School of BioSciences - Theses
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ItemDeciphering how CELLULOSE MICROTUBULE UNCOUPLING 1 (CMU1) controls pavement cell shape and root skewing via microtubule (MT) stability( 2023-07)The cell wall is a specialised structure that contributes not only to plant life but also to practical uses for our society. One of the most vital components in the cell wall is cellulose, synthesised by Cellulose Synthase (CESA) proteins that form large complexes that move along cortical microtubules (MTs) at the plasma membrane. The MT-steered trajectories of the CESAs are assisted by several MT-binding proteins, including Cellulose-Microtubule Uncoupling (CMU) 1 & 2, Companions of Cellulose synthase (CC) and Cellulose Synthase Interactive (CSI) proteins. Our lab has previously published studies on the in vivo interaction between CMU1 and MTs (Liu et al., 2016). However, many questions remained about the mechanism of interaction between CMU1 and MTs, and how these interactions contribute to plant growth and development. Chapter 1 offers a concise introduction to plant cell wall cellulose and the cytoskeleton, focusing specifically on the connection between the cellulose synthase complex and microtubules. There are four basic features that are presented, including a quick overview of the primary and secondary cell walls, the cytoskeleton in planta, the relationship between microtubule-associated proteins (MAPs) and cellulose, common phenotypes caused by microtubule disruption, and the goals of this research project are all topics that are covered in this thesis. In Chapter 2, the phenotypes of cmu mutants were investigated in vivo using a variety of techniques, including a root skewing assay, a root penetration experiment, root length and angle measurements, and cotyledon pavement cell analysis. Circularity and relative completeness of the pavement cells were significantly different between Col-0 and cmu mutants. In addition, the rate of microtubule polymerization was three times faster in the cmu1cmu2 mutants than in wild type. These findings imply that alterations in microtubules may cause pavement cells to become more round. In Chapter 3, two proteins, His-tag CMU1 full length (FL) and His-tag CMU1-FL-GFP, were expressed and purified. The His-CMU1-FL-GFP protein was discovered to attach to microtubules (MTs) in vitro. Using SDS-page and microscopy, I documented that the His-CMU1-FL protein caused MTs to bundle together. GFP served as a visual indicator to validate the binding and bundling of CMU1 to the MTs and a photobleaching experiment indicated that CMU1 proteins were capable of binding and bundling MTs. These results suggest that, when interacting with microtubules, CMU1 proteins are capable of both binding and bundling. In Chapter 4, to discover whether part of the CMU1 FL protein was capable of binding and bundling MTs, the variants of CMU1 FL, including the N terminus, Tetratricopeptide repeat (TPR) domain, and C terminus, with or without GFP and a His-tag were purified. The findings of the MT binding experiment indicated that the CMU1 TPR domain had the greatest binding capacity. The MT bundling test further showed that the TPR domain of CMU1 has greater bundling capacity than the N or C termini. In addition, the GFP version demonstrated that the TPR domain of CMU1 decorated MTs. This suggests that the TPR domain of CMU1 is primarily responsible for CMU1 MT binding and bundling in vitro. In Chapter 5, studies conducted in vitro and in vivo indicate that the MAP IQ-domain 2 (IQD2) protein interacts with CMU1. The phenotypes of the cmu1 and iqd2 mutants were examined in vivo using similar techniques as Chapter 2. Col-0 wild type, cmu mutants, and iqd2 mutants vary significantly in terms of the circularity and relative completeness of the pavement cells. Experiments conducted in vitro further showed that the presence of IQD2 promotes the binding and bundling of CMU1 to microtubules. In Chapter 6, the results of this study are summarized into a model of CMU1 and IQD2 function in microtubule regulation, cell wall synthesis, and plant growth and development. Some suggestions for future research possibilities are also proposed to test this model.
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ItemAnalysis on the role of Enteroblasts in epithelial homeostasis in the adult Drosophila midgut( 2023-06)Epithelial tissue functions as a protective barrier to the outside world, thus proper maintenance of epithelial homeostasis is essential for animal health and wellbeing. In response to damage or programmed cell death, the functional and structural integrity of an epithelium is maintained through cell turnover. In this project, we utilised the adult Drosophila midgut as a model to study homeostatic maintenance in an adult tissue, with particular focus on the role of the Enteroblast (EB), as it was reported to function as a reservoir for damage-induced tissue repair (Antonello et al., 2015). The process whereby EBs differentiate and give rise to new Enterocytes (ECs) is known as a Mesenchymal-Epithelial Transition (MET), and is an essential component for their proper incorporation into the existing epithelium. In this project, we built upon the current knowledge of the transcription factors and signalling pathways that drive EB MET and focused our investigations on the complex morphogenetic behaviour of EB cells. To this end, we have established an analysis pipeline that combines machine learning segmentation with cellular morphometric and spatial distributions analysis. Using this pipeline, we first uncovered a previously unreported bimodal distribution pattern for wildtype EBs which is associated with midgut regeneration status. In “quiescent” midguts, EBs were evenly spread out and new ECs were rare. In “regenerative” midguts, EBs were often found in groups, made up of EBs in various stages of their MET as well as new ECs, which we termed “MET regenerative clusters”. Next, using time-lapse imaging, we have established that EBs are motile in nature, and exhibit increased cytoplasmic movements when subjected to artificial tissue damage. With these findings, we proposed a working model linking midgut damage (i.e. EC loss) to regenerative cluster formation through the directional migration of nearby EBs. Furthermore, by applying the pipeline to EBs with target gene RNAi knockdowns, we identified Septate Junction (SJ) component Discs Large (dlg) to be a novel EB MET regulator. The presence of dlg-deficient cells disrupts normal midgut epithelial structure (i.e. a multilayered epithelium was observed), and such cells often exhibit polarity and junctional defects. Loss of dlg is also linked to apical membrane thinning and subsequent luminal extrusions. Signalling pathways linked to midgut stress responses were examined and we found the dlg-/- phenotype to be associated with JAK/STAT and JNK activation. Finally, to make further progress on our working model, we present the design of the TALGAR system (Targeted Apoptosis using LexA with GAL4 Analysis of Response) which allows for the induction of reproducible and temporally-regulated site-specific damage, as well as the ability to examine live EB cell dynamics and perform target gene manipulations. The TALGAR system was not completed due to time constraints but progress towards the goal was made and is described. Our work highlights the utility of the Drosophila model to further our understanding of epithelial homeostasis, and the importance of quiescent basal progenitors such as EBs in response to damage. The discovery of regenerative clusters opens up a new avenue for studying the triggers of regeneration, an area that has previously been difficult to address. As mechanisms of regeneration are conserved across evolution, this can pave the way for further research, and highlights the importance of MET in wounding and repair. It is also intriguing that the function of a junctional protein includes a role in the regulation of cell MET status. One of the many interesting questions left to be explored is whether other junctional proteins also regulate MET.
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ItemEcological genomics, behaviour and control of Aedes notoscriptus, a neglected disease transmitting mosquito from Australia( 2023-07)Mosquitoes are significant disease vectors, spreading pathogens that cause diseases like malaria, dengue, and Zika worldwide. In Australia, local mosquito species play a crucial role in disease transmission. Effective control strategies must consider the ecological characteristics of the target species and the surrounding environment. The first part of this thesis focused on studying the population structure and dispersal patterns of the Australian Backyard mosquito (Aedes notoscriptus) at fine and global scales. Notably, this species displays higher dispersal capabilities compared to the primary dengue-transmitting mosquito, Ae. aegypti. By combining mitochondrial DNA (mtDNA) and genome-wide single nucleotide polymorphisms (SNPs), I found evidence for cryptic mtDNA lineages of Ae. notoscriptus with varying geographical distributions. Additionally, SNP analysis identifies six distinct genomic groups that do not consistently align with the mtDNA lineages. The discordance between mtDNA and nuclear DNA is not attributed to Wolbachia infections but may be influenced by factors such as sex-specific asymmetries and human-assisted displacement. Furthermore, insights into invasive Ae. notoscriptus populations in New Zealand and the USA suggest their likely origin in Victoria, Australia. Additionally, I tested a novel mosquito trap design by evaluating its effectiveness through field and laboratory studies. The findings indicate that this system effectively reduces the number of Ae. notoscriptus eggs in the field. Furthermore, by reviewing existing literature, conducting human-baited field trials, and performing laboratory experiments, I found that the levels of male mosquito attraction to humans seems to be highly species specific, which is important for the effective and sustainable management of male mosquito releases to control population sizes and disease transmission.
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ItemRestoration of kelp beds on degraded temperate rocky reefs( 2023-04)The loss of kelp beds has been observed across the globe, often as a result of overgrazing by urchins, but also facilitated by other factors that reduce kelp bed resilience. While kelps are restricted to temperate waters, their local distribution and abundance are limited by the availability of suitable substrate, light conditions, and wave climate. Natural recovery of kelp beds can be limited by short dispersal distances for spores, and the stabilizing feedbacks of alternate reef states. The loss of ecosystem services provided by kelp beds and the lack of natural recovery is driving interest in restoration among land managers and researchers. Restoration attempts for these temperate rocky reef ecosystems to date have had mixed success, and lessons learned may be location specific, requiring further work to inform restoration efforts in different systems. The study area for this thesis is Port Phillip Bay, part of the Great Southern Reef (GSR) which stretches across southern Australia. Ecklonia radiata is the dominant kelp across the GSR, and Heliocidaris erythrogramma is the urchin of concern in Port Phillip Bay. Ecklonia radiata is a relatively small kelp with large morphological diversity across environmental gradients. Port Phillip Bay has a history of anthropogenic environmental disturbances including catchment-based pollution, introduction of invasive species, and removal of several large fishes through overfishing. In recent years, overgrazing by H. erythrogramma has been blamed for vast losses of E. radiata cover in the Bay. I begin this thesis by conducting a baseline survey of reef condition, urchin density, and urchin roe marketability across Port Phillip Bay. This allowed identification of specific management actions at a small (1 ha) spatial scale. I then explore three specific components of kelp restoration: 1) the use of kelp mimics to reduce sediment accumulation in the absence of an adult kelp bed; 2) the spatial scales over which natural recruitment can occur, and preferences for different substrate orientations and levels of shading; and 3) factors affecting success of transplanting juvenile kelp for restoration. The final chapter draws together the lessons from these four chapters in a management plan for kelp forests and urchins. Specific management actions were categorized at a 1 ha scale, allowing areas at highest risk to be identified, and actions to be prioritized based on management priorities and available resources. I observed divergent effects on sediment accumulation across sites, suggesting the artificial kelp behaves differently in slightly different environmental conditions. I observed most new kelp recruited within tens of meters from the nearest kelp bed, but densities and distances could not be explained by modelled local currents. I confirm the reduced sediment accumulation and low light conditions under a kelp canopy are associated with higher recruitment, and found vertical surfaces saw a tenfold increase in recruit density. However, I did not separate the effects, so the higher recruitment may have been due to one factor and in spite of the other. My transplant study demonstrated a potentially scalable restoration technique and highlighted the sensitivity of restoration spatial and temporal context. I also identified shallow exposed bommies as a potential source of juvenile sporophytes where high recruitment is coupled with very low likelihood of survival to maturity. Insights from this thesis can inform small scale interventions within the broader context of the Bay. My thesis identifies specific small scale kelp management actions appropriate within the broader context of Port Phillip Bay, but the lessons can be translated for application more broadly. My findings, some of which contrasts with previous studies, highlight the nuances in management of complex temperate reef ecosystems.
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ItemThe effects of chitin and chitosan on Cannabis sativa defense systems( 2023-05)Cannabis sativa is an emerging agricultural crop worldwide, with rapid expansion in both planting area and processing capacity in the past decade. The global market size of C. sativa production is projected to increase approximately 8–10% annually till 2030. More recently, to increase quality and consistency of C. sativa products, the plant cultivation has been moved indoors using hydroponic system. However, C. sativa is susceptible to several fungal diseases in both outdoor and indoor conditions, causing reduction in plant yield and product quality. Chitin and chitosan are natural elicitors, applied to promote plant growth and induce plant defense. Several studies have shown their capability to control plant diseases and suggested them as alternatives to chemical fungicides. However, their effects on C. sativa have not been investigated. In addition, only a few studies have tested chitin and chitosan effects on plant root systems in hydroponic settings. This is likely because root morphology and activities are difficult to monitor in natural conditions as roots are hidden underground. Therefore, in the first part of this project (Chapter 2), a new plant growth device, Root-TRAPR system, was developed to aid C. sativa root study. The system consists of two main parts, external structural frames and internal root growth chamber. The external frame is created using 3D printing techniques and the internal part is made of an acrylic sheet combined with a PDMS layer and a microscope slide. The system was applicable to handle C. sativa growth and to perform root morphology visualization and plant sample collections. Besides, experimental workflow, comprising plant growth and sample collection procedures and biochemical assays was successfully optimized and subsequently used for the main experiments. The effects of chitin and chitosan on C. sativa root systems were tested in hydroponic system using the Root-TRAPR device and the workflow developed earlier (Chapter 3). Chitosan was capable of inducing plant defense. Increased levels of defense hormones and enzyme activities were detected in root tissues. Defense proteins such as pathogenesis-related (PR) proteins, chitinases and peroxidases were, for the first time, identified in plant root exudate upon chitosan treatment. Chitosan was also found to inhibit root growth, but shoot growth was not interrupted. However, chitin had much lower effects than chitosan on both growth and defense responses of C. sativa. The subsequent experiment was set to further examine the protective effect of chitosan against fungal infection (Chapter 4). A fungal pathogen, Athelia rolfsii, causing southern blight disease, was selected for pathogenic study. The pathogen largely affected shoot growth, where the infected plants showed drooping and wilting leaves, but root development was less influenced. Chitosan priming was unable to protect plants from the disease, where all shoot growth parameters were not different from the infection condition. Nonetheless, proteomics analysis on root exudate showed a promising outcome. Chitosan-treated plants secreted defense enzymes, such as PR proteins, chitinases and peroxidases into exudate. While the pathogen secreted cell-wall degrading enzymes, indicating pathogenesis on plant root tissues. In chitosan-primed infected plants, enzyme and protein inhibitors were further detected, implying that plants counteract the pathogen attack via protein secretion. These protein inhibitors, secreted after pathogen infection, would be different types of plant defense proteins as compared to the proteins formerly secreted according to chitosan treatment. In Chapter 5, the findings of root growth-defense tradeoff as a consequence of chitosan treatment were compared with other studies to conclude the advantage and drawback of chitosan treatment on root growth and defense systems. The discussion also provides future directions for further research to fill the knowledge gap, which could be useful to promote chitosan application in practical agriculture, especially in hydroponic scenarios, to potentially replace chemical usages in plant disease management programs. In the last chapter (Chapter 6), information from all chapters above is summarized, showing key findings of this research and advising further study to advance the knowledge regarding chitosan applications in C. sativa cultivation to manage fungal diseases.
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ItemExpanding Our View of Algal-Bacterial Interactions Through Analysis of Molecular Data( 2023-04)Microalgae and bacteria have co-existed for millions of years and their interactions have significant bottom-up effects on ecosystem scale processes. Microalgae are highly diverse and innumerable, making it difficult to compile information on their interactions with equally ubiquitous and diverse bacteria. Furthermore, what we know so far about specific microalgal- bacterial interactions reflects only a small fraction of what potentially occurs in nature. In chapter 2, I performed a global screening for microalgae-bacteria associations using publicly available 16S rRNA metabarcoding data. The study aimed to discover previously unknown associations between microalgae and bacteria by using co-occurrence networks built from existing 16S rRNA gene-based metabarcoding datasets. This study identified a range of known and unknown associations between microalgae and bacteria, demonstrating the promise of 16S rRNA gene-based co-occurrence networks as a framework for hypothesis generation. In chapters 3 and 4, I studied the bacterial microbiome of Ostreobium, an alga living in coral skeleton. Ostreobium remains understudied despite extensive research on the coral holobiont. I hypothesized that the enclosed nature of the coral skeleton might reduce the dispersal and exposure of residing bacteria to the outside environment. This would allow stronger associations between Ostreobium and the bacteria making it an ideal algal-bacterial system to investigate evolutionary concepts such as codivergence and phylosymbiosis.Phylosymbiosis captures the correlation between the host phylogeny and the relationships of microbial communities associated with those hosts. Phylosymbiosis may arise from stochastic processes as well as through long-term host-microbe associations such as codiversification or coevolution. Codiversification, which is usually an indicator of coevolution, occurs when the speciation of one organism leads to the speciation of the associated organisms. In chapter 3, I investigated the bacterial microbiome associated with several Ostreobium strains in cultures using 16S rRNA metabarcoding, characterizing the taxonomic composition, diversity, likely physical associations of the bacterial associates and phylosymbiotic signatures. The results of chapter 3 indicated that Ostreobium is associated with taxonomically diverse bacteria. However, Ostreobium consistently associated with only a few of these bacterial groups (34 bacterial taxa), revealing a core microbiome. The data showed that while some bacteria may be loosely attached, others are tightly attached or potentially intracellular. Together with the core microbiome these potentially tightly attached or intracellular bacteria were defined as the closely associated microbiome of Ostreobium. I discovered that the Ostreobium-bacterial associations prevalent in cultures are likely to appear in their natural environment by analysing co-occurrence networks of 16S rRNA datasets from Porites lutea and Paragoniastrea australensis skeleton samples. The results also indicated significant congruence between the Ostreobium phylogeny and the community composition of its tightly associated microbiome, largely due to the phylosymbiotic signal originating from the core bacterial taxa. In chapter 4, I used metagenome-assembled genomes (MAGs) to investigate the functional potential of the bacteria associated with Ostreobium and addressed the codiversification concept. Here, the genomes representing the closely associated bacterial taxa of Ostreobium were specifically studied. Their genomes harboured dissimilatory nitrate/nitrite reduction, urea hydrolysis and methylamine degrading genes showcasing the potential to provide Ostreobium with ammonia. In addition, the results indicated that bacterial genomes possess vitamin B12 synthesis potential. A significant number of MAGs contained genes to metabolize C1 compounds like methylamine, methanol, and methane suggesting C1 metabolism may be a common trait among bacteria. Additionally, the presence of eukaryotic-like proteins in these MAGs indicated that they have the genetic machinery to maintain putatively stable host associations. Finally, the cophylogenetic analyses to assess the phylogenetic congruence between Ostreobium hosts and the bacterial associates provided evidence for codiversification. Independent cophylogenetic assessment of microbial orders showed significant congruence between the Phycisphaerales and Ostreobium phylogenies and identified family SM1AO2 to be potentially co-diverging with Ostreobium. This PhD expands our knowledge of microalgal-bacterial interactions by revealing previously unknown associations and providing insights into their evolutionary and functional relationships.
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ItemNo Preview AvailableIntrinsic and extrinsic modulators of Müller glia behaviour during retinal regeneration( 2022-12)Loss or dysfunction of the neurons in the retina leads to significant and irreversible vision impairment. Due to the variety of causes of this vision loss, ranging from genetic disease, environmental perturbations as well as ageing, a one-size-fits-all therapeutic approach does not currently exist. However, an exciting potential treatment involves activation of resident stem cells that already exist in the retina to replace these lost neurons. Muller glia are an abundant cell type in the retina of all vertebrate species, and in the established model system zebrafish (Danio rerio), Muller glia can successfully contribute to retinal regeneration and thus restoration of visual function. This is achieved through cellular reprogramming, whereby zebrafish Muller glia adopt a stem cell-like state and give rise to daughter progenitors through cell division, which can differentiate into mature retinal neurons. Despite an inability for human Muller glia to intrinsically regenerate the retina, factors crucial for driving this zebrafish Muller glia activation have been able to improve the neurogenic ability of mammalian Muller glia as seen in rodent systems. Therefore, enhancing our understanding of factors that influence Muller glia-driven regeneration in the zebrafish is crucial to designing future therapeutic strategies to treat neuron loss in the human retina. This thesis examines the current efforts determining factors that influence Muller glia-driven regeneration in the vertebrate retina, summarises research investigating key aspects of Muller glia behaviour in zebrafish retina regeneration, and discusses future directions for this research field to consider. Chapter 1 introduces the retina with an emphasis on the photoreceptor cells, the zebrafish model system, and the current understanding of Muller glia-driven regeneration in zebrafish, chick, and rodent systems. Chapter 2 provides evidence into the heterogeneity that exists among Muller glia in the zebrafish retina, how this heterogeneity can influence Muller glia activation, and the gene expression changes that drive this activation. Chapter 3 investigates external factors that can influence the behaviour of Muller glia following neuronal injury, with a focus on immune cells and debris clearance. Chapter 4 focuses on the fate of daughter progenitor cells derived from Muller glia in the regenerating zebrafish retina and how this progenitor fate can be influenced by the injury environment. Chapter 5 discusses future avenues to pursue from this research, as well as relevance in designing future therapeutic strategies to stimulate effective Muller glia-driven regeneration.
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ItemConflict and concordance in the evolutionary process: exploring the effects of separate sexes in Drosophila melanogaster( 2023-03)Differences in how selection acts on males and females are crucially important to many questions in evolutionary biology, including the evolutionary origins of sexual reproduction, and the maintenance of genetic variance in the face of selection. Theory predicts stronger total selection (i.e. natural and sexual) on the phenotypic effects of alleles when they are expressed in males, because males are typically subject to more intense sexual selection than females. However, selection among males does not always increase the fitness of their daughters, because alleles can differentially affect the sexes. Because selection acts on phenotypes produced by many alleles, this can result in mutually non-exclusive, conflicting fitness outcomes for females. For example, selection among males may favour alleles that increase male reproductive success and female fitness, such that natural and sexual selection align (‘good genes’ effects). In this case, selection among males reduces genetic load and increases population viability. Simultaneously, selection can also favour alleles that increase male reproductive success but reduce female fitness (intralocus sexual conflict). This latter scenario increases genetic load in females and may increase the risk of population extinction. Teasing apart the joint action of sexually concordant and antagonistic selection is a considerable challenge. However, new community resources, genetic tools and quantitative genetic methods provide the opportunity to resolve this confound. In this dissertation, I investigated the evolutionary ramifications of males, and the sexes sharing a genome, using multiple approaches. In Chapter 1, I summarise my main findings, provide a short primer on the evolutionary process and detail how this is affected by selection acting on separate sexes. I particularly focus on how differences between the sexes produce intralocus sexual conflict and the purging of mutation load through sexual selection. In my first data chapter (Chapter 2), I collated sex-specific breeding values for hundreds of traits in the Drosophila Genetic Reference Panel and used the Secondary Theorem of Natural Selection to estimate how these traits would respond to selection in a laboratory environment. I found that the expected response to selection is stronger in males than it is in females, and that very few traits are expected to respond to selection in opposite directions in males and females. I then explored the fitness consequences of intralocus sexual conflict in greater detail (Chapter 3), using an experimental evolution approach in Drosophila melanogaster that enforced female-limited, male-limited and unconstrained inheritance of the major autosomes, using an innovative breeding design. Contrary to earlier research, limiting the selective response to males did not increase male fitness. Moreover, a female-limited selection response also resulted in no increase to female fitness. I then capitalised on these ‘sex-limited evolution’ populations in Chapter 4, where I explored whether passing the autosomal genome through males reduced the load of deleterious mutations in the population. I found that a male-limited selection response history reduced mutation load relative to control and potentially also female-limited selection histories. In a final experiment, I investigated whether sexual selection enhances purging of a particularly deleterious gene complex that is associated with a sperm-killing selfish allele. Through manipulative experiments and a population genetic model, I tested the prediction that sperm competition and cryptic female choice could curtail the spread of the selfish allele complex. I showed that sexual selection acting among males can reduce the frequency of the selfish allele, which likely has a positive effect on population viability (Chapter 5). My findings suggest that the selective response is greater in males than it is in females, and that this extends beyond traits with direct links to male reproductive success. Furthermore, this stronger selection among males appears to purge deleterious mutations from the gene pool, to a greater extent than occurs in response to selection among females. Finally, the benefits of an evolutionary response in males extend to females, and these benefits appear to outweigh the relatively benign fitness consequences of segregating sexually antagonistic alleles (or lack thereof) in my experimental Drosophila populations.
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ItemMove it or lose it: how and when to use targeted gene flow( 2023-04)A myriad of threats are currently assailing threatened and soon to be threatened populations. Habitat removal, climate change, wildlife disease and the invasion of non-native species are all placing increased pressure on the systems we undervalue for their role in providing clean air, water, carbon sequestration and natural beauty. Many populations, however, contain individuals expressing traits that enable them to survive and reproduce even in the presence of these existential threats. Unfortunately, many of these adaptive traits are rare in endangered populations. But useful variation in traits doesn't only occur within populations: geographic trait variation is ubiquitous and can arise from a number of different processes, including natural selection, spatial sorting, and drift. Natural selection allows populations to adapt to local environmental conditions and can lead to locally adapted variants. Spatial sorting, the spatial analogue to natural selection, can also lead to predictable geographic variation in traits across space, as invasion and recolonisation select for increased dispersal. Both of these forms of selection result in predictable trait variation that conservation biologists can harness to promote conservation benefits. Geographic variation might also arise through drift, but such variation is much less predictable. Once geographic variation exists, it is possible to harness such variation to affect conservation goals. This idea -- targeted gene flow -- has the objective of identifying and harnessing geographic trait variation to promote conservation benefit. Current work on targeted gene flow has focused on understanding how species respond to certain threats and on exploring the use of targeted gene flow to boost adaptive potential in threatened populations. In this thesis, I explore the utility of applying targeted gene flow to two different conservation problems: i) can targeted gene flow be deployed to facilitate evolutionary rescue in response to rapid (and flexible) environmental change? and ii) can targeted gene flow be deployed to directly mitigate a threatening process? To answer these questions, I use a blend of field studies and simulation models focused on the introduced pest, the Cane Toad (Rhinella marina). This thesis starts by providing the reader with background information and current applications of targeted gene flow, as well as identifying and framing the need for novel measures to reduce the impacts of cane toads in Australia. To explore this use case, we first need to understand how to apply targeted gene flow in an optimal fashion, as well as how to measure any benefits. In the opening data chapter of my thesis, I set out to understand how to optimally deploy targeted gene flow and build upon initial work in this area to examine how targeted gene flow should be deployed against differing threat profiles. I find that the optimal timing and size of a targeted gene flow action is highly sensitive to the maximum rate of change of the threat across time, and that if conducted correctly, targeted gene flow can provide enough adaptive potential to stave off extinction whilst retaining almost all of the genetic diversity of the population under threat. This measure, the expected benefit, is a novel metric to benchmark the effectiveness of targeted gene flow applications. In the subsequent chapters of this thesis, I extend the notion of targeted gene flow to a different context: reducing the dispersal ability of invasive species. Cane toads are one of the most harmful introduced species in Australia. Decades of sustained investment in cane toad control, research and management has unearthed a number of effective strategies for the local control of cane toad populations, but no landscape level solution currently exists. In chapter three, I quantify the financial benefit of keeping areas toad-free. I conduct field studies to generate estimates of toad density and detectability, before combining these with removal and cost models to provide an estimate of the value of cane toad quarantine across offshore islands as well as a potential toad-free haven on the mainland: the Pilbara region of Western Australia. My final chapter explores how targeted gene flow can be deployed to increase the effectiveness of a landscape barrier designed to contain the toad invasion and so create a toad-free haven over 265,000 km2 of the Australian mainland. This chapter provides the first case study of how targeted gene flow can be deployed to directly mitigate a threatening process. In doing so, I provide the first evidence that targeted gene flow can be used to directly reduce the negative impacts of an invasive species, through driving down their dispersal ability, and in doing so render landscape barriers substantially more effective. This thesis shares the process of exploring a new application of targeted gene flow, from theoretical conception to an applied trial. I provide the first evidence that targeted gene flow can be used to reduce the ability of an invasive species to move across the landscape, alongside extensions to the current framework surrounding how to optimally implement targeted gene flow to aid threatened populations. The resulting strategies are not limited to the impacts of cane toads but instead have application to a wide range of conservation scenarios. Generally, my thesis develops the under-appreciated idea that, by being creative with geographic trait variation, we have a powerful and cost-effective tool for conserving biodiversity.
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ItemGenomically informed gene drive modelling( 2022)CRISPR/Cas gene drives are a focus of genetic biocontrol for pest species. They have the potential to radically affect pest species, by making them more manageable or by eradicating them. However, it is not yet fully understood how the elements of a gene drive interact to guide the progression of a gene drive. We explored how we can design gene drives that are safer, either by being temporally limiting or spatially limiting, through a modelling framework. Our modelling included a range of variables, with the addition of genomic information to infer the homing efficiency of the gene drive. We showed that there was no single variable that differentiated between the outcomes of a gene drive. Granted some variables were more influential in determining the outcome than others. The degree of dominance of the selection coefficient was shown to be strongly influential on the equilibrium outcome. While the interaction between conversion efficiency and resistance was shown to strongly influence the Temporary outcome. Furthermore, we showed that internal dynamics of a gene drive can be regulated by the variables of the gene drive. This provided insight into where effort should be directed in gene drive design to achieve the intended outcome of a gene drive, as well as controlling the progression to that outcome. The inclusion of genomic data in CRISPR gene drive modelling allowed for localisation of the gene drive due to genetic variation alone. Finding loci in the genome where there were allele frequencies differences allowed us to model gene drives that were highly efficient in the target population and poorly efficient in off-target populations. This conversion efficiency differential allowed for sustained gene drive localisation in spite of migration and selection. Population suppression was explored in our modelling to better understand how we could create sustained localised suppression. We showed sustained population suppression was possible through incomplete distortion of the sex ratio of the progeny. A deterministic gene drive model was developed to solve for equilibrium points for a range of migration rates and selection coefficients. These equilibria can be used as thresholds for gene drive design and monitoring. This work aims to further develop our understanding of how gene drives are likely to progress when released. We focussed on characterising which aspects of a gene drive were most important in determining both their progression and outcome. The inclusion of genetic information in our modelling revealed a new avenue that can be exploited to achieve gene drive localisation. This modelling work will aid in the design process of gene drives to increase our confidence that gene drives will work as intended.