Theses (Biology)

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Theses from the Dept. of Biology.

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    Chemistry and biochemistry of Populus leaf bud resin
    (2024-01-29) Piirtola, Eerik-Mikael; Constabel, Carsten Peter
    Poplar trees, such as black cottonwood (Populus trichocarpa), balsam poplar (Populus balsamifera), and eastern cottonwood (Populus deltoides) are known to secrete resinous exudate from their leaf buds as their adaptation to temperate climate. The leaf bud resin protects the developing leaf buds from frost during winter dormancy. During bud break, the sticky resin coats the young leaves, protecting against insect herbivory during the early stages of leaf development. Leaf bud resins from different poplar species contain diverse phenolic secondary metabolites, especially hydrophobic flavonoids, which are biologically active. Due to their flavonoid-rich composition, poplar bud resins have been used widely in traditional medicine for their antimicrobial properties. Poplar leaf bud resins are also essential for honeybees, which utilize them as a building material and antibiotic protection for their hives in the form of propolis. In this thesis, I characterized seasonal patterns of leaf bud resin accumulation, as well as genes involved in the biosynthesis of secreted flavonoids in leaf buds of P. trichocarpa, P. balsamifera, and P. deltoides using a combination of metabolomic analysis and transcriptomics. I used targeted and non-targeted analysis of the chemical composition of poplar bud resins to identify and quantify characteristic flavonoids in each poplar species. In parallel with the metabolomic analysis, transcriptomics and biochemical techniques were used to identify and characterize novel genes associated with the production of methoxylated and acylated flavonoids. The identified candidate genes were tested as recombinant proteins to characterize and verify their function. This work provides insight into the dynamic nature of poplar leaf bud resin biosynthesis and the enzymes involved in synthesizing characteristic flavonoids of poplar bud resin.
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    The effects of anthropogenic noise on the behaviour and vocalizations of plainfin midshipman fish, Porichthys notatus
    (2024-01-11) Woods, Mackenzie; Juanes, Francis
    Noise pollution in the ocean has been accelerating at an alarming rate, drastically altering underwater soundscapes and negatively affecting marine life in myriad ways. Many marine animals, including fishes, rely heavily on sound for communication, navigation, and environmental perception, all of which can be negatively affected by anthropogenic noise pollution from activities such as recreational boating, commercial shipping, marine construction, and seismic exploration. While the effects of noise on marine mammals are well documented, far fewer studies have investigated the effects of noise on fishes, and even fewer have been conducted in the field using realistic noise stimuli. In this thesis, I use a combination of field and laboratory studies to investigate how anthropogenic noise affects nesting plainfin midshipman fish (Porichthys notatus), which breed in the intertidal zone and rely on acoustic communication for mate attraction and defence. I demonstrate that plainfin midshipman sometimes alter nest defence behaviour in response to boat noise, but that testing context, such as whether they are tested in the laboratory or the field, the presence of eggs, and the noise stimulus used, can greatly affect experimental outcomes. I also found that males in the wild significantly reduced the number of agonistic vocalizations but increased the amplitude (loudness) of these calls when a motorboat was driven continuously near their nests. Males also increased the frequency (pitch) of their mating hums. Such vocal adjustments in response to noisy environments are referred to as the Lombard effect—a widespread phenomenon typically studied in mammals and birds but rarely tested in fishes. This study is amongst the first to report changes in vocalization frequency and amplitude in fishes, and it is the first to demonstrate this effect in wild fish using experimentally introduced noise from a real motorboat. The results in my thesis contribute to our growing understanding of how noise affects fishes and demonstrate the need for additional field studies using realistic noise stimuli and ecologically relevant contexts.
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    Feeding and bioenergetics of Chinook Salmon during the first winter at sea
    (2024-01-02) Innes, Katie; Juanes, Francis
    It is hypothesized that winter is a period of nutritional stress and elevated mortality for juvenile Chinook Salmon (Oncorhynchus tshawytscha). However, little is known about the winter ecology of this species. To address this gap in knowledge, first ocean winter Chinook Salmon were sampled systematically by microtrolling (hook-and-line capture) over three consecutive winters from late September to early April in 2020-2023 in the Strait of Georgia, British Columbia. Sampling regions included the Discovery Islands, Northern Strait of Georgia, and Southern Gulf Islands. Chinook Salmon were weighed and measured, and scales were collected for genetic stock identification. Winter diet samples were collected by gastric lavage and intact prey were preserved for energy density determination. By mass, Chinook Salmon consumed primarily Pacific Herring (Clupea pallasii), euphausiids, squid, and Primno spp. amphipods, and diets differed by region and season with some interannual variability observed. Both diet energy content and body condition declined throughout the winter, although the decline in diet energy content was not significant. The presence of Pacific Herring in the diets had a significant positive effect on diet energy content. A subset of sampled Chinook Salmon was also retained for energy density and organosomatic index analyses, both of which had significant seasonal trends that may be associated with allocating energy to storage in autumn and reduced rations in mid-winter. Using field-derived data as inputs, I developed bioenergetics models to estimate differences in overwinter growth, consumption, and feeding rates over two years and between two regions in the Strait of Georgia. These inputs included diet composition, prey and predator energy density, temperature at depth of capture, and predator weight. Regional and interannual differences in model estimates were observed, and January and February were consistently estimated to be periods of reduced consumption rates. Bioenergetic model estimates also provided possible evidence of the occurrence of size-selective processes, although this result should be interpreted with caution. The models based on our longitudinal sampling framework were then compared to ‘seasonal’ models which mimicked a field sampling design wherein discrete sampling events occurred once prior to winter and once following winter to highlight the temporal variability in fish bioenergetics which may be missed using a seasonal approach. Overall, the data presented in this thesis suggest that some degree of food limitation occurs during winter but does not provide strong evidence that supports the plausibility of winter as a period of nutritional stress for overwintering juvenile Chinook Salmon.
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    Lingonberry (Vaccinium vitis-idaea L.) species origin and subspecies divergence through genome sequencing and assembly
    (2023-08-25) Hirabayashi, Kaede; Owens, Gregory Lawrence
    Lingonberry (Vaccinium vitis-idaea L.) produces tiny red berries that are tart and nutty in flavour. It grows widely in the circumpolar region, including Scandinavia, northern parts of Eurasia, Alaska, and Canada. Although cultivation is currently limited, the plant has a long history of cultural use among indigenous communities. Given its potential as a food source, genomic resources for lingonberry are significantly lacking. To advance genomic knowledge, the genomes for two subspecies of lingonberry (V. vitis-idaea ssp. minus and ssp. vitis-idaea var. ‘Red Candy’) were sequenced and de novo assembled into contig-level assemblies. The assemblies were scaffolded using the bilberry genome (V. myrtillus) to generate chromosome-anchored reference genome consisting of 12 chromosomes each with total length 548.071 Mbp (contig N50 = 1.170 Mbp, BUSCO (C%) = 96.5%) for ssp. vitis-idaea, and 518.704 Mbp (contig N50 = 1.400 Mbp, BUSCO (C%) = 96.9%) for ssp. minus. RNA sequencing based gene annotation identified 27,243 genes on the ssp. vitis-idaea assembly, and transposable element detection methods found that 45.82% of the genome was repeats. Phylogenetic analysis confirmed that lingonberry is most closely related to bilberry and is more closely related to blueberries than cranberries. Estimates of past effective population size suggested a continuous decline over the past 1-3 MYA, possibly due to the impacts of repeated glacial cycles during Pleistocene leading to frequent population fragmentation. The genomic resource created in this study can be used to identify industry relevant genes (e.g., flavonoid genes), infer phylogeny, and call sequence-level variants (e.g., SNPs) in future research.
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    Post-transcriptional regulation of Vsx1 in the developing mouse retina
    (2023-08-15) Anderson, Christopher; Chow, Robert L.
    Post-transcriptional regulation encompasses many different mechanisms that impact mRNA stability and mRNA translation. It is one of several ways to fine tune the spatial and temporal components of gene expression necessary for proper development. I am studying post-transcriptional regulation in the mouse retina, which is an ideal experimental model for central nervous system development due to its simple anatomy, cellular organization, and ease with which it can be manipulated experimentally. My work is focused on Vsx1, a gene that is necessary for the terminal differentiation of a subset of retinal bipolar cells. VSX1 protein is first detected in the mouse retina exclusively in newly born bipolar cells between postnatal day 4 (P4) and P5, while its mRNA is first detectable three days earlier at P2. To determine whether the Vsx1 3’ untranslated region (UTR) mediates this apparent post-transcriptional regulation, an in vivo, dual reporter assay was developed which utilized the expression of an mCherry reporter carrying the Vsx1 3’UTR and a control GFP reporter for within-cell normalization. Vsx1 3’UTR-mediated repression of the mCherry reporter protein was observed in presumptive retinal progenitor cells electroporated at P0. Bioinformatic analysis of the Vsx1 3’UTR identified a 206 nt conserved region which contains microRNA recognition elements (MREs) for miRNAs known to be expressed in the developing retina, along with adenosine-uracil rich elements (AREs) that act as binding sites for various adenosine-uracil rich element binding proteins. Deletion of the conserved region from the Vsx1 3’UTR derepressed mCherry reporter expression suggesting that it mediates Vsx1 post-transcriptional repression. In addition, the 206 nt conserved region alone is sufficient to drive partial repression of mCherry suggesting it needs other regions of the 3’UTR for full repression. Mutations in the MREs for miR-17~92 and the AREs de-repressed mCherry expression suggesting these elements play a role in the post-transcriptional regulation of Vsx1. Using a real-time polymerase chain reaction approach and immunolabelling I detected precocious expression of gal mRNA in mice which have the LacZ gene (utilizing an SV40 poly-adenylation sequence) inserted into the Vsx1 locus. This suggested that Vsx1 mRNA is being post-transcriptionally regulated in post-natal development, with no evidence of precocious protein expression. Mice that contained a Vsx1-CreERT2 transgene (CreERT2 inserted into the Vsx1 locus) were crossed with the mice containing the Ai14 locus (Rosa26 locus with loxp sites and tdTomato (TdT) downstream) that allowed for TdT expression in cells that endogenously express Vsx1. TdT expression was restricted to post-mitotic bipolar cells and amacrine cells and was determined to be expressed in postnatal amacrine cell subtypes until ~P6-P8. Altogether, my results indicate that Vsx1 mRNA is under post-transcriptional regulation in retinal progenitors and bipolar cell precursor cells in early postnatal development through miRNA – MRE and ARE – ARE binding protein interactions within its 3’UTR. Additionally, Vsx1 mRNA is under regulation in newly born amacrine cells, and its expression appears to be regulated until late postnatal development.
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    Ectomycorrhizal fungal endemism and rainforest nutrition in Pacific Northeast
    (2023-05-01) McPolin, Marie Claire; Hawkins, Barbara J.
    Considerable research exists on how niche processes and spatial trade-offs structure the species richness observed in ectomycorrhizal fungal (EMF) communities, but little attention has been paid to how this might relate to the high levels of endemism reported in these communities. As endemism is typically associated with habitat specialization, I anticipated that EMF species endemic to the distinct high available nitrogen (N), low available phosphorous (P) soils of the Pacific Northeastern (PNE) temperate rainforests would display greater macronutrient concentration, indicative of superior nutrient exploitation. I measured both the sporocarp nutrition and the root tip abundance of EMF species on a mature forest (CWHVm biogeoclimatic zone) of Sitka spruce and western hemlock and determined fungal endemism using UNITE database. Endemic species, representing close to 50% of species found on root tips, had significantly higher sporocarp N, K and Mg concentrations than cosmopolitan species, but comparable P levels. Sporocarp N and P were strongly correlated, and species with higher N levels showed an increasing N:P ratio, supporting existing evidence for the N cost of organic P-acquiring enzymes. Endemics were more likely to occur on western hemlock (a coastally restricted genus) plots than Sitka spruce (a circumpolar genus) plots and became more frequent on root tips as inorganic P levels in the soil decreased. Endemics represented a diverse group, with moderate but non-random dispersion across the phylogeny; The Inocybaceae family were predominantly endemic, while Cortinariaceae was largely cosmopolitan, highlighting some role of phylogenetic niche conservatism in certain lineages, but not as an overall pattern. I conclude that endemic EMF account for a significant portion of fungi most well adapted to PNE coastal soils, have preference for coastally restricted Tsuga, and are expected to provide superior N nutrition to their tree hosts.
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    Orientation selectivity in the population of ON-OFF direction-selective ganglion cells in the mouse retina
    (2023-04-28) Ravi Chander, Prathyusha; Awatramani, Gautam
    In the mammalian retina, the orientation-selective (OS) and direction-selective (DS) information are generally thought to be relayed to higher visual centers via distinct ganglion cell types. Contrary to this notion, here I report that classic ON-OFF direction-selective ganglion cells (DSGCs) that are known to encode the four cardinal directions, also encode orientation of static stimuli. The DSGC’s preferred orientations was always orthogonal to its preferred-null axis defined by moving stimuli. To evaluate the synaptic mechanisms underlying orientation selectivity a combination of electrophysiological, optogenetic, and gene knock-out techniques were used to assess the functional properties of all four types of ON-OFF DSGCs. Cumulative results from multiple approaches revealed that the glutamate input to all four types of DSGCs was tuned to the vertical axis. This relies on signals from a specific presynaptic source (the bipolar cell type 5A; BC5A), which appear to be electrically coupled to vertically oriented processes of wide-field amacrine cells. By contrast, the GABAergic inhibition mediated largely by starburst amacrine cells was tuned either along the horizontal or vertical axis, consistent with their well-defined asymmetric wiring pattern. Thus, distinct combinations of inhibition and excitation underlie orientation selectivity in the nasal/temporal and dorsal/ventral coding DSGC populations, only the latter critically relying on the starbursts. Together, my work provides novel insights into how feature selectivity emerges in the hierarchical network in the retina.
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    Fine-scale Prey and Foraging Behaviour of Humpback Whales in Southern British Columbia
    (2022-12-23) Reidy, Rhonda; Juanes, Francis; Cowen, Laura Louise Elizabeth
    The North Pacific humpback whale Megaptera novaeangliae is showing strong recovery from commercial over-exploitation, and is recolonizing traditional feeding areas in Pacific Canadian waters now occupied by shipping lanes and high concentrations of people in coastal regions. Meeting and maintaining recovery conservation objectives, therefore, will require accurate information on important prey species and whale foraging behaviour. My dissertation evaluates three data-intensive sampling tools for collecting subsurface information in humpback whale-selected feeding areas in southern British Columbia. Systematic, small-vessel surveys, using active acoustics, enabled comparison of the spatial distribution of prey proximal to, and in areas without, humpback whales off Vancouver Island, British Columbia (BC). My objective was to use active acoustics to broadly separate fish from zooplankton in areas used by humpback whales in southern BC, and to determine if one of these functional prey groups is associated with whale presence more than the other. Surveyed areas in which humpback whales were present were associated with higher zooplankton than fish biomass. I recommend using 38, 125, and 200 kHz frequencies with concurrent net-sampling to improve acoustical classification of co-existing taxa in whale feeding areas. Kinematic diversity in rorqual feeding is manifest over space and time because different prey types are encountered by individual whales. I use a CATS Diary suction cup tag attached to a humpback whale in Juan de Fuca Strait, concurrently with acoustic prey mapping, to describe the prey and estimate the feeding performance of the whale. The tag sensor data suggested that the whale was feeding on krill, while the prey data determined that the whale was in fact feeding on fish, likely walleye pollock, using a “krill-like” lunge-feeding behaviour. A faecal sample from the whale revealed high DNA read abundance and bones from walleye pollock. Depending on the nature of the prey, inferences based solely on whale tag data may be vulnerable to incorrect assumptions about the prey type being targeted. Prey species actually ingested by rorquals are extremely difficult to determine, and therefore a large gap persists in understanding rorqual feeding ecology. I use molecular and visual analyses of faecal samples to illustrate a complementary approach to humpback whale diet analysis, with each method providing unique insight into prey diversity. DNA-metabarcoding of 14 humpback whale faecal samples revealed a fine-scale diversity of prey species detected in the faeces, but DNA detections from exogenous contaminants and secondary predation may influence the results. Accumulating evidence indicates that humpback whales are highly adaptable predators, which often associate with complex prey communities. Data collection must therefore be adaptable to changing spatial and temporal dimensions and include most of the water column. In my experience and opinion, calibrated multifrequency echosounders on small vessels enables data collection on a regular basis, and is among the most promising quantitative tool to meet the challenges of subsurface prey assessments for large whales in BC.
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    Unexpected Connections: Salicinoid Biosynthesis in Poplar
    (2022-12-22) Gordon, Harley Oliver William; Constabel, Carsten Peter
    Populus is a genus distributed across the northern hemisphere. Poplars (Salicaceae) are subject to stresses in their environment such as herbivory, drought, and fire. These perennial hardwoods produce abundant phenylpropanoid derived anti-herbivory molecules called salicinoids. Understanding salicinoid function and biosynthesis is crucial for understanding the chemical ecology, carbon balance, and adaptability of poplars to changing ecosystems. The full biosynthetic pathway of salicinoids is unknown; however, recent progress has identified a biosynthetic gene for salicinoids. The gene is a UDP-dependent glycosyl transferase called UGT71L1. Using CRISPR/Cas9 genome editing, UGT71L1 was disrupted in the hybrid poplar Populus tremula x Populus alba. Through metabolomic, transcriptomic, and biochemical techniques the co-dependent nature of growth, defence and salicinoid biosynthesis in poplars was explored. Following the elimination of UGT71L1, the exogenous application of deuterated benzenoids and mass spectroscopic analysis was used to examine biochemical connections across metabolic pathways. A carbon limited growth experiment was used to assess the capacity for glucosylated salicinoids to contribute to carbon reserves in resprouting trees. In addition, a second glycosyltransferase gene, UGT78M1, was disrupted in genome-edited poplars. Interruption of UGT71L1 disrupted salicinoid biosynthesis. UGT71L1 knockout plants had small crinkled leaves, reduced growth, and were preferred by insect herbivores. Growth impacts were caused by the abundance of salicylic acid, which increased in concentration following salicinoid biosynthesis interruption. Furthermore, we determined that benzyl benzoate is a precursor to salicortin biosynthesis. Salicinoids are also an inaccessible carbon sink in poplar that cannot be remobilized during carbon starvation. The hypothesized salicinoid biosynthetic gene UGT78M1 does not contribute to salicinoid biosynthesis; however, UGT78M1 is crucial for salicyl benzoate glucoside homeostasis. This dissertation highlights the small molecule trichotomy of plant biochemistry and identifies connections between specialized metabolites, phytohormones, and primary metabolites.
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    A deep dive into the sablefish (Anoplopoma fimbria) opsin repertoire: insight into melanopsin expression, localization and function in an unlikely demersal model.
    (2022-09-29) Barnes, Hayley; Taylor, John
    Light regulates many biological processes through light-sensitive proteins called opsins. Opsins are involved in vision, but they are also expressed in extraretinal tissue, where their roles are far less clear. Fish have large opsin repertoires, derived from a long history of gene duplication and divergence, making them useful models to study opsin diversity and function. I introduce the deep-sea sablefish (Anoplopoma fimbria) as a model for opsin research for three main reasons: i) the availability of a draft genome and transcriptome, simplifying the characterization of this species’ opsin repertoire, ii) the proximity of the only sablefish aquaculture facility in the world, providing exclusive access to a large number of individuals at all developmental stages, iii) the observation that sablefish occupy very different light environments during the course of development, ranging from well-lit shallow waters to the aphotic zone, which provides a light environment context for opsin gene expression data. My survey of the genome showed that sablefish have 36 distinct opsin genes (7 visual and 29 non-visual), even though they spend most of their lives in the dark. The sablefish opsin sequences and repertoire are similar to those of other teleost fish. To test the hypothesis that the sablefish opsin repertoire is being expressed/transcribed during the comparatively brief period of time when this species is exposed to light (the free-swimming larval stage through to the juvenile stage), I quantified the expression of five paralogous genes from a well-studied non-visual opsin family (OPN4’s) in the brain across life stages. Data show statistically stable expression of Opn4m1 and Opn4m3 among life stages, a rough association of Opn4x1 and Opn4m2 expression with age and light environment, and little-to-no expression of Opn4x2. I localized proteins encoded by the most highly expressed class of OPN4 genes in the brain, the Opn4m genes, to the surface of the optic tectum just below a cranial ‘window’; a zone that has been shown to express dozens of opsins in zebrafish (a distant relative, with their ancestor diverging more than 230 million years ago). Thus, in some cases, expression appears to be correlated with light exposure not only temporally, but also spatially. By studying non-visual opsins in sablefish, I have challenged and broadened the current understanding of opsin evolution and function in fish and provided the foundation for future studies to test brain regions for light-sensitivity, perform opsin gene knock-outs, and explore potential light-independent processes.
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    Assessing the contribution of Red Alder (Alnus rubra) to forest stand nitrogen budgets
    (2022-09-29) Nehring, Lise; Hawkins, Barbara J.
    Red Alder (Alnus rubra) is a native coastal hardwood in British Columbia and has evolved a symbiotic relationship with the nitrogen-fixing actinomycete, Frankia. This research uses δ15N signatures in soils, wood and litter to assess the contribution of nitrogen-fixing Red Alder to the components of stand nitrogen budgets. The stands used in this study are part of the B.C. Ministry of Forests’ long-term Experimental Project 1121.01 which examines the interactions between conifers and Red Alder. Planted in 1994, the Holt Creek site contains stands of Douglas-fir and Red Alder in five proportions (Red Alder: Douglas-fir proportions: 100/0, 50/50, 25/75, 11/89, 0/100). Increment cores from 5 trees per species per plot were taken along with soil and litter samples and analyzed for essential mineral elements and δ15N. I hypothesized that Red Alder would enhance soil nitrogen stocks and elevate δ15N signatures and that these changes would be observable in the δ15N signature of the tree rings of both species. Forest floor soil under Red Alder in the 100/0 plot was enriched in total nitrogen, and δ15N was elevated. This was due to the addition of nitrogen-rich litter, like followed by nitrogen discrimination in the forest floor during the process of nitrate leaching or denitrification. The litter of the two species did not differ in δ15N. The effect of forest floor nitrogen enrichment was visible in the tree rings of Douglas-fir in the 50/50 stand confirming that the effect of fixed-nitrogen can be observed in non-fixing species. Red Alder tree ring δ15N exhibited an unexpected non-linear relationship with time that could be due to reduced nitrogen fixation associated with declining tree vigour or negative feedback from low soil pH. This research provides insight into nitrogen fixation by Red Alder over time and its influence on pure and mixed stand nitrogen budgets.
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    The mechanism underlying bipolar cell subtype specification
    (2022-09-07) Ruiz de Chavez Ginzo, Alberto; Chow, Robert Lewis
    The mammalian central nervous system (CNS) has a high degree of complexity and cell type diversity that enables sophisticated processing of sensory information, circuit formation, and behaviour. While much is known about the patterning and specification of the major neuronal classes in the CNS, through processes such as morphogen gradient signaling and transcription factor combinatorial coding, much less is known about how subtypes within each cell class are specified. Bipolar cells are one of the main classes of interneurons in the vertebrate retina and consist of fifteen different subtypes based on their physiological function, morphology, and unique gene expression. The cellular mechanisms behind the specification of these subtypes are not fully known. In this thesis, I examine these mechanisms by investigating the role of extrinsic and intrinsic factors on the specification and differentiation of bipolar cell subtypes. We hypothesize that the specification of bipolar cell subtypes occurs in a multi-step manner and is dependent on non-cell autonomous (extrinsic) signals. To test this hypothesis, I conducted a series of experiments on the early postnatal mouse retina, which is the period when bipolar cells are generated. First, I examined whether bipolar cell marker onset was temporally ordered as would be predicted in a multi-step model. Postnatal day 3 (P3) mice were injected with EdU (5-ethynyl-2’-deoxyuridine), a thymidine analog that labels proliferating cells and then dissociated and fixed the retinal cells 24-120 hours after injection. My results show that Vsx2-5.3-PRE-Cre, a marker of pan-bipolar cells specification, is first detected 36 hrs after cell cycle exit, whereas specialized bipolar subtype-specific markers are expressed 48-60 hrs post-EdU injection. This observation is consistent with the idea that bipolar cells develop in a stepwise manner, first as an unspecified, pan-bipolar cell intermediate and then into one of the 15 subtypes. To further investigate this possibility, I developed a novel dissociated retinal culture assay that enabled me to accurately track retinal progenitor cells and postmitotic precursor cells and determine the requirement of cell autonomous and non-cell autonomous mechanisms during bipolar cell subtype specification. This assay involves culturing dissociated retinal cells from P3 EdU-injected mice at high density (abundant cell contact) or low density (scarce cell contact) at various timepoints, thereby allowing me to probe the role of these mechanisms in RPCs, early postmitotic cells, and late postmitotic cells. My findings revealed the first 24-48 hrs post cell cycle exit to be a critical, cell contact-dependent period for the specification of bipolar cell subtypes. This assay also allowed us to test the effect of blocking or activating the Notch and the Sonic Hedgehog (Shh) signal transduction pathways by using pharmacological compounds and recombinant ligands. Co-activation of Notch and Shh pathways increased the specification of Vsx1+ subtypes suggesting they play a role in their specification. Altogether, our results suggest that bipolar cell subtype specification follows a multi-step model, through an undifferentiated bipolar cell intermediate, and that cell contact plays a role in the specification mechanisms of bipolar cell subtype development. This is a novel finding that provides insight into the mechanisms underlying retinal neuronal subtype development and possibly in other neuronal cell types throughout the CNS.
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    Nearshore habitat use, estuarine residency, and conservation priorities for Pacific salmon in the Fraser River, British Columbia
    (2022-05-02) Chalifour, Lia; Baum, Julia Kathleen
    Cumulative effects from multiple anthropogenic stressors over the past three centuries have severely impacted estuarine and coastal habitats, with cascading effects on the species that rely upon them. Pacific salmon (Oncorhynchus sp.) are migratory species that use estuaries as juveniles and as adults and deliver critical nutrients to coastal ecosystems as they move between fresh and marine waters. Many once abundant salmon populations have been extirpated or are in severe decline relative to historic levels, yet the strength of the relationship between habitat loss and population productivity has been challenged. In this dissertation, I applied field studies, otolith analyses, and conservation decision science tools to investigate the relative importance of estuarine habitat to salmon populations, with the aim of advancing effective management solutions for these species and their habitats. First, I conducted a two-year field survey of fish communities in the Fraser River estuary, British Columbia, Canada comparing the species richness and relative catch amongst three distinct habitats. I found that this impacted estuary still supported a rich community of migratory marine and anadromous fishes, as well as resident estuarine fish species. Each habitat supported some unique fish assemblages, with eelgrass supporting the highest catch and diversity of fishes overall but brackish marsh supporting the highest and most consistent catch of salmonids. Next, I used otolith analyses to quantify the residency and growth of juvenile Chinook salmon in the estuary. I found that for one of the only two remaining Chinook salmon stocks abundant enough to still support limited harvest in the Fraser River, the estuary provides vital rearing habitat, with juveniles residing in the estuary for an average of 6 weeks, during which time they had mean daily growth rates of 0.57 mm fork length, approximating growth in healthier estuarine systems. The use of these habitats by juvenile Chinook salmon had not been quantified previously, so these findings directly inform management of this population, which was recently designated as Threatened by the Committee on the Status of Endangered Wildlife in Canada. Finally, I applied Priority Threat Management, a conservation decision science framework, to predict the future status of Pacific salmon in the lower Fraser River and identify the most cost-effective conservation solutions out of a suite of alternative management strategies. On our current trajectory none of these populations were predicted to be assessed as ‘green’ or healthy status at the end of 25 years. In contrast, implementation of broad scale habitat restoration, protection, and watershed management could considerably improve the viability of the lower Fraser to support these salmon, such that many (14/19) of these populations would have a >50% likelihood of being assessed as healthy. Together, this research provides novel evidence of active and selective use of estuarine habitats by juvenile salmon, reliance on estuarine habitat for early marine growth by juvenile Chinook salmon, and a direct link between habitat health and population status for lower Fraser River salmon populations.
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    CRISPR/Cas9 mutation of MYB134 and MYB115 to study regulation and functions of proanthocyanidins in poplar roots
    (2022-05-02) Liu, Yalin; Constabel, Carsten Peter
    Secondary metabolites play important roles in tree defense. Proanthocyanidins (PAs), one of the most common secondary metabolites, are widely distributed in trees and woody plants, and are abundant in poplar. In my research, molecular biology and biochemistry techniques were used to investigate the function of two important transcription factors, MYB115 and MYB134, in regulating the PA pathway in hybrid poplars. The importance of these transcription factors in regulating PA synthesis in leaves has recently emerged, but their roles in roots are not known. MYB134- and MYB115-overexpressing transgenic poplars showed a strong high-PA phenotype in leaves, but how these two regulators interact in vivo is still a mystery. This research aims to test the function of both MYBs in the regulation of PAs in poplar roots, and to explore the antimicrobial functions of root PAs. Both alleles of the MYB genes were sequenced in wild type poplars to design gRNAs for creating transgenic poplars with knocked-out (KO) MYB115 and MYB134 using the CRISPR Cas9 system. Both hairy root and whole plant transgenics with respective single- and double knock-outs were generated. Chemical and genetic characterization of both mutant types showed reduced PA content and down-regulated flavonoid genes in leaves. In poplar roots, only double-KOs showed a significant change in PA and salicinoid metabolism. These results indicated that the regulatory pathways for PA biosynthesis may differ in poplar leaves and roots. Significant PA concentrations remained in double-KO plants, suggesting other transcription factors for PA regulation are active. Because poplars accumulate large amounts of PAs in roots, potential functions of root tannins were also investigated. Antimicrobial activity of PAs was tested by disc inhibition assay in vitro and mycorrhizal co-culture sandwich assay in vivo. Pure PAs showed no inhibition towards the pathogenic fungi Armillaria ostoyae and A. sinapina but displayed slight inhibition to the mycorrhiza fungus Laccaria bicolor. These results provide preliminary insight into the functions of PAs in roots.
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    Holocene fire frequency and links to climate and vegetation history on Pender Island, British Columbia, Canada
    (2022-04-28) Giuliano, Camille; Lacourse, Terri
    Contiguous macroscopic charcoal analyses were performed on a 9.03 m long lake sediment core from Roe Lake on Pender Island in the Gulf Islands National Park Reserve of British Columbia, Canada to reconstruct the island’s fire history over the last 10,000 years. Charcoal particles >150μm were counted to quantify charcoal concentrations, charcoal accumulation rates and mean fire return intervals. Results show that the early Holocene was characterized by high charcoal accumulation rates and frequent low-severity fire with a mean fire return interval of 100 ± 29 years. Forests at the time were dominated by Pseudotsuga menziesii with an open canopy and fern taxa, particularly Pteridium aquilinum, being common in the understorey. This open vegetation, coupled with warm and dry summer climate, likely created conditions conducive to this fire regime. Charcoal accumulation rates decreased in the middle to late Holocene, and fire frequency decreased, resulting in a mean fire return interval of 167 ± 43 years. Climate cooled and moistened along with a decrease in seasonality during this time and the canopy closed, establishing closed-canopy Pseudotsuga menziesii forests. Climate appears to be the primary factor controlling fire regimes near Roe Lake for most of the Holocene. At times, shifts in the fire regime cannot be explained by changes in climate. Fire frequency increased between 7000-5000 cal yr BP, coincident with a peak in Quercus garryana pollen, despite cooling and moistening climate. Fire likely maintained patches of Q. garryana savanna during this time. Fire again became more common contrary to trends in climate after ~2500 cal yr BP. This late Holocene increase in fire is also seen elsewhere in the Pacific Northwest and may be a reflection of increased climate variability due to more frequent El Niño events or an increase in human-lit fires. Indigenous populations on southern Vancouver Island commonly used fire as a resource management tool and it is likely that people on Pender Island did as well. As fire management practices shift from fire suppression to more sustainable practices, this study offers the Gulf Islands National Park Reserve important baseline information on the area’s natural fire regime to help guide future conservation efforts.
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    Intertidal resource cultivation over millennia structures coastal biodiversity
    (2021-12-22) Cox, Kieran D.; Juanes, Francis; Dudas, Sarah Elizabeth
    Cultivation of marine ecosystems began in the early Holocene and has contributed vital resources to humans over millennia. Several more recent cultivation practices, however, erode biodiversity. Emerging lines of evidence indicate that certain resource management practices may promote favourable ecological conditions. Here, I use the co-occurrence of 24 First Nations clam gardens, shellfish aquaculture farms, and unmodified clam beaches to test several hypotheses concerning the ecological implications of managing intertidal bivalve populations. To so do, in 2015 and 2016, I surveyed epifaunal (surface) and bivalve communities and quantified each intertidal sites’ abiotic conditions, including sediment characteristics and substrate composition. In 2017, I generated three-dimensional models of each site using structure-from-motion photogrammetry and measured several aspects of habitat complexity. Statistical analyses use a combination of non-parametric multivariate statistics, multivariate regression trees, and random forests to quantify the extent to which the intertidal resource cultivation structures nearshore biodiversity Chapter 1 outlines a brief history of humanity's use of marine resources, the transition from extracting to cultivating aquatic taxa, and the emergences of the northeast Pacific’s most prevalent shellfish cultivation practices: clam gardens and shellfish farms. Chapter 2 evaluates the ability of epifaunal community assessment methods to capture species diversity by conducting a paired field experiment using four assessment methods: photo-quadrat, point-intercept, random subsampling, and full-quadrat assessments. Conducting each method concurrently within multiple intertidal sites allowed me to quantify the implications of varying sampling areas, subsampling, and photo surveys on detecting species diversity, abundance, and sample- and coverage-based biodiversity metrics. Species richness, density, and sample-based rarefaction varied between methods, despite assessments occurring at the same locations, with photo-quadrats detecting the lowest estimates and full-quadrat assessments the highest. Abundance estimates were consistent among methods, supporting the use of extrapolation. Coverage-based rarefaction and extrapolation curves confirmed that these dissimilarities were due to differences between the methods, not the sample completeness. The top-performing method, random subsampling, was used to conduct Chapter 4’s surveys. Chapter 3 examines the connection between shellfish biomass and the ecological conditions clam garden and shellfish farms foster. First, I established the methodological implications of varying sediment volume on the detection of bivalve diversity, abundance, shell length, and sample- and coverage-based biodiversity metrics. Similar to Chapter 2, this examination identified the most suitable method, which I used during the 2015 and 2016 bivalve surveys. The analyses quantified several interactions between each sites’ abiotic conditions and biological communities including, the influence of substrate composition, sediment characteristics, and physical complexity on bivalve communities, and if bivalve richness and habitat complexity facilitates increases in bivalve biomass. Chapter 4 quantifies the extent to which managing intertidal bivalves enhance habitat complexity, fostering increased diversity in the epifaunal communities. This chapter combines 2015, 2016, and 2017 surveys of the sites' epifaunal communities and habitat complexity metrics, including fractal dimension at four-resolutions and linear rugosity. Clam gardens enhance fine- and broad-scale complexity, while shellfish farms primarily increase fine-scale complexity, allowing for insights into parallel and divergent community responses. Chapter 5 presents an overview of shellfish as a marine subsidy to coastal terrestrial ecosystems along the Pacific coast of North America. I identified the vectors that transport shellfish-derived nutrients into coastal terrestrial environments, including birds, mammals, and over 13,000 years of marine resource use by local people. I also examined the abundance of shellfish-derived nutrients transported, the prolonged persistence of shellfish subsidies once deposited within terrestrial ecosystems, and the ecological implications for recipient ecosystems. Chapter 6 contextualizes the preceding chapters relative to the broader literature. The objective is to provide insight into how multiple shellfish cultivation systems influence biological communities, how ecological mechanisms facilitate biotic responses, and summarize the implications for conservation planning, Indigenous resource sovereignty, and biodiversity preservation. It also explores future work, specifically the need to support efforts that pair Indigenous knowledge, and ways of knowing with Western scientific insights to address conservation challenges.
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    Impacts of local and global stressors on coral biodiversity
    (2021-08-31) Maucieri, Dominique; Baum, Julia Kathleen
    Global biodiversity losses are being driven by human actions, and coral reef communities are not immune. Local anthropogenic stress and global climate change are rapidly changing coral reefs, through coral bleaching and mortality. How these stressors impact the biodiversity and community structure of corals on tropical reefs will not only affect the communities of fish and invertebrates that rely on coral reefs, but they could have lasting impacts on ecosystem functioning. The record-breaking marine heatwave caused by the 2015/2016 El Niño was superimposed on a strong local human disturbance gradient on Kiritimati, Kiribati, allowing for the investigation of how these combined disturbances affect coral communities. In Chapter 2, I investigated how soft coral cover varies with these disturbances and natural environmental factors, using benthic photoquadrats collected on Kiritimati’s forereefs from 2007 to 2019. Additionally, I conducted a literature review to establish what is already known about soft coral and disturbances, to compare Kiritimati data to that found in the literature. I show that soft corals are grossly understudied, with only a fifth (19/94) of coral studies presenting any results of heat stress effects on soft corals, and even fewer (5%) presenting taxonomic-specific results. On Kiritimati, prior to the 2015/2016 El Niño, soft corals were more common at sheltered sites with lower net primary productivity, but no effect of local disturbance was found. Soft corals were, however, highly vulnerable to heat stress, with a documented complete loss after the heatwave. I also show that soft coral skeletons persisted for years after the heatwave and provided substrate for hard coral recruitment. In Chapter 3, I examined how local and global stressors affected coral diversity, using community composition photoquadrat data collected from 2013 to 2017, and developed a conceptual framework for understanding effects of multiple stressors, when there are both discrete and continuous stressors. Coral alpha diversity (assessed as Hill diversity) exhibited a non-linear relationship with local anthropogenic stress, peaking at intermediate levels, and was negatively impacted by the marine heatwave, such that sites tended to decrease in both coral richness and evenness. Coral beta diversity (assessed as community composition turnover) was significantly impacted by both stressors, but sites exposed to higher levels of anthropogenic stress tended to experience less turnover during the heatwave. Explicitly considering the relationships between the two stressors, I found that it varied depending on the intensity of anthropogenic stress and the diversity metric (i.e., richness vs. composition) examined. For Hill-Richness, I found a tipping point at moderate levels of local anthropogenic stress, below which there was an additive response and above which the response tended towards synergy. In contrast, for Hill-Shannon and Hill-Simpson the responses were additive and there was an antagonistic effect between stressors for community composition. By using the frameworks outlined in this thesis for reporting changes to soft coral due to disturbances, and examining relationships between discrete and continuous stressors, we may better predict how reefs will look in the future and what actions will conserve and assist in the recovery of coral reef ecosystems.
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    Factors influencing the intriguing persistencce of a Wolbachia symbiont in spotted wing Drosophila
    (2021-05-31) McPherson, Audrey E.; Perlman, Steven John; Abram, Paul K.
    Wolbachia is a maternally inherited, endosymbiotic bacterium that infects at least 40% of terrestrial arthropods. As a facultative symbiont in the majority of its hosts, Wolbachia commonly act as a reproductive parasite; however, there are a number of Wolbachia strains that do not cause reproductive manipulations in their hosts and have no apparent fitness enhancement, yet are stably maintained in populations at low to intermediate frequencies. How these strains of Wolbachia persist in nature has been a long-standing question and is still unresolved. One explanation for the persistence of such strains is that they provide a context-dependent fitness advantage to their hosts. In this thesis, I investigate one such strain of Wolbachia, wSuz, which infects the agricultural pest, Drosophila suzukii, also known as spotted wing Drosophila. To explore the possibility that wSuz may be involved in pathogen protection, I screened wild flies for Wolbachia and two naturally occurring RNA viruses, Teise Virus and a recently discovered virus related to Motts Mill Virus. I did not find an association between Wolbachia and virus infection. Additionally, I designed an experiment to test whether Wolbachia increases host fitness at high larval densities. Intriguingly, although there was no effect of density, the frequency of Wolbachia infection changed dramatically in just one generation, but in opposite directions in replicate experiments that were performed a month apart. These results support the hypothesis that Wolbachia frequencies can change quickly across generations and provide some type of condition-dependent benefit. The maintenance of Wolbachia remains a mystery, but my study provides some exciting clues about what conditions may be playing a role.
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    Fitness and transmission of a selfish X chromosome in female Drosophila testacea
    (2021-05-26) Powell, Candice; Perlman, Steven John
    Selfish genetic elements break the rules of Mendelian inheritance to bias their transmission to following generations, often with negative fitness consequences. A striking example involves selfish X chromosomes that operate in males and interfere with the production of sperm that carry a Y chromosome. Only X chromosome-bearing sperm are produced, and this can result in extraordinary female-biased sex-ratio distortions. Most studies have focused on how selfish X chromosomes operate in and affect males, and there has been relatively little work on their consequences in females. In this thesis, I characterize fitness effects and transmission in females, in a recently discovered selfish X chromosome system in Drosophila testacea, a common woodland fly. I show that females with two copies of the selfish X chromosome have reduced fitness compared to females carrying zero, or one copy. Specifically, these females have a lower hatch rate and lifetime fecundity. Additionally, I show that heterozygous females are more likely to transmit the selfish X chromosome than the wildtype copy to their offspring. I observe this transmission bias in eggs, larvae, and adults, which suggests that the selfish X chromosome is preferentially segregating into the egg, rather than the polar bodies, during oogenesis. We believe this is the first documented case of a selfish X chromosome acting through both sexes. The negative fitness effects and the biased transmission in males and females will have important consequences on the evolutionary dynamics of the selfish X chromosome. In addition, the phenomenon of biased transmission in both sexes has the potential to yield interesting insights in the mechanism of meiotic drive.
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    Asymmetry in the lateral line of threespine stickleback, Gasterosteus aculeatus: ecology, evolution and behaviour
    (2021-05-18) Planidin, Nicholas; Reimchen, T. E.
    Behavioural asymmetry (laterality) is widespread among conspicuously bilaterally symmetrical organisms, playing a part in many aspects of life history from reproduction to feeding. Laterality is typically thought to occur due to morphological asymmetry within the brain, in which one hemisphere becomes specialized for a given task. However, the influence of sensory receptor asymmetry on the development of lateralized behaviour has undergone little investigation. The role of inconspicuous receptor asymmetry in behavioural laterality is particularly important, given the ubiquity of small deviations from symmetry. Here I have investigated morphological asymmetry in the lateral line, a series of mechanoreceptors called neuromasts that comprise one of the major sensory modalities of fishes. I examined a subset of the lateral line of 3,987 threespine stickleback from 64 populations from coastal British Columbia, characterizing neuromast count and asymmetry among habitats. Furthermore, I scored 657 stickleback from four experimental transplant populations relocated from stained lakes to unstained ponds, to determine whether or not neuromast count or asymmetry changes in a novel habitat. Neuromast count did not differ between oceanic and freshwater stickleback, or between sympatric lake-stream pairs but did differ among clarity regimes, ranging from a complete lack of neuromasts to a doubling of neuromasts compared to oceanic stickleback. Loss of neuromasts was associated with reduced light transmission, lower pH and a lack of piscivorous fishes. Stickleback with more lateral plates developed more neuromasts and males bore more neuromasts than females. One transplant pond underwent a 70% increase in neuromast count within just a couple of generations, whereas the other three transplant populations underwent more gradual change, suggesting both phenotypically plastic and genetic mechanisms underlying difference in neuromast counts among populations. Asymmetry was widespread among individuals, differing by up to seven neuromasts between the two sides on a single bony plate. However, no populations exhibited a strong directional bias. The degree of absolute asymmetry differed among clarity regimes, with stickleback in stained habitats having less asymmetry in their neuromasts counts. Asymmetry did not differ between oceanic and freshwater populations or sympatric lake-stream pairs. Males exhibited greater asymmetry than females, particularly in large-bodied populations. As with neuromast count, neuromast asymmetry quickly changed in some transplant populations and more gradually in others, increasing by up to 14% in just a couple of generations. To assess the functional consequences of my geographic survey, I experimentally tested 40 stickleback for their response to a simulated predator, localization of vibrations in the dark and rheotaxis. I compared behaviour and laterality to neuromast count and asymmetry measured by fluorescent microscopy. Stickleback with fewer neuromasts were more likely to respond to simulated predator strikes, but other non-lateralized behaviours were independent of neuromast count. The strongest laterality I observed was the ‘hugging’ of the arena wall with the right side 57% of the time, with laterality being present in other behaviours, albeit weakly. While some behaviours correlated with lateral line asymmetry, there was no consistent association between lateralized behaviour and asymmetry in the lateral line. I found that ecological factors such as predation landscape and photo-regime shape both mechanoreceptor count and asymmetry in the lateral line, with potential phenotypic plasticity in both traits. The lateral line’s role in response to a model predator and lateralized behaviour supports the influence of mechanosensory asymmetry in eco-evolutionary dynamics.