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M.Sc. Biological Sciences
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Determining the effect of different cover crops on nematode communities in vegetable productionAgricultural soil environments contain different types of nematodes, part of several trophic levels that aid in balancing the soil food web. Beneficial free-living nematodes (FLNs) consist of bacterivores, fungivores, predators and omnivores that help in the mineralization of the soil and the top-down control of damaging plant-parasitic nematodes (PPNs). Conversely, PPNs feed directly on plant roots and cause $125 billion in worldwide crop losses annually, making them a plant pathogen of great concern for growers. Farmers have started to implement the use of cover crops in agricultural systems for the protection and enrichment of soil but research on how different covers affect nematode populations is lacking and in demand. This study aims to determine the effect of legume and grass cover crops, cow pea and pearl millet, as well as their mixture on the abundance and diversity of FLN and PPN populations. Soil samples were taken prior to, during, and after cover crop implementation to analyze nematode communities using morphological and DNA metabarcoding techniques. Results showed that the implementation of cow pea and pearl millet covers as well as their mixture in a spinach agricultural system led to the control of PPNs and the proliferation of FLN communities. Specifically, we found that the mixture of both cover crops and the use of pearl millet as a single species generated healthier nematode populations with significantly less PPNs and more FLNs when compared to the control and fallow plot treatments using both nematode identification methods. However, the choice of cover crop to implement by growers should be based on specific soil health needs to promote a healthy soil and nematode population. Ultimately, understanding how different cover crops impact nematode populations can inform sustainable agricultural management practices. By strategically selecting legume and grass cover crops, farmers can optimize soil health, reduce crop losses caused by PPNs, and promote a balanced soil ecosystem to improve crop productivity.
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Influence of cover crops on invertebrate diversity in Niagara organic vineyardsCover crops can play different functions in vineyards, such as preventing soil erosion, improving soil health, reducing the need for pesticides, and increasing nitrogen fixation in soils. Despite the potential of cover cropping, little research has been done on the influence of diverse vegetation on invertebrate functional groups. This study aimed to examine the influence of different cover crop species in the diversity of pests, neutral, and beneficial invertebrate species. The first objective of this study was to assess diversity in the invertebrate community and their functional groups in six different cover crop treatments (Trifolium incarnatum, Vicia villosa, Pennisetum glaucum, mixture of T. incarnatum, V. villosa, P. glaucum, Lolium perenne, and spontaneous vegetation) across two different organic vineyards in the Niagara region. The second objective was to test the use of sentinel plants to identify direct interactions between cover crops and invertebrates. These sentinel plants were grown in greenhouse conditions before being introduced into the field. To access the invertebrate communities involved, the deployment of sticky traps, vine sweeps and cover sweeps in June, August and September 2022 was used to obtain a comprehensive idea of invertebrate communities. The results showed that cover crop species, whether grown in monoculture or mixture, did not significantly influence invertebrate diversity and relative proportion of functional groups. Instead, factors such as vineyard management, environmental conditions, and landscape complexity may play a key role in shaping invertebrate community diversity. Additionally, temporal variation played an integral role in influencing the abundance of pest and beneficial morphospecies in both vineyards. Invertebrate interactions on sentinel plants were nonconclusive, leaving potential interactions between cover crops and invertebrates uncertain in terms of sentinel plant deployment. Also, cover crops grown in the interrow of the grapevines were not the sole vegetation species present in the area, making it challenging to attribute performance solely to a singular species in the study. The results of this study indicate how complex and intricate invertebrate community diversity can be in operating applications. Further investigation on cover crop performance in the field is warranted to better understand how they may influence invertebrate community structure dynamics.
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Investigating Vitis sp. cold stress tolerance with abscisic acid (ABA) analogsThe sustainability of Vitis sp. grapevine cultivation is threatened by climate change with pressure mounting on low temperature stress adaptations. Abscisic acid (ABA) is hypothesized to act as a central mediator of dormancy status and cold tolerance of grapevine buds. With one or more modifications to the chemical structure of ABA, synthetic analogs can improve its bioactivity in planta. The current research is founded in a comprehensive investigation on dormant grapevine bud cold stress responses following the administration of 8’-acetylene ABA (ABA-1017) and tetralone ABA (ABA-1016). Using V. vinifera (cv. Merlot) and interspecific Vitis hybrid (cv. Marquette) as freeze sensitive models, this thesis set out to determine: (1) the seasonal effect of ABA analogs on the resistance to losing cold tolerance; and (2) the impact of these products on bud dormancy physiology in relation to ABA catabolism. A record of five consecutive seasons (2017-2022) of differential thermal analysis data (DTA) was utilized to perform correlational test on the estimated low temperature exotherm (LTE) to the average minimum field temperature in deacclimation. Water content was measured for untreated, ABA-treated, and ABA analog-treated buds throughout the 2020-2021 and 2021-2022 dormant seasons. Samples were collected in alignment for hormone profiling (LC-MS/MS) to quantify the relative levels of ABA, its metabolites, and the degree of analog uptake/residual following application. Single-node cuttings from treated vines were maintained in a controlled environment (25°C, 16-hour light) to monitor the progression of dormancy against a fixed time-to-event threshold of 50% bud break over a 60-day observation window. Results suggest: (1) ABA analogs can improve deacclimation resistance and delay bud break, but their effect is inconsistent from year-to-year and varies in response to extreme cold stress; and (2) water availability and hormonal status are impacted by the application of ABA analogs unpredictably, and results from dormancy status evaluations are inconclusive to determine the impact of these products on the endo-ecodormancy transition of buds. This research ultimately lays the foundation to continue to explore the hallmarks of Vitis sp. bud dormancy and cold tolerance coordinated by ABA and develop an optimized freeze/frost-mitigation strategy for cool climate grape growers.
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Characteristics of dominance: behaviours and hormones in a facultatively social bee species (Xylocopa virginica)In facultatively social species such as Xylocopa virginica (eastern carpenter bee), individuals can breed either on their own or in groups, where resources are monopolized by a single dominant female. As such, we would expect that natural selection would act strongly in this species to promote traits that would make females more dominant. This thesis sets out to determine the characteristics that make female X. virginica dominant both through behavioural assays and hormonal manipulation. I paired females with non-nestmates in dyads consisting of two dominants, two subordinates or one of each, during two separate years. I found higher levels of tolerance in dyads without a dominant female in them, but no difference in aggression between dyad types, which would have been expected if dominants tended to be the most aggressive. Behaviours also differed between years with the high population density year having higher levels of aggression and tolerance overall. Clearly many factors contribute to how female X. virginica interact with potential rivals, beyond simply their level of dominance. I also treated females with a Juvenile Hormone (JH) analogue, methoprene, to determine whether it increases ovarian development in this species, as it does in other Hymenoptera. Increased dose of methoprene lead to increased ovarian development in X. virginica, making treated individuals better able to lay eggs and therefore to take advantage of being in a dominant social position. There was no lethal or negative sublethal effects on treated bees meaning that this method could be used in the future for field testing in this species. I suggest that X. virginica dominance is not defined solely by aggression. There are many ways for females to gain a dominant position, and females that are not aggressive enough on their own to fight their way to a dominant position are likely to use other behaviours such as nestmate feeding to gain dominance. The outcomes of this study highlight why dominance and the factors leading to it should be considered closely for the specific species being studied, because aggression is shown to not be the main source of dominance in X. virginica.
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A comparison of blue vane trap, timed targeted netting, and timed photographic collection methods for evaluating Canadian bumble bee diversityBumble bee (genus Bombus) populations across Canada are experiencing increases and decreases in abundance; some species are becoming more common while others are at risk of extirpation or extinction. It is important to monitor population changes so that extirpation and extinction can be prevented. Current population assessments for bumble bees, when conducted, use many different collection methods, but this limits our ability to compare across studies and understand trends. There is a call within the scientific community to create a national standard method for collecting bees. The goal of this research was to provide a recommendation for which collection methods could be used across Canada for bumble bee assessments, including assessments of species at risk. Three collection methods, blue vane traps (BVTs), timed targeted netting, and timed targeted photography, were compared with the objective of determining which method provided good diversity information, detected at-risk species, and required low sampling effort. To assess the universality of method performances across the country, surveys were conducted in three different regions of Canada, the Carolinian portion of the Mixedwood Plains Ecozone (southern Ontario), the Prairies Ecozone (Saskatchewan), and the Boreal Shield Ecozone (Newfoundland and Labrador). With some exceptions, the general structure of surveys was that BVTs were deployed for 1 week at a time, and multiple 30-minute netting and photographic surveys were conducted during each collection week. Regional differences were apparent. In the Prairies Ecozone BVTs collected the most specimens while in the other regions BVTs collected the fewest. BVTs detected the most species in the Carolinian and Prairies Ecozones, but netting detected the most species in the Boreal Shield Ecozone. For all regions, BVTs were the most efficient method at low sample sizes when compared using rarefaction. BVTs also detected the most species at risk. Distinct species compositions produced by BVTs compared to netting and photos demonstrated complementarity between these methods. Netting and photo species distributions also differed from each other in most regions. The overall recommendation when assessing Canadian bumble bee populations is to use BVTs in week-long durations with either netting or photo surveys to complement them.
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The Role of Retinoic Acid in the Formation and Modulation of Invertebrate Electrical SynapsesCommunication between cells in the nervous system is dependent upon structures known as synapses. Synapses are broadly characterized as either chemical or electrical in nature, owing to the type of signals that are transmitted across them. Factors that can affect chemical synapses have been extensively studied. However, the factors that can influence the formation and modulation of electrical synapses are poorly understood. Retinoic acid, a vitamin A metabolite, is a known regulator of chemical synapses, yet its capacity to regulate electrical synapses is not as well established. Preliminary evidence from the central neurons of both invertebrates and vertebrates suggests that it is also capable of regulating the strength of electrical synapses. In this study, I provide further insights into how retinoic acid can act as a neuromodulator of electrical synapses. My findings suggest that retinoic acid is capable of rapidly altering the strength of electrical synapses in a dose- and isomer-dependent manner. Further, I provide evidence that this acute effect might be independent of either the retinoid receptors or a protein kinase. In addition, I provide novel findings to suggest retinoic acid is also capable of regulating the formation of electrical synapses. Long term exposure to two isomers of retinoic acid, all-trans-retinoic acid and 9-cis-retinoic acid, reduces both the proportion of cell pairs, and the average synaptic strength between cells that form electrical synapses. In summary, these investigations provide novel insights into the role that retinoids play in the both the formation and modulation of electrical synapses in the CNS.
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Interactions between caffeic and caftaric acids, furan-derived aroma compounds, and chitosan during base wine ageingDuring sparkling wine ageing, chemical changes take place that alter the composition of the wine, creating nutty, roasted, and caramel aromas and flavours not previously detected in young wines. Furan-derived aroma compounds, a group of odor-active chemicals formed during the long-term storage of wine, contribute to these empyreumatic aroma changes. Hydroxycinnamic acids (HCAs) such as caffeic and caftaric acid, known to be responsible for oxidative browning in wine, also influence the formation of furan-derivative aroma compounds in model wine solutions. Additionally, sparkling wine research using chitosan has been shown to influence the formation of furfural, a furan-derivative thought to contribute a caramel aroma to aged wine. This project had three aims: To determine (1) the impact caffeic and caftaric acid have on furan-derived compound formation during base wine storage, (2) the ability of mushroom-derived chitosan to reduce caffeic and caftaric acid concentrations in Pinot noir juice, and (3) to establish whether chitosan added to base wine influences the generation of furan-derived aroma compounds during storage. For Aim 1, Chardonnay (2019) base wine was stored at 15 and 30 °C for 90 days with additions of fructose, caffeic acid, and caftaric acid independently, or in combinations. Results showed that at 15 and 30 °C, furfural increased slightly with 5 mg/L additions of caffeic and caftaric acid. For Aim 2, Pinot noir juice (2021) was stored at 10 °C for 18 hours with 1 g/L additions of < 3 kDa, 250 kDa, and 422 kDa molecular weight (MW) chitosan, before being racked off prior to fermentation. In the finished wine, < 3 kDa chitosan treatment decreased caftaric acid and increased the amino acids, while the 250 kDa chitosan decreased total HCA estimation, turbidity, and brown pigmentation compared to the control. For Aim 3, Pinot noir (2021) base wine was stored at 15 and 30 °C for 90 days with the aforementioned chitosan treatments. All three MW chitosan treatments at 30 °C substantially increased furfural, homofuraneol, and 5-methylfurfural generation relative to the control. At 15 °C, furfural and homofuraneol were also found at greater concentrations in the chitosan-treated wine. Overall results show (1) Slight modulation of furan-derived compounds due to increased caffeic and caftaric acid concentrations, (2) The reduction of caftaric acid by a low MW chitosan prior to fermentation, and (3) An increase in furan-derived compounds in base wine treated with chitosan during short-term storage. The findings of this thesis determined for the first time the influence of HCAs and chitosan on the concentrations of aroma and flavour compounds, with real applications into the acceleration of ageing in sparkling wines.
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Role of Viral Infections in Rapid Prunus Decline in the Niagara RegionPlant diseases associated with viral infections are a serious issue for growers, causing significant reductions in yield and plant death. Rapid Prunus Decline (RPD) is an emerging disorder resulting in rapid and unexplained death of Prunus species in southern Ontario. In order to better understand RPD, disease symptoms in all major Prunus species in the Niagara region were surveyed, along with identification of major viruses in diseased Prunus trees. Our survey showed that diseased trees often showed symptoms of dehydration, leaf chlorosis, reduced new growth, and death. The average mortality rate in the Niagara region was 20% among all Prunus species with the highest site-specific mortality rate of 67%. In order to understand the contribution of viruses to RPD, RNA sequencing was employed to identify viruses in diseased trees and characterize viral sequence diversity. In total, 20 unique virus species and one viroid were identified in 42 diseased trees, demonstrating a wide diversity of viruses in diseased Prunus trees. Tomato ringspot virus (ToRSV) infection can result in similar symptoms to those observed in RPD trees. Based on these similar symptoms, a total of 507 Prunus trees were surveyed and the frequency of ToRSV infection was determined to be 13%. No direct correlation was identified between the presence of ToRSV and incidence of RPD, suggesting that other factors are involved. Multiple ilarviruses were identified in diseased trees including Grapevine associated ilarvirus (GaIV), Tobacco streak virus (TSV), Tomato necrotic spot virus (ToNSV), Prunus necrotic ringspot virus (PNRSV), and Prune dwarf virus (PDV). GaIV and ToNSV have never been previously reported in Prunus trees, or in Canada. GaIV was the most commonly detected virus and is an emerging concern for stone fruit production. Mixed infection of ilarviruses could have synergistic pathological effects, and the interactions between ilarviruses and other viruses are poorly understood. In summary, over the course of this study, many different viruses were identified in Prunus species exhibiting decline, yet no single virus was identified in all Prunus spp. or in all instances of the disease, suggesting RPD is a complex disease that could have multiple contributing factors.
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Evaluating and targeting mitochondrial dynamics and plasticity in conditions of mitochondrial dysfunctionMitochondria represent dynamic structures which undergo various changes in morphology in order to rapidly respond to changes in cellular physiology. Changes in mitochondrial dynamics are observed in various pathological events and represent a potential target for therapeutics. In this thesis, I first investigated the role of zinc supplementation on rat aortic endothelial (RAENDO) and smooth muscle (RASMC) cell mitochondrial form and function to elucidate the underlying mechanisms by which zinc prevents the development of smooth muscle neointimal hyperplasia in vivo. I discovered that zinc differentially alters the gene expression of several mitochondria-associated genes in cells treated with ZnSO4 for 7 days. In agreement with this, an increase in mitochondrial network fragmentation (fission) is observed in RAENDO cells, whereas fusion is observed in RASMC cells with zinc treatment. I determined that cell-type specific differences in zinc storage and buffer capacity necessitating different mitochondrial quality control mechanisms may underscore differential responses to zinc exposure in vivo. These results highlight mitochondria as an important target for the treatment or prevention of neointimal hyperplasia in vivo. The potential to modulate mitochondrial network morphology to alter cellular function lead to the second study of this thesis, in which a mitochondrial-targeting moiety, triphenylphosphonium (TPP), is employed to target mitochondrial lipid composition. Here, I evaluated the use of TPP-linoleic acid (TPP-LA) to modulate mitochondrial cardiolipin (CL) composition in a tafazzin knockout (TAZKO) mouse myoblast model of Barth syndrome, a disease characterized by abnormal CL composition. My results indicate that TPP-LA non-significantly enhances mitochondrial membrane potential and apparent fusion in TAZKO and isogenic wildtype (WT) cells. CL analyses reveal that TPP-LA administration for 14 days during differentiation may rescue the CL profile of TAZKO cells. TPP-LA treatment also enhances biochemical differentiation in WT cells but fails to rescue differentiation in TAZKO cells. These results highlight the complex relationship between mitochondrial lipid composition and modulation of mitochondrial dynamics. Taken together, these two projects highlight the plastic nature of mitochondria, their role in pathological conditions and potential as therapeutic targets.
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AN EXAMINATION OF NOTCH SIGNALLING IN THE REGENERATING SPINAL CORD OF THE AXOLOTL, Ambystoma mexicanum: ESTABLISHMENT AND CHARACTERIZATION OF AN AXOLOTL OPTOGENETIC CONSTRUCT FOR UPREGULATION OF NOTCH1 SIGNALLING IN VIVO.The Notch signalling pathway is a juxtacrine signalling pathway conserved across vertebrate and invertebrate species and is known to be a potent regulator of progenitor cell fate decisions during nervous system development. The dysregulation of the Notch pathway has been implicated in the establishment of an anti-neurogenic environment following spinal cord injury in mammals that ultimately prevents functional recovery. In regeneration-competent species, where both neurons and glia are produced by resident progenitor cells in response to trauma, Notch appears to be regulated differently. In the regeneration-competent axolotl, very little is known regarding the role of the Notch signalling pathway in the establishment of a regeneration permissive environment. Here I report that the axolotl possesses a homolog of the Notch1 receptor and qPCR data indicate that its expression decreases significantly at 7 days post injury in caudal spinal cord tissue. I further report that the Notch downstream target gene, Hes1, is expressed in the spinal cord 3 days following injury and that bath application of the indirect Notch inhibitor, tert-Butyl (S)-{(2S)-2-[2-(3,5 difluorophenyl)acetamido]propanamido} phenylacetate (DAPT), impairs spinal cord regeneration. Finally, I have modified an existing optogenetic plasmid construct that allows for light-dependent temporal and spatial Notch receptor-independent signalling through the axolotl Notch intracellular domain (NICD). In vitro characterization has included the determination of an effective membrane anchor for this construct and the production of an appropriate light-insensitive negative control plasmid. This research has provided evidence for a role of Notch during spinal cord regeneration in the axolotl and provided a unique optogenetic tool to facilitate the determination of the in vivo role that Notch signalling plays during spinal cord regeneration in the axolotl.
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Identification of clone x rootstock effects on cold hardiness and relevant dehydrins changes in Cabernet franc (Vitis vinifera L.)Cold injury can harm grapevines and severely reduce yields in cool-climate regions. Previous studies indicate bud cold hardiness differences among Vitis vinifera cultivars, but comparatively, little attention has been given to the effect of clone and rootstock. This study investigates the bud cold hardiness of Cabernet franc with different clones and rootstock combinations throughout dormancy during the stages of cold acclimation, maximum hardiness and deacclimation. The research also looks at the protein expression of dehydrins which may impact cold hardiness. Four clone × rootstock combinations of Cabernet franc (214 × 101-14, 327 × 101-14, 327 × 3309, and 327 × Riparia Gloire) were assessed over two dormant seasons in 2018-19 and 2019-20. Buds were sampled every two to four weeks and were measured for cold hardiness by differential thermal analysis (DTA). Cold hardiness was reported as low temperature exotherms (LTE). Vine performance including phenological stages, yield components, vine balance, environmental resistance, and fruit composition including titratable acidity (TA), soluble solids, and pH were measured in 2019 and 2020. SDS-PAGE profiles and immunoblots measured dehydrins intensity during two dormant seasons in 2018-19 and 2019-20. Rootstock was found to impact cold hardiness of Cabernet franc. No significant differences were observed in bud hardiness between clones of Cabernet franc; despite significant differences in bud hardiness of clone × rootstock combinations of Cabernet franc. Dehydrin bands of five molecular weights (62 kDa, 57 kDa, 41 kDa, 30 kDa, and 23 kDa) were detected in the 2018-19 dormant season, and dehydrin bands of four molecular weights (62 kDa, 41 kDa, 30 kDa, and 23 kDa) were detected in the 2019-20 dormant season. The development of dehydrin bands varied for clones, rootstocks, and different molecular weights. No consistent correlations were observed between dehydrin intensity and LTE50 values, despite the increase of dehydrin intensity and the decrease of LTE50 values observed during acclimation. This study reveals the effect of clone and rootstock on cold hardiness, which is important for clone and rootstock selection in cool climate regions to reduce cold injury, and the complexity of the relationship between cold hardiness and dehydrins. In the future, studies should continue to focus on cold hardiness of clone and rootstock in different cultivars. Change of dehydrin levels during dormant seasons in more cultivars should also be studied to reveal the relationship between dehydrins and cold hardiness.
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Investigating the Modulation of Drosophila melanogaster Body-wall Muscle Contraction by the Neuropeptide DPKQDFMRFamideThe 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.
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Selecting cover crop species for vineyards of the Niagara regionOrganic 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.
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The development of a new genetic test for grapevine cultivars using a computational genomics approachDue 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.
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The ecological and physiological consequences of sun vs. shade nesting for the small carpenter bee, Ceratina calcarata, and their offspringCeratina 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.
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Crawford Lake Consumers: Water Column and Palynological StudiesDespite 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.
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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 profilingRNA 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.
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INVOLVEMENT OF THE ENDOCANNABINOID SYSTEM IN THE EPENDYMOGLIAL RESPONSE TO SPINAL CORD REGENERATION IN THE MEXICAN AXOLOTL, Ambystoma mexicanumResearch 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.
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Demography and molecular ecology of the solitary halictid Lasioglossum zonulum: with observations on Lasioglossum leucozoniumHalictid 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.
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Investigating how Notch, and JAK/STAT Signaling Synergistically Regulate Intestinal Stem Cell Homeostasis Using Engineered Optogenetic AllelesStem 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.
