Ph.D. Biology

Permanent URI for this collectionhttps://hdl.handle.net/10464/2238

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Open Access
Investigating Neuromodulatory Mechanisms of Cotransmitter Proctolin in Drosophila Neuromuscular Junction
(Brock University) Jung, Jae Hwan; Department of Biological Sciences
Proctolin is a neuropeptide that induces or increases muscle contraction in nearly all arthropods. In the fruit fly, Drosophila melanogaster, proctolin is released onto skeletal muscles as a cotransmitter with the excitatory neurotransmitter, L-glutamate (Glu). This thesis examines mechanisms of action through which proctolin modulates contractions of Drosophila muscles. Chapter 1 includes a literature review of neuromodulation, cotransmitters, neurohormones and effects of proctolin, and it gives a rationale for the scientific questions addressed in subsequent chapters. Previous work showed that increasing neural activity reduces the threshold and EC50 values for proctolin’s ability to increase nerve-evoked contractions. Chapter 2 provides evidence that this activity-dependence cannot be attributed solely to an increase in Glu release with increasing neural activity. Proctolin potentiates contractions elicited by directly applying Glu to larval muscles but does not consistently shift EC50 values. Effects of proctolin and Glu on contraction are supra-additive but do not appear to be synergistic. Chapter 3 demonstrates that proctolin increases calcium-induced fluorescence in muscle cytoplasm by itself and enhances calcium signals elicited by Glu. The effect of proctolin is blocked by nifedipine, an inhibitor of voltage-gated, L-type calcium channels in the plasma membrane, suggesting a role for calcium influx. Proctolin failed to increase calcium-induced fluorescence changes induced by caffeine in calcium-free saline, suggesting that proctolin may not act directly on ryanodine receptors (RyRs) that release calcium from the sarcoplasmic reticulum (SR) into the cytosol. Chapter 4 examines a hypothesis in which activating the proctolin receptor inhibits calmodulin, since calcium-calmodulin complexes are reported to inhibit RyRs at high calcium concentrations, limiting calcium release from the SR. A calmodulin inhibitor, W7, mimicks some but not all effects of proctolin, suggesting that although calmodulin inhibition may contribute to proctolin’s effects, other mechanisms must be involved. Data are presented implicating the possible involvement of protein kinase C (PKC), the cAMP-dependent protein kinase (PKA) and the plasma membrane calcium ATPase (PMCA), which normally removes calcium from the cytosol. This thesis adds to our understanding of how cotransmission of proctolin with Glu modulates muscle performance in Drosophila, and it suggests an experimental framework for investigating effects of cotransmitters generally.
Open Access
Mechanistic Insights into the Notch signalling pathway through Molecular Condensation
(Brock University) Foran, Gregory; Department of Biological Sciences
The ability of a cell to undergo cellular decisions is ingrained in the integration of cues from its local environments. One such cue is relaying information based on local cell density, neighbouring cell identity, and cell positional information. A key signalling pathway in interpreting this information is the Notch signalling pathway. It is currently poorly understood how Notch is able to play a titratable role in signal transduction as our current model does not effectively translate how fluctuations in Notch signalling lead to dynamic changes in Notch target gene expression. One potential mechanism that has recently been shown to play a role in titratable gene expression of several signalling pathways is the ability for proteins to undergo molecular condensation within living cells. To date, Notch signalling has been difficult to study because of the lack of live cell systems that allow us to study both Notch protein localization and Notch target gene expression in real-time. Using PlayBack, a system I developed that allows for cost-effective plasmid cloning, I was able to develop both a light controlled Notch construct, OptoNotch, and a Live Notch target gene promoter reporter, called Hes1-Live-RNA, to allow for both the visualization and quantification of Notch activity within living cells. Using these tools, I discovered that the Notch 1 intracellular domain forms molecular condensates which in turn positively facilitates both Notch1 target gene expression as well as facilitating Notch’s ability to regulate enhancer looping. This is the first instance of N1ICD undergoing condensation within live cells and offers a mechanism by which Notch can have a titratable effect on target gene expression.
Open Access
The Maillard reaction in traditional method sparkling wine
(Brock University) Charnock, Hannah M.; Department of Biological Sciences
The development of “aged” aromas in sparkling wine is often an indicator of quality and is characterized by caramel and toasted qualities. Compounds responsible for these aromas are derived via the Maillard reaction (MR), a non-enzymatic condensation between sugars and amino acids capable of generating a myriad of aroma compounds in a complex reaction cascade. During the production of traditional method sparkling wines, several major aging intervals take place, including the storage of reserve base wines, after the second alcoholic fermentation, and during the storage of finished wines. The work presented in this dissertation investigated the influence of amino acid and sugar precursors, plus the potential catalytic role of metal ions, on the formation of MR-associated products (MRPs) during base wine and sparkling wine aging. Four primary research objectives were addressed. The metal ion content of commercial sparkling wines produced in Niagara was established, and to our knowledge, this study represents the first report on sparkling wine metal profiles. Differences in metal composition were identified between production methods and styles, and calcium and magnesium were confirmed to be the most abundant divalent metal ions, highlighting their candidacy for involvement in MR pathways in wine. In a subsequent study, the influence of calcium and magnesium on the formation of MRPs was assessed in modified base wine treated with varying sugar and amino acid combinations during accelerated aging at 50 degrees C. Aging duration and amino acid additions were primary drivers of variation among MRPs, with calcium and magnesium having a lesser effect. A separate set of studies aimed to identify the impact of different sugar-types in dosage, the final sugar addition during production, on the formation of both MRPs and metabolite levels in sparkling wines during cellar aging. Aging duration had a greater influence on MRP and metabolite composition compared to sugar type, demonstrating that aging conditions for sparkling wine are central to the evolution of the wine matrix. This work contributes novel information to understanding the MR in mild conditions and can inform future research focused on optimizing sparkling wine composition and aging to enhance flavour in accordance with consumer preferences.
Open Access
Environmental and social influences of foraging behaviour and maternal investment with a note on the mating frequency of the eastern carpenter bee, Xylocopa virginica
(Brock University) Duff, Lyndon; Department of Biological Sciences
This thesis studies how weather and sociality influence the foraging effort input costs of brood production, and that multiple mating contributes to low-group relatedness and non-kin sociality in the Eastern carpenter bee, Xylocopa virginica. In Chapter 2, using a natural experiment which included a drought and three normal weather years, we found that mothers foraged more in a drought but had smaller offspring than most normal weather years. Droughts conditions lead to fewer flowers, and it follows that less abundant food resources should lead to a trade-off of more work for fewer resources. Indeed, we found that during the drought, the size of pollen loads that mothers carried were smaller than in every other normal year showcasing the impact of the drought. In Chapter 3, I assess the foraging effort in both solitary and social foundresses showing that the foraging input costs are much higher for social primary foundresses than for solitary foundresses. That foraging costs are higher for social foundresses is atypical compared to most literature, which usually suggests that solitary behaviour is more costly than social behaviour. Lower social costs implies that social behaviour should become the more predominant phenotype in the population, however, in our Niagara population there was always a mix of solitary and social phenotypes. A mix of solitary and social phenotypes suggests balanced selection for both phenotypes. That the social phenotype is more costly is interesting and we suggest that social primary foundresses must feed their nestmates throughout the foraging period. Nestmate feeding has been documented in this bee before, so it seems like the most plausible explanation. In Chapter 4, we assessed the mating frequency of female carpenter bees using behavioural observations and microsatellite markers. We found using both sets of data that X. virginica mates multiply. Multiple mating has direct implications on the genetic relatedness of already low relatedness in non-kin social groups. Non-kin sociality is understudied, and we hope that these findings spur additional studies.
Open Access
The influence of grapevine cultivar, clone, and rootstock on cold hardiness and dehydrin proteins
(Brock University) Hébert-Haché, Andréanne; Department of Biological Sciences
Grapevine cold hardiness is a critical phenotype that is impacted by genetic and environmental factors. The extent of intra- and inter-cultivar differences within the Vitis vinifera L. specie are not well characterized. The dehydrins are a family of proteins that is upregulated by cold temperatures and have seldom been studied in overwintering grapevines. This thesis contains four research articles on these topics. First, cold hardiness of different cultivars was compared using the Vine Alert database containing data from ten winters from more than twenty vineyards. We report average and maximum mid-winter hardiness for Cabernet franc, Cabernet Sauvignon, Chardonnay, Merlot, Pinot noir, Riesling, Sauvignon blanc and Syrah, showing that cultivar and year have a larger impact than site within a viticultural region. Acclimation and deacclimation rates were weakly influenced by cultivar and site, but significantly influenced by the time of the year and the year itself. Cold hardiness was then studied for different clone and rootstock combinations of Riesling (clones 49 and 239 on Riparia gloire and SO4, and 239 on 3309) and Sauvignon Blanc (clones 242, 297, 376, 530) over three winters (2016 to 2019). We demonstrated that clone and clone x rootstock interaction can influence cold hardiness to a greater extent than rootstock alone. We then studied the presence and relative concentration of dehydrin proteins. We identified six new dehydrin bands (23 kDa, 26 kDa, 35 kDa, 41 kDa, 48 kDa, 90 kDa), showed that their concentration peaked either mid-winter or immediately before deacclimation, and demonstrated that they were all correlated to cold hardiness. For the final chapter, we demonstrated intra- and inter-cultivar differences in the hardiness-dehydrin correlations, particularly for the 23 kDa band which is negatively correlated to the % maximum hardiness in Riesling but not in Sauvignon blanc. Riesling cold hardiness was strongly correlated to temperature before sampling, but dehydrin accumulation was strongly correlated to the temperature before sampling in Sauvignon blanc, a key difference in cold-exposure response. This thesis contains findings that are actionable for the industry and relevant for the ongoing research efforts in the field of cold hardiness.
Open Access
Feasibility study using remote sensing technologies to improve zonal vineyard management
(Brock University) Lee, Leeko Hyun Suk; Department of Biological Sciences
The primary purpose of this research was to examine the feasibility of using remote sensing data to improve efficiency of zonal vineyard management. To achieve this goal, correlation analysis between the significant vineyard management variables and different remote sensing data analysis tools were undertaken. The variables included leaf water potential, soil moisture, canopy size, vine health, vineyard yield, and fruit composition, which further impacts wine quality. The remote sensing data analysis tools included normalized difference vegetation index (NDVI), and other indices extracted from electromagnetic reflectance data of grapevine leaves and canopies. In each site, sentinel vines (i.e., 72-81) were identified in a grid form. GPS-based geolocation was carried out for six Cabernet Franc vineyards in Ontario's Niagara wine country. Even though remote sensing data analysis tools were not associated with several other important variables for quality grape production, this research still confirmed that remote sensing data analysis has significant potential to differentiate specific zones of canopy size, water stress, yield, some superior fruit compositions, and the resulting wine sensory attributes within a single vineyard site. This study also confirmed that the mechanism of plant defense systems against biotic stress could have impacts on the spectral behaviour of grapevine leaves and hyperspectral remote sensing technologies could be applied as a tool to identify the spectral behaviour changes due to stress. Overall, this study verified the feasibility of remote sensing technologies to enhance the efficiency of vineyard management in the correlation of data from various remote sensing data-analysis techniques and viticulturally important variables for plant health and growth, and fruit and wine quality. As a first step to develop a site-specific crop management (SSCM) model for vineyard management, it also proposes future research opportunities to test and develop an efficient vineyard management decision making model.
Open Access
Modulation of Voltage-Gated Ca2+ Channels by Retinoic Acid Signaling
(Brock University) de Hoog, Eric; Department of Biological Sciences
Retinoic acid (RA), the active metabolite of vitamin A, has an established role in development of the nervous system, but has recently emerged as a critical regulator of adult nervous system function. RA is important for learning and memory in both vertebrates and invertebrates. It is also important for synaptic plasticity at rodent hippocampal synapses and is known to interact with Ca2+ signaling to regulate receptor expression and influence synaptic transmission. How RA signaling might mediate plasticity in an invertebrate nervous system has not yet been studied. Voltage-gated Ca2+ channels (CaV) represent a ubiquitous means of Ca2+ entry into a cell, and which regulate neuronal functioning (such as gene expression and neurotransmitter release). As such, CaV channels represent a potentially important locus for inducing plasticity in both vertebrates and invertebrates. It is not known in any species, whether RA might mediate changes in neuronal communication by influencing the functional properties of CaV channels. Here I show, utilizing cultured neurons from the molluscan pond snail, Lymnaea stagnalis, that RA signaling modulates CaV channel function. RA signaling inhibited CaV channel function by shifting the voltage dependence of channel activation to more depolarized potentials. I found that RA also produced spike broadening and activity-dependent complex spiking, an effect I determined was mediated by RA-induced inhibition of delayed rectifier voltage-gated K+ channels (KV), (and enhanced inactivation of these KV channels). Paradoxically, the effect of RA to induce spike broadening and activity-dependent complex spiking, enhanced Ca2+ influx through CaV channels. However, the concurrent inhibition of CaV2 channels limited this enhanced Ca2+ influx. I also provide evidence that constitutive/basal retinoid receptor signaling upregulates CaV channel function. I show that a retinoic acid receptor (RAR) antagonist produced G-protein-mediated voltage-dependent inhibition of CaV channels, a ubiquitous form of presynaptic plasticity that occurs at vertebrate synapses. I also show that a retinoid X receptor (RXR) antagonist produced a novel G-protein-independent form of voltage-dependent inhibition of CaV channels. Overall, my study indicates that retinoid signaling diversely regulates CaV channel function in this invertebrate species and provides insights into the mechanisms by which RA signaling might mediate neuronal and/or synaptic plasticity.
Open Access
Investigating the Effect of Cell Culture Compositions on Mitochondrial Metabolism, Dynamics, and Transcriptome and Proteome of cells
(Brock University) Moradi, Fereshteh; Department of Biological Sciences
The phytoestrogen Resveratrol (RES) is a natural polyphenol that has been detected in more than 70 plant species. RES has structural similarity to mammalian estrogens and can bind to estrogen receptors, eliciting genomic and non-genomic effects. Both RES and physiological estrogens like 17-β-estradiol (E2) have wide-ranging effects on mitochondria. In this thesis, I began by investigating RES’s effects on mitochondrial network dynamics (Chapter 2) and discovered a pro-fusion activity apparently mediated by the mitofusin enzyme Mfn2. RES stimulated mitochondrial network hyper-fusion morphology in all three cell lines investigated (C2C12 (mouse myoblast), PC3 (prostate cancer), and MEFs (mouse embryonic fibroblast)), but the effect was absent in Mfn2-null MEFs. As this work was being completed; several research groups introduced ‘physiologic cell culture media’ that are modeled on the human plasma metabolome. I co-authored a study (not in this thesis) demonstrating that RES’s effects on mitochondrial dynamics are dependent on cell culture conditions. To follow up on this, I investigated whether E2’s mitochondrial effects might also be dependent on the cell culture environment, and showed conclusively that this is indeed the case, using C2C12 cells as a model system (Chapter 3). These results and those published by others in 2017-2019 suggested that medium composition can profoundly affect cellular functions. In Chapter 4, I followed this up by studying how culture conditions affect mitochondrial bioenergetics and network morphology using four cancer cell lines and showed that this is a significant issue. Finally, to gain a more complete understanding of this phenomenon, I completed a full transcriptomic and proteomic analysis of media effects using MCF7 breast cancer cells as a model (Chapter 5). I showed that hundreds of transcripts and proteins are affected according to culture conditions. Taken together, the results presented in this thesis emphasize the significant extent to which the cell culture environment affects experimental outcomes, particularly with respect to mitochondrial bioenergetics and dynamics. This information contributes to the development of cell culture experiments providing results that can be translated in vivo.
Open Access
Thermal tasting: methodological considerations and implications for alcohol behaviour.
(Brock University) Thibodeau, Margaret; Department of Biological Sciences
Thermal tasting is a phenomenon whereby some individuals perceive thermally-induced taste sensations when their tongue is warmed or cooled. These individuals, known as thermal tasters (TT), report a variety of thermally-induced tastes and the tastes reported can vary with temperature regime used and location on the tongue tested. TT are typically compared to thermal non-tasters (TnT), individuals who do not experience thermally-induced sensations. The literature suggests that TT give higher intensity ratings to orosensory stimuli than TnT; however, small sample sizes and differences in classification schemes between studies confound our understanding of TTS (thermal taste status). It is unknown whether the increased orosensory responsiveness of TT is universal or whether it is driven by a subgroup of TT. Furthermore, up to 50% of individuals are non-classifiable (NC). The largest database of individuals who have undergone TTS screening was compiled to address the literature gaps. Findings indicate that TT are more responsive than TnT to orosensory stimuli, regardless of the classification scheme used. The orosensory responsiveness of NC is not homogeneous, suggesting that NC are not a separate group but rather misclassified TT and TnT. Sweet TT are more likely than non-sweet TT to experience thermally-induced sensations during lingual warming. Similarly, sour TT are more likely than non-sour TT to report thermally-induced tastes during cooling. However, no differences in orosensory responsiveness based on these or other subgroups are identified, suggesting that the heightened orosensory responsiveness of TT is universal across this phenotype. The final study sought to characterize the binary interactions between ethanol and four orosensory stimuli (fructose, quinine, tartaric acid and alum sulphate) both overall and by comparing TT and TnT. In general, TT are more responsive than TnT to all stimuli in the study. Few interactions between TTS and stimulus intensity exist suggesting that TT and TnT perceive the sensations elicited by alcoholic beverages similarly, albeit at different intensities. Together, the thesis helps inform best practices for TTS screening and classification, provides insights into TTS mechanisms and furthers our understanding of alcoholic beverage perception.
Open Access
Metarhizium robertsii interactions with Phaseolus vulgaris (Haricot Bean)
(Brock University) Hu, Shasha; Department of Biological Sciences
Metarhizium is an insect pathogenic fungus, as well as a plant root symbiont. During symbiotic interactions, it can benefit the plant by improving plant growth, antagonizing plant pathogens and herbivores, and enhancing plant tolerance to abiotic stresses. In this thesis, the interactions between Metarhizium robertsii and Phaseolus vulgaris (haricot bean) were studied from two aspects. First, a phenotypically degenerated (low conidia production) strain of Metarhizium was serially passaged through bean plant. Second, the immune responses of haricot bean during endophytic colonization were assessed. Commercial application of Metarhizium for insect biocontrol requires optimal production of conidia as infective propagules. It was demonstrated that conidial production and virulence of phenotypically degenerated Metarhizium were restored by serial passages through bean roots, as well as switchgrass roots, and wax moth larvae. A decrease in the expression of fungal DNA methyltransferase was observed in the phenotypically degenerated Metarhizium strain through bean passages. Whole genome bisulfite sequencing analysis showed differences in the distribution of differentially methylated regions in the degenerated and subsequently recovered strains. Metarhizium can antagonize the plant pathogen, Fusarium solani f. sp. phaseoli during bean root colonization. Using comprehensive plant hormone analysis, transcriptional expression, and stomatal size analysis, bean immune responses to colonization by Metarhizium and/or Fusarium were assessed. In comparison to un-inoculated bean, root colonization by Metarhizium resulted in reduction of abscisic acid (ABA), increased stomatal size, and decreased expression of plant immunity genes in bean leaves, which is different from those in bean colonized by Fusarium. Furthermore, exogenous application of ABA resulted in reduction of bean root colonization by Metarhizium but increased colonization by Fusarium, compared to corresponding plants without ABA application. Therefore, ABA was implicated in differential responses of bean plants to root colonization by Metarhizium and Fusarium. In conclusion, this thesis provided new insights into the study of the interactions between Metarhizium and haricot bean. Some novel findings were that fungal DNA methyltransferase was implicated in the recovery of phenotypically degenerated Metarhizium and a plant hormone, abscisic acid was implicated in differential interactions of endophytic colonization by Metarhizium when compared to a pathogenic interaction by Fusarium.
Open Access
Investigating the Role of MicroRNAs in Regeneration and Axonal Pathfinding
(Brock University) Walker, Sarah; Department of Biological Sciences
During both development and regeneration, axons must navigate through a complex and changing environment to reach their proper synaptic target. To do so, axons utilize a specialized structure, the growth cone, which senses and interprets guidance cues in its surrounding environment to change the direction of axonal outgrowth. MicroRNAs, which regulate mRNA translation, have recently been shown to regulate both neurite outgrowth and growth cone guidance in response to classical guidance cues during vertebrate development. However, little is known of their regulation of neuronal regeneration in an invertebrate. Thus, the main aim of this thesis was to study the role of microRNAs during CNS regeneration of the pond snail, Lymnaea stagnalis. Specifically, I determined the expression patterns and relative abundance of microRNAs in the regenerating CNS in response to retinoic acid (RA). Using miRNA-Sequencing, I identified one neuronally enriched microRNA, miR-124, that was up-regulated in RA-induced regenerating CNS. Using PCR and in situ hybridization, I characterized its distribution in the snail CNS, and discovered it shared similar expression patterns to that of vertebrates. In cell culture, I found miR-124 was abundant within regenerating motorneurons and was localized to their growth cones. I next determined that miR-124 contributed to RA-induced growth cone turning behaviour. During attractive growth cone turning to RA, the abundance and distribution of miR-124 was altered, in both a cue and context-dependent manner. Finally, I demonstrated that miR-124 targeted the Rho kinase, ROCK, during turning responses to RA, likely to promote the formation of a neurite shaft, or to maintain growth cone polarity. Together, these findings provide the first evidence for a role of microRNAs in mediating growth cone behaviours to RA in regenerating motorneurons.
Open Access
The role of mobile elements in recent primate genomes
(Brock University) Tang, Wanxiangfu; Department of Biological Sciences
Mobile elements (MEs), which constitute ~50% of the primate genomes, have contributed to both genome evolution and gene function as demonstrated by ample evidence discovered over the last few decades. The three studies in this thesis aims to provide a better understanding of the evolutionary profile and function of MEs in the primate genomes by taking a computational comparative genomics approach. The first study represents a comprehensive analysis of the differential ME transposition among primates via identification of species-specific MEs (SS-MEs) in eight primate genomes from the families of Hominidae and Cercopithecidae using a comparative genomics approach. In total, 230,855 SS-MEs are identified, which reveal striking differences in retrotransposition level in the eight primate genomes. The second study represents a more focused analysis for the identification of a new type of MEs, which we term “retro-DNA” for non-LTR retrotransposons derived from DNA transposons, in the recent primate genomes. By investigating biallelic DNA transposons that have both the insertion and pre-integration alleles in ten primate genomes, a total of 1,750 retro-DNA elements representing 750 unique insertion events are reported for the first time. The third study provides an analysis of the mechanism underlying the differential SINE transposition in the primate genomes. In this study, Alu profiles are compared and the Alu master copies are identified in six primate genomes in the Hominidae and Cercopithecidae groups. The results show that each lineage of the primates and each species owns a unique Alu profile exclusively defined by the AluY transposition activity, which is determined by the number of Alu master copies and their relative activity. Overall, work in this thesis provides new insights about MEs and their impact on the recent primate genomes by revealing differential ME transposition as an important mechanism in generating genome diversity among primate lineages and species through discovering a new type of MEs and preliminary analysis of the mechanism underlying the differential ME transposition among primates. Furthermore, taking advantage of the recently available primate genomes and transcriptomes data, the work in this thesis demonstrates the great potential of the comparative genomic approach in studying MEs in primate genomes.
Open Access
Influence of adolescent social instability stress on the intake of ethanol and sucrose in a rodent model
(Brock University) de Lima Marcolin, Marina; Department of Biological Sciences
Adolescence is a sensitive period in which the effects of stress and alcohol can have long-lasting impacts. Social instability stress in adolescent rats (SS; postnatal day 30-45, daily 1 hour isolation + new cage partner) alters behavioural responses to psychostimulants and increases anxiety-like behaviour, but differences in voluntary consumption of natural and drug rewards are unknown. The main goal of my thesis was to investigate the effects of adolescent social instability stress (SS) on immediate and long-lasting changes on reward-related behaviours in male rats using voluntary alcohol intake paradigms. Another goal was to investigate the influence of social context on the propensity to drink alcohol, as well as the influence of these factors on sucrose intake. In chapter 2, I found that adolescent SS increased alcohol intake irrespective of social context, and adolescents drank more alcohol than adults. The intake of sucrose was not altered by stress, except during context of competition. In chapter 3, I found that history of alcohol drinking reduced synaptic plasticity markers in the dorsal hippocampus and prefrontal cortex, and this reduction was sometimes further reduced by SS. The propensity to drink alcohol was found not to differ between SS and CTL rats in the first experiment, and reduced among SS rats in the second experiment. After nine days of alcohol absence, the propensity to drink alcohol was not increased by previous alcohol access, and SS increased intake only in alcohol-naïve rats. History of alcohol drinking reduced anxiety-like behaviours and blunted SS-induced reduction in social interactions. Both SS and alcohol decreased corticosterone levels at baseline and after fear recall without changing freezing behaviour. My findings indicate that using a model of mild social stressor can have great impact on adolescent rats, but moderate effects in adult rats. The behavioural changes caused by stress can be enhanced later in life by history of alcohol drinking, but that does not necessarily cause an increase in the propensity to drink during adulthood, as other studies have shown. Adolescent stressed rats drink more alcohol than other groups, but they don’t seem to continue drinking more when they reach adulthood. These results indicate that the effects of social instability stress are transient in regards to propensity to drink, and can be the basis for alterations caused by both alcohol and stress.
Open Access
Functional antagonism of the mesolimbic dopaminergic system on mesolimbic cholinergic system in the vocal expression of an emotional state
(Brock University) 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.
Open Access
Proximate influence on eusocial caste behaviour
(Brock University) Awde, David N; Department of Biological Sciences
Queens and workers of eusocial sweat bee species are morphologically and developmentally similar, which means that each female is capable of behaving as a queen or a worker. However, few females lay eggs and behave as queens, while the majority of females provision the queen’s offspring, rarely lay eggs, and behave as workers. This makes eusocial sweat bee species, such as Lasioglossum laevissimum, excellent models to study the underling environmental (social) and genetic factors that contribute to variation in caste behaviours. My research focused on describing some of the proximate mechanisms that influence caste behaviours in L. laevissimum females. The social environment of a sweat bee colony, specifically the behaviour of a queen, can have a dramatic impact on worker behaviour. Queens suppress worker reproduction by physically bullying their workers. In a nesting aggregation at Brock University, almost half of L. laevissimum nests became queenless, which provided me with a natural experiment to assess the direct influence by queens on worker behaviour. Dissection data showed that a small proportion (17%) of workers developed their ovaries in both queenright and queenless nests. This suggests that L. laevissimum queens exert an early, negative, and strong influence on worker egg-laying behaviour, which lasts after she is gone. Next I assessed the relationship between gene expression and L. laeivsismum caste behaviours. I predicited that queens would express a gene associated with egg-laying, vitellogenin, more than workers, and that workers would express genes associated with foraging, the foraging gene, more than queens. Lasioglossum laevissimum queens had higher vitellogenin expression levels than workers, and females with high ovarian development had high vitellogenin expression, regardless of caste. On the other hand, queens and workers had similar foraging expression levels. Gene expression comparisons between queens and workers highlight two important behavioural characteristics of sweat bee castes. First, in eusocial sweat bees, both queens and workers actively provision brood at some point during the breeding season, which is reflected in their similar foraging expression levels. Secondly, queens lay eggs while a small proportion of workers have queen-like ovarian development, reflected in vitellogenin expression differences between castes.
Open Access
ENDOCANNABINOID REGULATION OF ADOLESCENT DEVELOPMENT IN MALE AND FEMALE RATS
(Brock University) 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.
Open Access
Phage-mediated biological control of Erwinia amylovora: The role of CRISPRs and exopolysaccharide
(Brock University) Yagubi, Abdelbaset; Department of Biological Sciences
Fire blight, caused by bacterium Erwinia amylovora, is a very serious disease affecting apple, pear and other fruit plants. The development of phage-based biopesticides is currently in progress in our lab. Emergence of phage-resistant bacteria is a valid concern. Two attributes of the bacterial host that may contribute to the development of resistance were studied, the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas) system and exopolysaccharide (EPS) interaction with phages. The structure of E. amylovora CRISPR/Cas system was determined in 8 E. amylovora isolates from different geographical regions. Three CRISPR-array sets named CR1, CR2 and CR3 were detected in 4 isolates, and only 2 arrays were determined in the rest of the isolates. No significant similarity was found between spacers in any of these systems to phage DNA sequenced in this study or from GenBank. Also the Cas level of expression was not stimulated during phage infection. Introduction of extra copies of Cas genes to enhance expression did not result in phage resistance. Nevertheless, E. amylovora CRISPR/Cas system was found to be efficient in blocking the transformation of plasmids carrying protospacers matched spacers in CRR1 and CRR2. Among phages that have been sequenced in this study are ΦEa9-2 and ΦEa35-70. ΦEa9-2 (Podoviridae) genome is 75,568 bp, and found to be related to coliphage N4. ΦEa35-70 (Myoviridae) genome is 271,084 bp, and found to carry a potential EPS depolymerase gene. Activity of ΦEa35-70 EPS depolymerase was only detected when cloned and expressed in E. coli, but His-tagged purified protein did not exhibit any EPS-depolymerase activities. This study offers critical information for the design of novel and effective phage-based biopesticides for the control of E. amylovora. It provides a new knowledge on the molecular structure and function of CRISPR/Cas system and EPS-phage interaction.
Open Access
Molecular ecology and social evolution of the eastern carpenter bee, Xylocopa virginica
(Brock University) Vickruck, Jessica L; Department of Biological Sciences
Bees are extremely valuable models in both ecology and evolutionary biology. Their link to agriculture and sensitivity to climate change make them an excellent group to examine how anthropogenic disturbance can affect how genes flow through populations. In addition, many bees demonstrate behavioural flexibility, making certain species valuable models with which to study the evolution of social groups. This thesis studies the molecular ecology and social evolution of one such bee, the eastern carpenter bee, Xylocopa virginica. As a generalist native pollinator that nests almost exclusively in milled lumber, anthropogenic disturbance and climate change have the power to drastically alter how genes flow through eastern carpenter bee populations. In addition, X. virginica is facultatively social and is an excellent organism to examine how species evolve from solitary to group living. Across their range of eastern North America, X. virginica appears to be structured into three main subpopulations: a northern group, a western group and a core group. Population genetic analyses suggest that the northern and potentially the western group represent recent range expansions. Climate data also suggest that summer and winter temperatures describe a significant amount of the genetic differentiation seen across their range. Taken together, this suggests that climate warming may have allowed eastern carpenter bees to expand their range northward. Despite nesting predominantly in disturbed areas, eastern carpenter bees have adapted to newly available habitat and appear to be thriving. This is in marked contrast to many other bee species, particularly in the genus Bombus, who appear unable to shift their ranges along with climate change. Facultatively social organisms are interesting species to study the evolution of social groups, and the remaining chapters address questions of sociality in X. virginica. I used observation nests and genetic relatedness to examined how females behave towards one another in the spring prior to the establishment of dominance hierarchies in social nests. In spring, females directed fewer aggressive behaviours and more cooperative behaviours towards familiar rather than related individuals, indicating that females use nestmate recognition rather that kin recognition when interacting with conspecifics. Overwintering groups often contain both related and unrelated individuals, indicating that many bees interacting with one another in the fall prior to overwintering may be unrelated, emphasizing the importance of recognizing nestmates. Within social carpenter bee nests three different types of female have been described: primary, secondary and tertiary. Primary females are the dominant foragers and egg layers in the nest while secondary and tertiary females appear to join a reproductive queue behind the primary. To understand the nature and flexibility of this reproductive queue I performed removal experiments across three different years. This study showed that secondary females always assumed the role of replacement primary, while tertiary females rarely opted to forage and reproduce even if they were the only female in the nest. Removal experiments demonstrated that social groups in X. virginica are complex and comprise two different reproductive strategies (breed in the current year or delay reproduction) as well as form dominance hierarchies among primary and secondary females. Several tertiary females were able to become primary or solitary females in their second summer, providing evidence for how each type of female may have evolved in social nests. Finally, I examined how competition influences the evolution and maintenance of social groups in eastern carpenter bees. In conditions of high population density significantly more social nests were present in the population, indicating that competition for limiting nesting resources drives individuals together into social groups. Within social groups relatedness was low, and siblings actually dispersed away from one another to other nests in the population, reducing competition among kin. Eastern carpenter bees appear to demonstrate an interesting evolutionary route to sociality, where very high levels of competition among kin lead to dispersal, while limited nesting substrate forces individuals back into unrelated social groups. While predicted by kin selection, social groups of this nature are previously undescribed in the Hymenoptera, and further study of eastern carpenter bees can provide novel insights into alternate routes to sociality.
Open Access
Etiology and Management of Grape Sour Rot
(Brock University) 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.
Open Access
Responses to Reflection in Two Invertebrate Species
(Brock University) May, Holly; Department of Biological Sciences
The present thesis investigates the responses to reflection in both the crayfish Procambarus clarkii and the fruit fly Drosophila melanogaster. Responses to reflection in crayfish depend on social status and the current work suggests that learning and memory consolidation are required for these responses to be altered. Crayfish were treated to either massed or spaced training fights prior to reflection testing. The results show that subordinate crayfish treated to spaced training display a response typical of subordinate crayfish but subordinate crayfish treated to massed training exhibit a response typical of dominant crayfish. Fruit flies are shown to be attracted to reflection and responses to reflection are described here for the first time. Responses in fruit flies are shown to be dependent on social status. The frequency of behaviours were altered in isolated flies but not socialized flies. The addition of pheromones cVA and 7,11-HD were used to investigate how the addition of chemical cues altered responses to reflection in fruit flies. Socialized fruit flies treated with cVA exhibited an increase in the frequency of behaviours on both mirrored and clear glass walls, while isolated flies exhibited a decrease. Socialized flies treated with 7,11-HD spent more time on mirrored walls compared to glass walls, whereas the frequency of all behaviours were decreased in isolated flies treated with 7,11-HD.

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