade bees, suggesting a lower metabolism at a moderate temperature. As a result, I conclude offspring from sunny nests displayed irreversible developmental thermal plasticity. Offspring from sunny nests experienced a thermal trade-off as a result of their developmental conditions; they had a capacity for higher thermal tolerance as evidenced by a shifted thermal breadth, but at the expense of a smaller adult body size. Such trade-offs may enhance their response to climate change and contribute to speciation.
    • Crawford Lake Consumers: Water Column and Palynological Studies

      Heyde, Autumn; Department of Biological Sciences
      Despite their important role in lake ecosystems, the fossil record of consumers has been underutilized compared to the remains of algae and plants in paleoenvironmental studies. Cladocerans, chironomids, and testate amoebae were found in palynological preparations of sediments throughout Crawford Lake (a unique meromictic lake in Ontario, Canada), but rotifer lorica and cysts of aloricate ciliates were only preserved in seasonally laminated sediments in the monimolimnion of this lake, demonstrating the exceptional preservation potential in this portion of the lake water column. Relatively diverse assemblages of consumer palynomorphs were associated with anthropogenic impact on this lake, and the annual chronological resolution afforded by varves allowed these to be related to historic events in the small watershed, the most notable being the operation of a lumber mill on the south shore of the lake, and to archeological and pollen evidence of several phases of agricultural settlement between the 13th and 15th centuries. Lower diversity of consumer palynomorphs between the Iroquoian and Euro-Canadian settlement phases (i.e., late 15th through early 19th centuries) mainly reflects the sharp decline in most rotifer taxa and the cladoceran Bosmina longirostris, but the persistence of the rotifers Keratella hiemalis and Kellicottia longispina is evidence that the lake ecosystem did not return to pre-human impact conditions after abandonment of the Iroquoian settlement. Understanding how the trophic level of consumers responded to natural and anthropogenic stressors relied heavily on rarely preserved rotifer lorica, but the observation that the cladoceran B. longirostris tended to thrive relative to the typically more common Daphnia at times of cultural eutrophication may have broader application in palynological studies of lakes. Contrary to long-standing assumption, the exceptional preservation of organic-walled microfossils in undisturbed seasonal laminae in the deep basin of Crawford Lake cannot be explained by anoxia. Observations of seasonal migration of zooplankton to and from the mixolimnion in conjunction with instrumental measurements of dissolved oxygen, temperature, and conductivity in the water column almost monthly from October 2019 through September 2020 demonstrated that this meromictic lake is uncharacteristically well-oxygenated below the chemocline. Instead, exceptional preservation is attributed to the lack of bioturbation and the suppression of bacterial decomposition in the cold, nearly brackish, highly alkaline bottom waters devoid of benthos larger than ostracods able to migrate into the deep basin via interstitial waters. The annual resolution possible in sediments deposited in a typically well-oxygenated setting is an attractive feature in the search for a potential GSSP to define the Anthropocene Epoch using plutonium from fallout of thermonuclear testing as a primary marker.
    • Analysis of cellular responses to microwave irradiation in E. coli and change of oxygen level and culture medium in human cancer cell lines using RNA-seq based transcriptomic profiling

      Hilson, Eunike Ilona; Department of Biological Sciences
      RNA sequencing (RNA-seq) is one of the applications of next-generation sequencing (NGS) with differential gene expression (DGE) analysis at the transcriptomic level as its primary objective. Among the NGS technologies, the Illumina platforms are the current standard for RNA-seq analysis for their best cost efficiency and sequencing accuracy. In this study, we employed Illumina-based RNA-seq to examine the gene expression profile change in E. coli cells after exposure to microwave irradiation (MWI) and in cancer cell lines in response to different culture conditions using breast cancer cell lines (MCF7) and prostate cancer cell lines (PC3) as the models. Our results in examining the gene expression change in E. coli showed that the non-thermal effects of MWI led to E. coli cells entering the stationary phase with most of the downregulated genes involved in metabolic and biosynthesis pathways. MWI also upregulated the expression of genes important for the maintenance of membrane integrity and adhesion associated with bacterial motility. In comparison with other similar studies, our methodology allowed us to observe the impact of non-thermal effects of MWI at 2.45 GHz via simultaneous cooling. Our results in examining the transcriptomic profile of MCF7 and PC3 cells in response to oxygen level and culture medium change showed that gene expression in MCF7 is highly affected by oxygen level and culture medium changes when compared to PC3, especially in DMEM at 18% O2. DNA replication, cell-cycle, and viral carcinogenesis are the most affected pathways observed from different culture conditions in both cell lines. In PC3, only the legionellosis seems to be most impacted by culture medium changes at 5% O2, involving 8 differentially expressed genes (DEGs), important for cancer cell development. DGE analysis also provides the transcriptomic profile of MCF7 and PC3, showing that different nutrient composition (between DMEM and Plasmax) and oxygen levels (5% O2 and 18% O2) changes the metabolism and various signaling pathways in both cell lines differently suggesting that the oxygen level and culture medium are important factors impacting the outcome of cell culture-based experiments in cell type-specific fashion.

      Tolentino, Michael; Department of Biological Sciences
      Research into the molecular mechanisms of the psychoactive effects of cannabis has led to the discovery of the endocannabinoid system (ECS), a neuromodulatory system conserved throughout the animal kingdom. Little is known about its function in mammals, but there is evidence suggesting its contributions in the cellular processes that are important in CNS development and are conserved during CNS regeneration. However, these studies focussed primarily on mammals, which display limited abilities to regenerate after traumatic CNS injury. Furthermore, nothing is known regarding the role of endocannabinoids in CNS regeneration-competent species like the Mexican axolotl, one of the few vertebrates that can regenerate their spinal cord. The current study investigates the potential role of the ECS in influencing the pro-regenerative response observed in the axolotl spinal cord. I provide evidence that the main ECS receptor in the CNS (CB1) is upregulated in the regenerating caudal spinal cord and tail tissues of larval axolotls at 4 hours post amputation, lasting until 14 days post amputation. By performing immunofluorescence studies on these tissues, I demonstrate the expression of this receptor mainly in the ependymal region. In addition, bath application of the CB1 inverse agonist, AM251, significantly inhibited caudal growth of the spinal cord and tail by 7 days post amputation. The current study also identified an upregulation in a second ECS receptor, CB2, at 7- and 14-days post amputation. Immunofluorescence analysis revealed the localization of this receptor to the subependymal regions within the spinal cord. Furthermore, inhibition with the CB2 inverse agonist, AM630, similarly demonstrated an inhibition in spinal cord and tail regeneration by 7 days post amputation. An assessment of CB1 and CB2 expression was performed by identifying their localization in bromodeoxyuridine-positive (proliferating) and doublecortin-positive (differentiating neuronal) cells in 7-day regenerate tissue. These studies are the first to examine the role of the ECS during spinal cord regeneration in a regeneration-competent vertebrate and may aid in developing novel therapies for human nervous system injuries or pathologies.
    • Demography and molecular ecology of the solitary halictid Lasioglossum zonulum: with observations on Lasioglossum leucozonium

      Proulx, Alex; Department of Biological Sciences
      Halictid bees are excellent models for questions of both evolutionary biology and molecular ecology. While the majority of Halictid species are solitary and many are native to North America, neither solitary nor native bees have been extensively studied in terms of their population genetics. This thesis studies the social behaviour, demographic patterns and molecular ecology of the solitary Holarctic sweat bee Lasioglossum zonulum, with comparisons to its well-studied sister species Lasioglossum leucozonium. I show that L. zonulum is bivoltine in the Niagara region of southern Ontario but is univoltine in a more northern region of southern Alberta. Measurements of size, wear and ovarian development of collected females revealed that Brood 1 offspring are not altruistic workers and L. zonulum is solitary. A large proportion of foundresses were also found foraging with well-developed ovaries along with their daughters, meaning L. zonulum is solitary and partially-bivoltine in the Niagara region. L. zonulum being solitary and univoltine in Calgary suggests that it is a demographically polymorphic and not socially polymorphic. Thus, L. zonulum represents a transitional evolutionary state between solitary and eusocial behaviour in bees. I demonstrate that Lasioglossum zonulum was introduced to North America at least once from Europe in the last 500 years, with multiple introductions probable. Most North American specimens share the same mitochondrial DNA haplotype as those in Europe, with a small portion from western North America possessing distinct sequences. Investigations using microsatellite markers found North American populations to have a deficit of heterozygosity, and Bayesian analysis suggests that there are 3-4 lineages of L. zonulum in North. It is theorized that introductions could also be from Europe, Asia, or could even represent a native population which arrived via the Bering Land Bridge. I suggest that the plasticity found in L. zonulum may have a genetic cause and exists in North America due to the multiple introductions and potentially diverse geographic origins of this species. The outcome of my studies highlight why Lasioglossum zonulum is a model organism for the study of how eusociality evolved and why it warrants further and more in-depth study.
    • Investigating how Notch, and JAK/STAT Signaling Synergistically Regulate Intestinal Stem Cell Homeostasis Using Engineered Optogenetic Alleles

      Lidster, Taylor; Department of Biological Sciences
      Stem cells are a class of undifferentiated cells that have the unique ability to give rise to a variety of specialized cell types during tissue development and growth. These cells communicate amongst one another by sending and receiving signals from multiple pathways that regulate cell fate decisions by promoting either self-renewal or differentiation. The Notch, and JAK/STAT signaling pathways are central regulators of multicellular development and are vital for tissue maintenance. The interplay between the Notch, and JAK/STAT pathways required for tissue homeostasis has not been fully elucidated, particularly as it relates to the intestinal epithelium. I utilize the Drosophila melanogaster midgut as a model system to study stem cell dynamics and more specifically, how Notch and JAK/STAT signaling cooperatively regulate intestinal stem cell turnover. In order to accomplish this, I employed both traditional and optogenetic methods to elicit Notch blockade and ectopic activation of JAK/STAT signaling in the midgut. First, I demonstrated that ectopic JAK/STAT signaling paired with Notch knockdown causes substantial ISC overproliferation, leading to the formation of large ISC tumors spanning the entire midgut. Quantitative assessment of ISC pools confirmed that Notch and JAK/STAT signaling work in a synergistic manner, rather than an additive manner, to regulate ISC homeostasis. I also utilized two optogenetic alleles, Opto-Delta and Opto-Domeless, to recapitulate Notch blockade and JAK/STAT hyperactivation using light. Opto-Delta was tested in the intestinal epithelium and demonstrated the ability to block Notch signaling in the ISCs, resulting in the formation of stem cell clusters. Subsequently, an Opto-Domeless construct was created and expressed in the nervous system during embryogenesis to assess its efficacy to activate JAK/STAT signaling. The embryos that expressed Opto-Domeless exhibited a significant decrease in viability when subjected to the light condition, suggesting possible light-responsiveness. Expression of Opto-Domeless in the midgut was also achieved, however, ISCs expressing Opto-Domeless appeared to have JAK/STAT activity in the absence of light exposure, suggesting that Opto-Domeless is not behaving in a binary fashion and will require further validation. Altogether, these findings reveal great potential for light-gated proteins, as they provide a powerful approach to disentangle dynamic cellular signals by using light.
    • Sleepers and Creepers, Colony Polymorphisms in Metarhizium robertsii

      Angelone, Steven; Department of Biological Sciences
      In this thesis I attempt to identify, describe, and explain the vast colony polymorphism differences observed in Metarhizium robertsii from a physical and genetic standpoint. These descriptions provide a framework to explain the colony polymorphisms seen in M. robertsii. This work is based on the observation of three distinct phenotypes: a highly conidiating phenotype (Sleeper), a primarily hyphal phenotype (Creeper) and an intermediary (Mixed) phenotype that represents the middle range between the Sleeper and Creeper phenotypes. Based on our findings, Sleepers typically have a higher fungal mass recovered from plant rhizospheres compared to Creepers and Mixed phenotypes. Creepers and Mixed strategy phenotypes are almost undifferentiable when grown on minimal nutrient medium whereas the Mixed and Sleepers tend to look very similar when grown on nutrient rich media. I concluded that the three phenotypes cannot be differentiated using artificial media. However, it was possible to differentiate between the Sleeper, Creeper and Mixed by infecting Great Wax Moth larvae (Galleria mellonella) with conidial suspension of M. robertsii. Key genes in the conidiation pathway, such as the methyltransferase MrDIM-2, may be relevant for differentiation between the Sleeper and Creeper/Mixed phenotypes. Expression levels of the Sleeper phenotype were found to be significantly lower than the Mixed or Creeper phenotypes.. The results have shown that MrDIM-2 expression influences either a Sleeper phenotype or a Mixed/Creeper phenotype presentation.
    • 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.