Theses (Biology)

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Theses from the Dept. of Biology.

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    Male fitness and the persistence of a selfish X chromosome in Drosophila testacea
    (2024) Radford, Julianne; Perlman, Steven John
    Selfish X chromosomes bias their own transmission during reproduction by interfering with Y-bearing sperm, a phenomenon best characterized in flies. Male flies carrying a selfish X chromosome produce almost exclusively female offspring, with potentially negative fitness consequences. Left unchecked, selfish X chromosomes may spread to fixation and result in extinction; therefore, one major question in the field is to understand how these chromosomes persist in nature. My research investigated one of two main hypotheses for selfish X persistence: the potential fitness consequences of harbouring the selfish element. The Palearctic woodland fly, Drosophila testacea, was recently found to carry an ancient selfish X chromosome, which serves as a great model for understanding how selfish X chromosomes persist over evolutionary time. My research investigated the contribution of male fitness effects in the persistence of this selfish X chromosome. I asked (1) if male age affects drive strength; (2) if males that carry a selfish X chromosome have reduced survival; (3) if female flies preferentially mate with wildtype or driver males; (4) if female mate choice interacts with male age; (5) if male mate success is affected by drive presence; and (6) if female propensity to remate is affected by male genotype. I found that male age does not alter strength of drive, driver males do not suffer reduced survival compared to wildtype males, females do not exhibit mate discrimination against driver males, and male mate success and female propensity to remate are not affected by male genotype. The present study suggests that while some male fitness effects play a role in drive persistence in D. testacea, male age, longevity, and mate success do not. Selfish X chromosomes are increasingly recognized as major players in organismal biology. Research on meiotic drive systems such as the selfish X chromosome in D. testacea will deepen our understanding of how gene drive persists over evolutionary time, and may provide insight on the prospect of synthetic gene drives.
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    Transparent Exopolymer Particles and Phytoplankton Nutrient Physiology in the North Pacific and Arctic Oceans
    (2024) Livingston, Michael; Varela, Diana Esther
    The export of organic carbon from the surface to the deep ocean is a key process that regulates the level of carbon dioxide (CO2) in the atmosphere, and is known as the biological carbon pump (BCP). The efficiency of the BCP is largely governed by biological influences in the upper layers of the water column, including the magnitude of primary production, and the sinking rate of particulate organic carbon (POC). However, these processes are still poorly understood in large parts of the ocean. A key factor affecting the BCP is the presence of transparent exopolymer particles (TEP) in the surface ocean. These particles originate from the exudation of organic exopolymeric substances by marine phytoplankton and form sticky carbon (C) gels that facilitate the aggregation of organic matter. These particles are less dense than seawater and affect the sinking of POC from the surface to the deep ocean, and therefore play an important role in the ocean’s C cycle. The overall objective of this thesis was to quantify key biological factors that affect surface ocean C cycling and to investigate the effects of variation in environmental conditions on the strengths of these factors. Over a 4-year study, I examined the concentrations and rates of production of TEP, phytoplankton nutrient physiology, and contributions of different sized phytoplankton to nutrient cycling in the Eastern Subarctic North Pacific (ESNP), and Pacific and Central Arctic regions. Measurements of TEP and a variety of biological and environmental variables were made across all regions in the ESNP and Arctic, and total and size-fractionated nutrient uptake rates of C, nitrate (NO3-) and silicic acid (Si(OH)4) were additionally measured across the ESNP. The concentrations of TEP were largely correlated with the amount of phytoplankton biomass and productivity, but also by environmental variables such as temperature, mixed layer depth, and nutrient concentrations. We used multivariate models derived from experimental observations measured during this study to produce novel estimates of surface TEP concentrations in the ESNP from 1998 to 2018. Model results show that the concentration of C in the form of TEP (TEP-C) in the surface of oceanic regions of the ESNP is estimated to be between 5-15μg C L-1 year-round. We further measured TEP concentrations relative to C fixation by phytoplankton and export potential (i.e., new production), providing the first quantitative comparison among these variables across large spatiotemporal gradients in the ESNP. Results show that low productivity regimes are characterized by higher concentrations of TEP-C (and higher estimates of TEP-C turnover) relative to C uptake and new production, which suggests these regions may undergo less efficient C export. This thesis presents the first field-based connection between excess C consumption by phytoplankton and TEP concentrations. Size-fractionated measurements reveal that small-sized phytoplankton (< 5 μm) are responsible for most of the nutrient uptake and TEP production in the oceanic and less-productive regions of the ESNP. The contribution of the small size-class to nutrient uptake rates did not appear to be influenced by environmental variations. Overall, this work provides new perspectives on surface ocean C cycling in the ESNP by shedding light on the main predictors of TEP, predicting TEP-C concentrations, and by relating TEP-C to total primary productivity and new production. It is also the first of its kind to measure total and size-fractionated Si uptake over large spatiotemporal scales.
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    Spatial and Seasonal Variability in Eukaryotic Phytoplankton Composition in the Arctic Ocean Revealed with Metabarcoding Analysis
    (2024) Crawford, Rebecca M. B.; Gawryluk, Ryan; Varela , Diana Esther
    Rapid environmental shifts in the Arctic Ocean have brought increased attention to its biodiversity. Environmental changes in the Bering and Chukchi Seas are especially concerning, as these regions are among the most productive in the world’s ocean, and their fisheries contribute to both local and global food systems. Predicting the impacts of climate change requires understanding biodiversity at the base of the food web, including eukaryotic phytoplankton, and the environmental drivers affecting them. This study evaluated the variability in the structure of phytoplankton communities at spatial and inter-annual scales (2021 and 2022) in the Bering and Chukchi Seas in the Pacific Arctic Region, and at a seasonal scale (September 2019 to July 2020) in Cambridge Bay in the Canadian Arctic using metabarcoding of the V4 18S rRNA gene. The community structure of eukaryotic phytoplankton exhibited both strong spatial and temporal trends, and was influenced by temperature and nutrient concentrations, as well as by sea ice extent and thickness in the Pacific Arctic Region. The chain-forming diatoms Chaetoceros and Thalassiosira were responsible for the high phytoplankton biomass of this region and were associated with temperatures below 1 degree C, and high nitrate concentrations, aligning with previous observations. The community composition also varied from year to year, with the picoprasinophyte, Micromonas, and the prymnesiophyte, Phaeocystis, contributing to a larger proportion of the overall phytoplankton community, particularly in surface waters, in 2022 compared with 2021. In addition, several potentially toxigenic algae, including Alexandrium, Aureoccocus and Pseudo-nitzschia were present in the Pacific Arctic Region and were generally associated with warmer temperatures. In Cambridge Bay, there were distinct seasonal changes in phytoplankton community structure, including a polar winter community dominated by dinoflagellates, a spring under ice community dominated by Phaeocystis, and a late spring/summer community composed largely of Micromonas with an increase in Chaetoceros by July with the onset of sea ice melt. Ultimately, findings from this work add to the growing body of knowledge on phytoplankton community structure in dynamic Arctic icescapes. Findings from metabarcoding analysis in the Pacific Arctic Region corroborate previous observations that diatoms are driving the phytoplankton biomass in July and provide a higher resolution of their taxonomic placements. The year-long investigation in Cambridge Bay indicates that continuous sampling strategies capture finer scale variation in phytoplankton community structure that was lacking from our Pacific Arctic interannual dataset. These types of sampling strategies should be incorporated to monitor other productive and vulnerable Arctic Regions.
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    The Role of Siliceous Rhizaria in the Silicon Cycle in the Northeast Subarctic Pacific Ocean
    (2024) Melville, Anna Olivia; Varela, Diana Esther
    Siliceous Rhizaria are zooplankton protists found in all ocean basins from the surface to the deep aphotic zone. Like diatoms, a group of phytoplankton with silica cell walls, siliceous Rhizaria utilize silicon (Si) to form their skeletons. While diatom production is known to strongly control the marine Si cycle, an important biogeochemical process that modulates global long-term stability, the role of Rhizaria on Si cycling is poorly understood. Therefore, the Si cycle is not fully constrained in the ocean. Furthermore, long-term shifts in community composition and seasonal changes to Si uptake rate (ρSi) in siliceous Rhizaria have never been studied. To address these knowledge gaps, field work was conducted in the Northeast Subarctic Pacific Ocean (NESP) and Pacific Arctic Region (PAR) between 2010-2023. Stations were sampled with bongo net tows which were vertically hauled from 250 m and 1200 m to the ocean surface. In this thesis, chapter 2 presents a historical time series (summers 2010-2020) of siliceous Rhizaria community composition in the NESP. The results showed that there were no yearly trends in species abundance, richness, or composition. Rhizaria abundance data from 2010-2020 were incorporated into a published dataset to update the estimated Si production by Rhizaria from 0.4 – 6.4 Tmol Si year-1 to 0.4 – 5.5 Tmol Si year-1 in global waters >40°N/S. Chapter 3 compares Rhizaria community composition in the NESP (March, July, and August 2022) and the PAR (July 2022). In the NESP, wintertime conditions promoted mixotrophic taxa of Rhizaria, while summer favoured phagotrophic Rhizaria. The sampling locations in the PAR displayed an absence of siliceous Rhizaria during this study. Chapter 4 presents the results from diatom and Rhizaria ρSi experiments conducted in August 2022 and May 2023 in the NESP. Overall, the contribution of siliceous Rhizaria to total daily ρSi was less than 0.12% and their contribution to total silica biomass was less than 4% confirming that diatoms are the main drivers of Si cycling in the NESP. However, it can be estimated that more than 38% of silica produced by Rhizaria could be buried along Line P suggesting that Rhizaria contribution to Si flux might be higher than expected. This work provides a novel contribution to our understanding of the ecology of Rhizaria in the NESP and PAR. Furthermore, this thesis provides new data which can be used to constrain the global Si cycle and predict how the Si cycle might shift with climate change.
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    Swiss Needle Cast and the Foliar Mycobiome of Coastal Douglas-fir (Pseudotsuga menziesii)
    (2024) Hayward, Emma; Ehlting, Jürgen
    Fungal pathogens of trees are an essential component of forest ecosystems as an ecological driver of diversity and natural selection; however, they can also have devastating effects. My research aims to understand better Nothophaeocryptopus gaeumannii, the causal agent of Swiss Needle Cast (SNC), a disease affecting Douglas-fir (Pseudotsuga menziesii). This pathogen infects the needles of its host, is associated with defoliation, and is endemic throughout the Douglas-fir range. In the Pacific Northwest of the US and Canada, a rise in the incidence and severity of SNC, which is thought to be linked to climatic changes and forestry practices, has been observed in the past few decades. There is a genetic component to SNC tolerance, enabling the selection of SNC resilient genotypes for reforestation. However, fungal load is not always correlated with needle loss, suggesting a more complex relationship. My thesis work collected SNC symptom severity data (measured as stomatal occlusion severity, needle loss severity, and relative growth rate) on a general combining ability population with a high incidence of SNC from the provincial Douglas-fir breeding program. Host phenotyping revealed a similar lack of correlation between disease signs and symptoms. Stomatal occlusion severity and relative growth rate were found to be low-moderately heritable traits within this breeding population (h2 = 0.19-0.34 and 0.28, respectively), whereas needle loss severity had low heritability (h2 = 0.11-0.12), and considerable variation within families and across locations in the plot. I hypothesize that this is due to this study site's endemic level of infection. SNC severity at endemic levels is likely not strong enough to drown out other biotic and abiotic factors contributing to needle loss. I propose stomatal occlusion incidence as a reasonable trait to breed for resistance under endemic-level conditions. The second part of my research investigated fungal community composition within Douglas-fir needles. Community analysis was done to determine if specific communities or taxa determine SNC symptom response or drive this lack of correlation between SNC signs and symptoms. I used ITS amplicon metagenomics supplemented by fungal culturing to characterize the foliar mycobiome of a subset of individuals from the same Douglas-fir breeding population. The Douglas-fir foliar mycobiome was diverse, including mostly rare OTUs and a few highly abundant OTUs, such as N. gaeumannii (64.5% of reads). N. gaeumannii was found to have a negative correlation with the second most abundant OTU, belonging to the genus Rhizosphaera, identifying it as a direct competitor with N. gaeumannii in the foliar environment for some host genotypes. The genus Rhizosphaera exhibited associations with particular families within the breeding population, suggesting some level of genetic determination. Foliar mycobiome community assemblages differed significantly among families but not between disease severity levels or groupings. The foliar mycobiome's composition, therefore, has implications for breeding programs in the context of synthetic communities ('SynComs') as seen in the context of white spruce and its pathogen spruce budworm. Despite no clear patterns in fungal community composition based on SNC symptom severity, fungal taxa that are known foliar pathogens, as well as taxa that are known to produce beneficial bio-active compounds (i.e., mycoparasitic taxa), were found within my dataset. This could explain some of the variation associated with needle loss, which was not correlated with the fungal load of N. gaeumannii at this site.
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    Impacts of marine shipping on the underwater soundscape and Chinook salmon behaviour
    (2024) Murchy, Kelsie; Juanes, Francis; Rountree, Rodney
    With the persistent growth of the shipping industry, the underwater soundscape across the globe has been changing. The prevalence of low-frequency noise from shipping activities has led to a noticeable increase in ambient underwater noise levels. Research to date has focused on large commercial vessels in motion, but commercial vessels are also anchoring in coastal ecosystems and there is a high presence of small commercial vessels that might be altering the marine soundscape. In this dissertation, these underrepresented anthropogenic noise sources are explored in Cowichan Bay, and Campbell River, British Columbia using underwater hydrophones. Results from these chapters (2 and 3) demonstrated significant changes to the underwater soundscape from anchored commercial bulk carriers and tugboats, highlighting the need to understand these noise sources further. Additionally, the growing body of literature on the impacts of anthropogenic noise on the underwater soundscape has generated concern about the impacts of the elevated noise on marine species. In the Northeast Pacific Ocean, many ecologically important species have been declining since the 1970s including Chinook salmon Oncorhynchus tshawytscha). Although behavioural changes including modified sound production behaviours have been documented in response to anthropogenic activities for some fishes, there has been little research on the impacts of shipping on Chinook salmon. Furthermore, Chinook salmon sound production has received little attention and only limited information on frequency and amplitude has been reported. To better understand the impacts of shipping on Chinook salmon vocal behaviour, understanding of salmon sound production, Chinook and other Pacific salmon, is required. Sound production in three species of Pacific salmon (Chinook, pink, O. gorbuscha and coho salmon, O. kisutch) was evaluated at Big Qualicum Hatchery (chapter 4). Chinook and coho salmon were found to produce air movement, hydrodynamic and pulse sounds, while pink salmon were shown to likely produce all these sounds. However, because pink salmon were always recorded in mixed schools with Chinook salmon, further work is required to validate their sound production. Additionally, the impacts of shipping on Chinook salmon movement and behaviour have never been evaluated, but vessel noise and other anthropogenic sources like pile driving have been shown to produce spatial displacement and behavioural changes in other salmonids. To begin to address this issue, changes in Chinook salmon behaviour in the presence of shipping noise were evaluated using acoustic tags in Cowichan Bay, British Columbia (chapter 5). Significant changes in depth for tagged salmon were observed with increased sound pressure levels and number of AIS-equipped vessels present. However, the influence of the number of AIS-equipped vessels present on the overall activity levels of Chinook did not appear linear and further work is needed to understand vessel noise effects on activity levels. This chapter represents the first study demonstrating the impacts of increased underwater noise levels on Chinook salmon. Collectively this dissertation highlights the impacts of anchored commercial vessels and tugboats on the underwater soundscape and the influence of shipping noise on Chinook salmon behaviour, demonstrating the importance of developing mitigation methods to help reduce the noise produced by shipping activities to protect these ecologically, and culturally important species.
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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.