• Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

      Abubaker, Kamal Salem; Department of Biological Sciences (Brock University, 2010-10-26)
      Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.
    • Cell-selective modulation of the neuromuscular system in Drosophila

      Ormerod, Kiel; Department of Biological Sciences
      The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.
    • Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

      Robb, Ellen; Department of Biological Sciences (Brock University, 2013-05-14)
      Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.
    • Characterisation and optimisation of the flavour of health-promoting, plantderived bitterants in functional beverages.

      Gaudette, Nicole J.; Applied Health Sciences Program (2012-07-30)
      Flavour is a combination of taste, odour, and chemesthetic sensations. Close associations exist between these sensory modalities, and thus, the overall flavour of a food or beverage product can change when the intensity of one or more of these sensations is altered. Strategies to modify flavour are often utilized by the food industry, and are central to the engineering of new and reformulated products. For functional food and beverages, flavour modification is particularly important, as fortifying agents can elicit high levels of less than desirable sensations, such as bitterness and astringency. The application of various flavour modifying strategies can decrease the perceived intensity of these sensations, and in tum, improve the sensory profile of the product. This collection of studies describes the sensory characteristics of experimental functional beverages fortified with trans-resveratrol, (+)-catechin, and/or caffeine, and examines the impact of novel flavour modifying strategies on the perceived flavour of these beverages. In the first study, results demonstrate that the flavour profile of Cabemet Sauvignon wines fortified with 20 mglL and 200 mg/L of trans-resveratrol is not perceived as different compared to control wine (0 mglL). However, Riesling wine fortified with 200 mg/L is perceived as significantly higher in bitterness compared to 20 mglL and control. For some functional food formulations, alternative strategies for flavour modification are needed. Traditional methods, such as the addition of sucrose and sodium chloride, may decrease the perceived 'healthiness' of a product, and thus, may be sub-optimal. In a second study, high and low concentrations of five different bitter inhibiting compounds - 'bitter blockers' - (B-cyclodextrin, homoeridictyol sodium salt, carboxymethylcellulose - low viscosity, zinc sulfate, magnesium sulfate) were tested for their efficacy towards decreasing the bitterness of high and low concentrations of caffeine and (+)catechin - two health-relevant, plant-derived bitterants. B-cyclodextrin and homoeridictyol sodium salt were the most effective blockers at decreasing (+ )-catechin and caffeine, respectively. In addition to bitter blockers, additional flavour modifying strategies, either alone or in combination - may also be successful in functional food formulations. Both sucrose and rebaudioside A - a plant-derived sweetener - were effective at decreasing the bitterness of (+)catechin. When added to (+)-catechin along with B-cyc1odextrin, both sweeteners provided the most effective decrease in bitterness compared to binary, ternary, or quaternary mixtures of (+)catechin together with bitter blockers, sweeteners, andlor odourants. The perceived intensity of sensations elicited by sweeteners and odourants was not affected by the addition of bitter blockers, and thus, their impact within these complex matrices is minimal. In addition, withinmodal (taste-taste) compared to cross-modal (taste-odour) sensory interactions were more effective at decreasing the bitterness of (+ )-catechin. Overall, results from these studies demonstrate that certain novel, alternative flavour modifying approaches may be successful towards lowering the bitterness and astringency elicited by (+ )-catechin and caffeine in aqueous solutions.
    • Competing demands for a complex system : photosystem II repair, photoprotection and quantum yield

      Veerman, John; Department of Biological Sciences (2012-04-04)
      Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge.
    • Cryptic species status of Anopheles (Diptera: Culicidae) mosquitoes in Canada using a multidisciplinary approach

      Thielman, Aynsley; Department of Biological Sciences (2012-04-04)
      Many species of Anopheles mosquitoes (Diptera: Culicidae) are now recognized as species complexes whose members are often indistinguishable morphologically but identifiable based on ecological, genetic, or behavioural data. Because the members of species complexes often differ in their vector potential, accurate identification of vector species is essential for successful mosquito control. To investigate the cryptic species status of Anopheles mosquitoes in Canada, specimens were collected from across the country and examined using morphological, molecular, and ecological data. Six of the seven traditionally recognised species from Canada were collected from locations in British Columbia, Quebec, Newfoundland and Labrador, and throughout Ontario, including Anopheles barberi, An. earlei, An. freeborni, An. punctipennis, An. quadrimaculatus s.l., and An. walkeri. Variation in polymorphic traits within An. earlei, An. punctipennis, and An. quadrimaculatus s.l. were quantified and egg morphology examined using scanning electron microscopy. Morphological identification of adult and larval specimens suggested that two described cryptic species, An. perplexens and An. smaragdinus, were present in Canada. DNA sequence data were analysed for evidence of cryptic species using three molecular markers: COl, ITS2, and ITS!. Intraspecific COl variation was very low in most species «1 %), except for An. punctipennis with 2% sequence divergence between those from British Columbia (BC) and Ontario (ON), and An. walkeri with 7% sequence divergence between populations from Manitoulin Island (NO) and Long Point Provincial Park (LP). Similar patterns were also seen using ITS2 and ITS 1. Therefore, molecular data revealed the presence of two putative cryptic species within two species examined (i.e., An. walkeri and An. punctipennis), corresponding to collection location (i.e., NO vs. LP and BC vs. ON, respectively). Surprisingly, there was no molecular support for the presence of either An. perplexens or An. smaragdinus in Canada despite the morphological assessments. Ecological data from all collection sites were recorded and are available in an online database designed to manage all collection and identification data. Current bionomic information, including regional abundance, larval habitat, and species associations, was determined for each species. This multidisciplinary study of Anopheles mosquitoes is the first detailed investigation of these potential disease vectors in Canada and demonstrates the importance of an integrated approach to anopheline systematics that includes molecular data.
    • Defence of Agaricus bisporus against toxic secondary metabolites from Trichoderma aggressivum.

      Sjaarda, Calvin; Department of Biological Sciences (Brock University, 2013-09-12)
      Trichoderma spp are effective competitors against other fungi because they are mycoparasitic and produce hydrolytic enzymes and secondary metabolites that inhibit the growth of their competitors. Inhibitory compounds produced by Trichoderma aggressivum, the causative agent of green mold disease, are more toxic to the hybrid off-white strains of Agaricus bisporus than the commercial brown strains, consistent with the commercial brown strain’s increased resistance to the disease. This project looked at the response of hybrid off-white and commercial brown strains of A. bisporus to the presence of T. aggressivum metabolites with regard to three A. bisporus genes: laccase 1, laccase 2, and manganese peroxidase. The addition of T. aggressivum toxic metabolites had no significant effect on MnP or lcc1 transcript abundance. Alternatively, laccase 2 appears to be involved in resistance to T. aggressivum because the presence of T. aggressivum metabolites results in higher lcc2 transcript abundance and laccase activity, especially in the commercial brown strain. The difference in laccase expression and activity between A. bisporus strains was not a result of regulatory or coding sequence differences. Alteration of laccase transcription by RNAi resulted in transformants with variable levels of laccase transcript abundance. Transformants with a low number of lcc transcripts were very sensitive to T. aggressivum toxins, while those with a high number of lcc transcripts had increased resistance. These results indicated that laccase activity, in particular that encoded by lcc2, serves as a defense response of A. bisporus to T. aggressivum toxins and contributes to green mold disease resistance in commercial brown strains.
    • Delineation of within-site terroir effects using soil and vine water measurement. Investigation of Cabernet Franc

      Hakimi-Rezaei, Javad; Department of Biological Sciences (Brock University, 2009-01-28)
      . The influence of vine water status was studied in commercial vineyard blocks of Vilis vinifera L. cv. Cabernet Franc in Niagara Peninsula, Ontario from 2005 to 2007. Vine performance, fruit composition and vine size of non-irrigated grapevines were compared within ten vineyard blocks containing different soil and vine water status. Results showed that within each vineyard block water status zones could be identified on GIS-generated maps using leaf water potential and soil moisture measurements. Some yield and fruit composition variables correlated with the intensity of vine water status. Chemical and descriptive sensory analysis was performed on nine (2005) and eight (2006) pairs of experimental wines to illustrate differences between wines made from high and low water status winegrapes at each vineyard block. Twelve trained judges evaluated six aroma and flavor (red fruit, black cherry, black current, black pepper, bell pepper, and green bean), thr~e mouthfeel (astringency, bitterness and acidity) sensory attributes as well as color intensity. Each pair of high and low water status wine was compared using t-test. In 2005, low water status (L WS) wines from Buis, Harbour Estate, Henry of Pelham (HOP), and Vieni had higher color intensity; those form Chateau des Charmes (CDC) had high black cherry flavor; those at RiefEstates were high in red fruit flavor and at those from George site was high in red fruit aroma. In 2006, low water status (L WS) wines from George, Cave Spring and Morrison sites were high in color intensity. L WS wines from CDC, George and Morrison were more intense in black cherry aroma; LWS wines from Hernder site were high in red fruit aroma and flavor. No significant differences were found from one year to the next between the wines produced from the same vineyard, indicating that the attributes of these wines were maintained almost constant despite markedly different conditions in 2005 and 2006 vintages. Partial ii Least Square (PLS) analysis showed that leaf \}' was associated with red fruit aroma and flavor, berry and wine color intensity, total phenols, Brix and anthocyanins while soil moisture was explained with acidity, green bean aroma and flavor as well as bell pepper aroma and flavor. In another study chemical and descriptive sensory analysis was conducted on nine (2005) and eight (2006) medium water status (MWS) experimental wines to illustrate differences that might support the sub-appellation system in Niagara. The judges evaluated the same aroma, flavor, and mouthfeel sensory attributes as well as color intensity. Data were analyzed using analysis of variance (ANOVA), principal component analysis (PCA) and discriminate analysis (DA). ANOV A of sensory data showed regional differences for all sensory attributes. In 2005, wines from CDC, HOP, and Hemder sites showed highest. r ed fruit aroma and flavor. Lakeshore and Niagara River sites (Harbour, Reif, George, and Buis) wines showed higher bell pepper and green bean aroma and flavor due to proximity to the large bodies of water and less heat unit accumulation. In 2006, all sensory attributes except black pepper aroma were different. PCA revealed that wines from HOP and CDC sites were higher in red fruit, black currant and black cherry aroma and flavor as well as black pepper flavor, while wines from Hemder, Morrison and George sites were high in green bean aroma and flavor. ANOV A of chemical data in 2005 indicated that hue, color intensity, and titratable acidity (TA) were different across the sites, while in 2006, hue, color intensity and ethanol were different across the sites. These data indicate that there is the likelihood of substantial chemical and sensory differences between clusters of sub-appellations within the Niagara Peninsula iii
    • Delineation of within-site terroir effects using soil and vine water measurements in Riesling vineyards within the Niagara Peninsula

      Willwerth, James Joseph; Department of Biological Sciences (2012-04-04)
      The major focus of this dissertation was to explain terroir effects that impact wine varietal character and to elucidate potential determinants of terroir by testing vine water status (VWS) as the major factor of the terroir effect. It was hypothesized that consistent water status zones could be identified within vineyard sites, and, that differences in vine performance, fruit composition and wine sensory attributes could be related to VWS. To test this hypothesis, ten commercial Riesling vineyards representative of each Vintners Quality Alliance sub-appellation were selected. Vineyards were delineated using global positioning systems and 75 to 80 sentinel vines per vineyard were geo-referenced for data collection. During the 2005 to 2007 growing seasons, VWS measurements [midday leaf water potential ('l')] were collected from a subset of these sentinel vines. Data were collected on soil texture and composition, soil moisture, vine performance (yield components, vine size) and fruit composition. These variables were mapped using global information system (GIS) software and relationships between them were elucidated. Vines were categorized into "low" and "high" water status regions within each vineyard block and replicate wines were made from each. Many geospatial patterns and relationships were spatially and temporally stable within vineyards. Leaf'l' was temporally stable within vineyards despite different weather conditions during each growing season. Generally, spatial relationships between 'l', soil moisture, vine size, berry weight and yield were stable from year to year. Leaf", impacted fruit composition in several vineyards. Through sorting tasks and multidimensional scaling, wines of similar VWS had similar sensory properties. Descriptive analysis further indicated that VWS impacted wine sensory profiles, with similar attributes being different for wines from different water status zones. Vineyard designation had an effect on wine profiles, with certain sensory and chemical attributes being associated from different subappellations. However, wines were generally grouped in terms of their regional designation ('Lakeshore', 'Bench', 'Plains') within the Niagara Peninsula. Through multivariate analyses, specific sensory attributes, viticulture and chemical variables were associated with wines of different VWS. Vine water status was a major contributor to the terroir effect, as it had a major impact on vine size, berry weight and wine sensory characteristics.
    • Development of a bacteriophage-based biopesticide for fire blight

      Lehman, Susan M.; Department of Biological Studies (Brock University, 2007-05-28)
      Fire blight is an economically important disease of apples and pears that is caused by the bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1 infection in the spring, and rapidly removing infected tissue. The possibility of using phages to control E.amylovora populations has been suggested, but previous studies have. failed to show high treatment efficacies. This work describes the development of a phage-based biopesticide that controls E. amylovora populations under field conditions, and significantly reduces the incidence of fire blight. This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE. amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of these phages on blossom surfaces during the period when the flowers are most susceptible to infection. Seven phages and one carrier isolate were selected for field trials from existing collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the . /' phages and carrier was based on characteristics relevant to the production and field perfonnance of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta assays showed that both the phages and the carrier make significant contributions to reducirig the development of fire blight symptoms in pear blossoms. Field-scale phage production and purification methods were developed based on the growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages in various liquid media. Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to simultaneously monitor the phage, carrier, and pathogen populations over the course of selected treatments. In all cases. the observed population dynamics of the biocontrol agents and the pathogen were consistent with the success or failure of each treatment to control disease incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment application. The phages then grew preferentially on the pathogen, once it was introduced into this blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention. The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossonls. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae. The genome ofErwinia phage <l>Ea21-4 was sequenced and annotated. Most of the 8-4.7 kB genome is substantially different from previously described sequences, though some regions are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to approximately 30% of the predicted open reading frames based on amino acid sequence comparisons and N-terminal sequencing of structural proteins.
    • The efficacy of anti-predator behaviour in the wood fog tadpole (Rana sylvatica) /

      Kerling, Candice L.; Department of Biological Sciences (Brock University, 2007-06-04)
      Activity has been suggested as an important behaviour that is tightly linked with predator avoidance in tadpoles. In this thesis I examine predator-prey relationships using wood frog tadpoles {Rana sylvaticd) as prey and dragonfly larvae {AnaxJunius) and backswimmers {Notonecta undulatd) as predators. I explore the role of prey activity in predator attack rates, prey response to single and multiple predator introductions, and prey survivorship. The data suggest that Anax is the more successful predator, able to capture both active and inactive tadpoles. In contrast, Notonecta strike at inactive prey less frequently and are seldom successftil when they do. A mesocosm study revealed that the presence of any predator resulted in reduced activity level of tadpoles. Each predator species alone had similar effects on tadpole activity, as did the combined predator treatment. Tadpole survivorship, however, differed significantly among both predator treatments and prey populations. Tadpwles in the combined predator treatment had enhanced risk; survivorship was lower than that expected if the two predators had additive effects. Differences in survivorship among wood frog populations showed that tadpoles from a lake habitat had the lowest survivorship, those from a shallow pond habitat had an intermediate survivorship, and tadpoles from a marsh habitat had the highest survivorship. The frequency of interactions with predators in the native habitat may be driving the population differences observed. In conclusion, results from this study show that complex interactions exist between predators, prey, and the environment, with activity playing a key role in the survival of tadpoles.
    • Elucidation of odour-potent compounds and sensory profiles of Vidal blanc and Riesling icewines from the Niagara Peninsula : effect of harvest date and crop level

      Bowen, Amy J.; Department of Biological Sciences (Brock University, 2011-10-14)
      I t was hypothesized that the freeze/thaw cycles endured by icewine grapes would change their chemical composition, resulting in unique chemical fingerprint and sensory properties, and would be affected by harvest date (HD) and crop level (CL). The objectives were: 1) to identify odour-active compounds using gas chromatographic and sensory analysis; 2) to determine the effect of CL and HD on these compounds; 3) to determine the icewine sensory profiles; 4) to correlate analytical and sensory results for an overall icewine profile. CharmAnalysis™ determined the Top 15 odour-potent compounds in Vidal and Riesling icewine and table wines; 24 and 23 compounds, respectively. The majority of the compounds had the highest concentrations in the icewines compared to table wines. These compounds were used as the foundation for assessing differences in icewine chemical profiles from different HD and CL. Vidal and Riesling icewine were made from grapes picked at different HD; HI : 19 December; H2: 29 December; H3: 18 January; H4: 11 February (Vidal only). HI wines differed from H3 and H4 wines in both Vidal and Riesling for aroma compounds and sensory profiles. - Three·CL [control (fully cropped), cluster thin at fruit set to one basal cluster/shoot (TFS), and cluster thin at veraison to one basal cluster/shoot (TV)] were evaluated for Riesling and Vidal cultivars over two seasons. Vidal icewines had the highest concentration of aroma compounds in the control and TV icewines in 2003 and in TFS icewines in 2004. In Riesling, most aroma compounds had the highest concentration in the TV icewines and the lowest concentration in the TFS wine for both years. The thinned treatments were associated with almost all of the sensory attributes in both cultivars, both years. HD and CL affected the chemical variables, aroma compounds and sensory properties of Vidal and Riesling icewines and freeze/thaw events changed their sensory profile. The most odour-potent compounds were p-damascenone, cis-rose oxide, 1- octen-3-ol, 4-vinylguaiacol, ethyl octanoate, and ethyl hexanoate. The role of Pdamascenone as a marker compound for icewine requires further investigation. This research provides a strong foundation for the understanding the odour-active volatiles and sensory profiles important to icewine.
    • ENDOCANNABINOID REGULATION OF ADOLESCENT DEVELOPMENT IN MALE AND FEMALE RATS

      Simone, Jonathan; Department of Biological Sciences
      The present thesis investigated the contributions of adolescent endocannabinoid signalling to brain and behaviour development in male and female rats. In chapter 2, daily administration of the CB1 antagonist AM251, alone or in tandem with a psychological stressor, increased social interactions, reduced dorsal hippocampal CB1 expression, and increased mPFC GAD67 expression in female rats 24-48 h after treatment, with no effects in males. In chapter 3, adolescent CB1 antagonism reduced anxiety in adult males, with no effects in females. Conversely, adolescent AM251 increased contextual fear in adult females, with no effects in males. In chapter 4, AM251 females spent more time initiating social interactions after a 5-day drug washout period than vehicle females, with no effects in males. To identify brain regions underlying the effects of AM251 on social behaviours, I repeated social interaction testing in vehicle and AM251 females and collected brains for immunohistochemical labelling of EGR-1 as a marker of neural activation in the CA1, CA2, and CA3 subfields of the dorsal hippocampus and the shell and core divisions of the nucleus accumbens (NAc). Consistent with my previous findings, AM251 females spent more time initiating social interactions and had greater EGR-1 cell counts in the NAc shell than vehicle females, with no group differences in the NAc core or in any of the hippocampal subfields investigated. EGR-1 cell counts in the dCA2 were negatively correlated with social interactions in vehicle and AM251 females. A positive correlation between NAc shell EGR-1 cell counts and social interactions was observed only in AM251 females. Regression analysis using drug treatment and EGR-1 cell counts in dCA2 and NAc shell resulted in a model with an adjusted R2 of 0.90. Both drug treatment and EGR-1 cell counts in the dorsal CA2 emerged as unique predictors of individual differences in social interaction, and drug and NAc shell EGR-1 cell counts interacted to significantly predict social interactions in AM251 females only. Together, these studies provide support for sex-specific contributions of endocannabinoid signalling to the development of brain and behaviour in adolescence in male and female rats.
    • Erwinia amylovora bacteriophage resistance

      Roach, Dwayne R.; Department of Biological Sciences (2012-04-04)
      It has been proposed that phages can be used commercially as a biopesticide for the control of fire blight caused by the phytopathogen Erwinia amylovora. The aim of these studies was to investigate two common bacterial resistance mechanisms, lysogeny and exopolysaccharide production and their influence on phage pathogenesis. A multiplex real-time PCR protocol was designed to monitor and quantify Podoviridae and Myoviridae phages. This protocol is compatible with known E. amylovora and Pantoea agglomerans rtPCR primers/probes which allowed simultaneous study of both phage and bacterial targets. Using in vitro positive phage selection, bacteriophage insensitive derivatives were isolated within sensitive populations of E. amylovora. Prophage screening with real-time PCR and mitomycin C induction determined that the insensitive derivatives harboured the temperate Podoviridae phage ΦEaTlOO. Lysogenic conversion resulted in resistance to secondary homologous phage infections. Prophage screening of environmental samples of E. amylovora and P. agglomerans collected from various locations in Canada, United States and Europe did not demonstrate lysogeny. Therefore, lysogeny is rare or absent while these bacterial species reside on the plant. Recombineering was used to construct exopolysaccharide deficient E. amylovora mutants. The EPS amylovoran mutants became resistant to Podoviridae and certain Siphoviridae phages. Increasing amylovoran production increased phage population growth, presumably by increasing the total number of bacterial cell surface receptors which promoted increased phage infections. In contrast, amylovoran did not playa role in Myoviridae infections, nor did production of the EPS levan for any phage pathogenesis.
    • Etiology and Management of Grape Sour Rot

      Huber, Cristina; Department of Biological Sciences
      Sour rot is characterized by increased volatile acidity (VA) in ripe grapes. VA is associated with spoilage organisms and wineries may reject grape crops based on their concentration of acetic acid. Our research associated Hanseniaspora uvarum, Gluconobacter oxydans, and to a lesser extent, Gluconobacter cerinus and Acetobacter malorum with sour rotted grapes in the Niagara Peninsula, designated viticultural area, Ontario, Canada, and the pathogenicity of these organisms was confirmed by laboratory assays. Only G. oxydans was shown to penetrate around the site of pedicel attachment to the grape. The yeasts required further wounding. Candida zemplinina was also associated with the sour rot microbial community. This species showed variable pathogenicity by strain and most strains were not highly pathogenic. C. zemplinina gained dominance in the microbial population of grapes only after sour rot symptoms were observed, indicating a succession which was studied in laboratory assays. There was a correlation between temperature, moisture, and berry ripeness and the development of sour rot when conditions were monitored in a Vitis vinifera cv. Riesling vineyard over four years, and this was confirmed in laboratory assays. Disease management options are limited since sour rot is caused by a complex of yeasts and bacteria, with symptoms developing just as grapes approach maturity. Post-veraison treatments for sour rot were investigated. Wineries routinely add potassium metabisulphite (KMS) to the surface of fruit in bins and to grape juice to kill spoilage organisms. Replicated field trials were conducted in V. vinifera cv. Riesling in 2010 and 2011 to determine the efficacy of KMS at different concentrations and pre-harvest timings as a fruiting-zone spray. Potassium bicarbonate (Milstop) was also evaluated for its efficacy against sour rot. Plots were rated for incidence and severity of sour rot and VA (g acetic acid/L juice). KMS treatments at concentrations above 5 kg/1000L and Milstop sprayed at the label concentration of 5.6 kg/1000L were able to reduce the severity of sour rot compared to untreated control plots which had a severity above 50% (2011). KMS was able to reduce VA to below the winery rejection threshold of 0.24 g acetic acid/L when sour rot severity reached 12% in untreated plots (2010). When tested in the laboratory in disk diffusion assays conducted on yeast peptone dextrose agar, KMS at a concentration of 10 g/L had the greatest efficacy against G. oxydans and H. uvarum. Grape incubation assays showed the potential of KMS acidified with tartaric acid to reduce sour rot symptoms. Acidification did not show as much potential in field trials, calling for further research.
    • Evolutionary Origin and Maintenance of Sociality in the Small Carpenter Bees

      Rehan, Sandra; Department of Biological Sciences (2012-07-31)
      Many arthropods exhibit behaviours precursory to social life, including adult longevity, parental care, nest loyalty and mutual tolerance, yet there are few examples of social behaviour in this phylum. The small carpenter bees, genus Ceratina, provide important insights into the early stages of sociality. I described the biology and social behaviour of five facultatively social species which exhibit all of the preadaptations for successful group living, yet present ecological and behavioural characteristics that seemingly disfavour frequent colony formation. These species are socially polymorphic with both / solitary and social nests collected in sympatry. Social colonies consist of two adult females, one contributing both foraging and reproductive effort and the second which remains at the nest as a passive guard. Cooperative nesting provides no overt reproductive benefits over solitary nesting, although brood survival tends to be greater in social colonies. Three main theories explain cooperation among conspecifics: mutual benefit, kin selection and manipulation. Lifetime reproductive success calculations revealed that mutual benefit does not explain social behaviour in this group as social colonies have lower per capita life time reproductive success than solitary nests. Genetic pedigrees constructed from allozyme data indicate that kin selection might contribute to the maintenance of social nesting -, as social colonies consist of full sisters and thus some indirect fitness benefits are inherently bestowed on subordinate females as a result of remaining to help their dominant sister. These data suggest that the origin of sociality in ceratinines has principal costs and the great ecological success of highly eusociallineages occurred well after social origins. Ecological constraints such as resource limitation, unfavourable weather conditions and parasite pressure have long been considered some of the most important selective pressures for the evolution of sociality. I assessed the fitness consequences of these three ecological factors for reproductive success of solitary and social colonies and found that nest sites were not limiting, and the frequency of social nesting was consistent across brood rearing seasons. Local weather varied between seasons but was not correlated with reproductive success. Severe parasitism resulted in low reproductive success and total nest failure in solitary nests. Social colonies had higher reproductive success and were never extirpated by parasites. I suggest that social nesting represents a form of bet-hedging. The high frequency of solitary nests suggests that this is the optimal strategy when parasite pressure is low. However, social colonies have a selective advantage over solitary nesting females during periods of extreme parasite pressure. Finally, the small carpenter bees are recorded from all continents except Antarctica. I constructed the first molecular phylogeny of ceratinine bees based on four gene regions of selected species covering representatives from all continents and ecological regions. Maximum parsimony and Bayesian Inference tree topology and fossil dating support an African origin followed by an Old World invasion and New World radiation. All known Old World ceratinines form social colonies while New World species are largely solitary; thus geography and phylogenetic inertia are likely predictors of social evolution in this genus. This integrative approach not only describes the behaviour of several previously unknown or little-known Ceratina species, bu~ highlights the fact that this is an important, though previously unrecognized, model for studying evolutionary transitions from solitary to social behaviour.
    • The Fast Regulation of Photosynthesis in Diatoms: an inquiry into the physiological and physical origins of non-photochemical chlorophyll fluorescence quenching.

      Derks, Allen Kimbell; Department of Biological Sciences (Brock University, 2015-01-06)
      Diatoms are renowned for their robust ability to perform NPQ (Non-Photochemical Quenching of chlorophyll fluorescence) as a dissipative response to heightened light stress on photosystem II, plausibly explaining their dominance over other algal groups in turbulent light environs. Their NPQ mechanism has been principally attributed to a xanthophyll cycle involving the lumenal pH regulated reversible de-epoxidation of diadinoxanthin. The principal goal of this dissertation is to reveal the physiological and physical origins and consequences of the NPQ response in diatoms during short-term transitions to excessive irradiation. The investigation involves diatom species from different originating light environs to highlight the diversity of diatom NPQ and to facilitate the detection of core mechanisms common among the diatoms as a group. A chiefly spectroscopic approach was used to investigate NPQ in diatom cells. Prime methodologies include: the real time monitoring of PSII excitation and de-excitation pathways via PAM fluorometry and pigment interconversion via transient absorbance measurements, the collection of cryogenic absorbance spectra to measure pigment energy levels, and the collection of cryogenic fluorescence spectra and room temperature picosecond time resolved fluorescence decay spectra to study excitation energy transfer and dissipation. Chemical inhibitors that target the trans-thylakoid pH gradient, the enzyme responsible for diadinoxanthin de-epoxidation, and photosynthetic electron flow were additionally used to experimentally manipulate the NPQ response. Multifaceted analyses of the NPQ responses from two previously un-photosynthetically characterised species, Nitzschia curvilineata and Navicula sp., were used to identify an excitation pressure relief ‘strategy’ for each species. Three key areas of NPQ were examined: (i) the NPQ activation/deactivation processes, (ii) how NPQ affects the collection, dissipation, and usage of absorbed light energy, and (iii) the interdependence of NPQ and photosynthetic electron flow. It was found that Nitzschia cells regulate excitation pressure via performing a high amplitude, reversible antenna based quenching which is dependent on the de-epoxidation of diadinoxanthin. In Navicula cells excitation pressure could be effectively regulated solely within the PSII reaction centre, whilst antenna based, diadinoxanthin de-epoxidation dependent quenching was implicated to be used as a supplemental, long-lasting source of excitation energy dissipation. These strategies for excitation balance were discussed in the context of resource partitioning under these species’ originating light climates. A more detailed investigation of the NPQ response in Nitzschia was used to develop a comprehensive model describing the mechanism for antenna centred non-photochemical quenching in this species. The experimental evidence was strongly supportive of a mechanism whereby: an acidic lumen triggers the diadinoxanthin de-epoxidation and protonation mediated aggregation of light harvesting complexes leading to the formation of quencher chlorophyll a-chlorophyll a dimers with short-lived excited states; quenching relaxes when a rise in lumen pH triggers the dispersal of light harvesting complex aggregates via deprotonation events and the input of diadinoxanthin. This model may also be applicable for describing antenna based NPQ in other diatom species.
    • Functional antagonism of the mesolimbic dopaminergic system on mesolimbic cholinergic system in the vocal expression of an emotional state

      Silkstone, Michael; Department of Biological Sciences
      The overarching goal of this thesis was to determine if the initiation of a positive emotional state could antagonize the expression of a negative emotional state in rats. The hypothesis of the thesis argued that the initiation of a positive emotional state would ameliorate the vocal expression of a negative emotional state. The subjective emotional state of the rat was indexed by the quantity and type of pharmacologically induced ultrasonic vocalizations (USVs). Adolescent and adult rats can emit vocalizations above the upper threshold of human hearing (>20 kHz) termed ultrasonic vocalizations (USVs). These USVs are broadly divided into 50-kHz, reflective of a positive emotional state, and 22-kHz USVs, reflective of a negative emotional state. Pharmacologically, injection of dopamine agonists into the nucleus accumbens shell is sufficient for the initiation of 50-kHz USVs, while injection of cholinergic agonists into the anterior hypothalamic-medial preoptic area (AH-MPO) or the lateral septum (LS) can initiate 22-kHz USVs. In chapter two of the thesis, I demonstrated that microinjection of the dopamine agonist, apomorphine, into the medial shell of the nucleus accumbens attenuated the extent of carbachol-induced 22-kHz USVs from the AH-MPO. I also demonstrated that this effect was dependent upon the microinjection of apomorphine into the central region of the nucleus accumbens shell. In chapter three, I demonstrated that apomorphine could also decrease the extent of carbachol-induced 22-kHz USVs from the LS providing evidence that the effect reported in chapter two was not isolated to the AH-MPO, but rather extending along the medial cholinoceptive vocalization strip. In the third chapter. I also demonstrated that the magnitude of the reduction in the number of 22-kHz USVs was correlated to the number of emitted frequency-modulated (FM) 50-kHz USVs induced by apomorphine. In the fourth chapter, I investigated whether blocking dopamine receptors, either systemically using the typical D2-antipsychotic agent, haloperidol, or microinjection of the D2 antagonist, raclopride, into the nucleus accumbens shell could increase the emission of carbachol-induced 22-kHz USVs from the LS. The results showed that antagonism of dopamine receptors, either systemically or intracerebrally, did not increase the number of 22-kHz USVs. Interestingly, it was also observed that after the prolonged recording of carbachol-induced 22-kHz USVs, some 50-kHz USVs spontaneously appeared after roughly 300 s into the recording. I argued that these 50-kHz USVs, which I defined as “rebound 50-kHz USVs” are not initiated by carbachol since they occurred when the carbachol-response weaned. It was also demonstrated these rebound 50-kHz USVs were dependent upon dopamine release within the nucleus accumbens since both systemic, and intracerebral application of dopamine antagonists into the central division of the nucleus accumbens shell blocked the occurrence of rebound 50-kHz USVs. Altogether, the data supports the thesis that activation of a positive emotional state decreases the expression of the negative emotional state in rats when measured using ultrasonic vocalizations.
    • Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

      Hall, Dawn.; Department of Biological Sciences (Brock University, 2007-05-28)
      Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.
    • HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

      Ahmed, Musaddeque; Department of Biological Sciences (Brock University, 2014-02-19)
      Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.