Genetics - Theses
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ItemNo Preview AvailableRoles of nicotinic acetylcholine receptors in development, viability, and insecticide response, in Drosophila melanogaster( 2021)The majority of excitatory neurotransmission in the insect brain occurs via nicotinic acetylcholine receptors (nAChRs), however knowledge of which nAChR subunits may be required in specific neurons is virtually absent. The ubiquity of nAChRs in the insect brain also makes them ideal molecular targets for many neuroactive insecticides. Mutations in specific nAChR subunits can confer high levels of resistance, but the fitness costs that may be associated with resistance alleles or with low-dose insecticide exposure are not fully understood. This thesis examines two Drosophila nAChR subunits with the most severe loss of function phenotypes, with a focus on their endogenous functions and their roles in insecticide response. Chapter Two investigates the role of the Da5 subunit in larval development. Preliminary observations had indicated that loss of Da5 causes larval mortality and is associated with precocious wandering and moulting phenotypes. Here, these phenotypes are quantified and found to be associated with loss of ecdysis triggering hormone. Cell types requiring Da5 were also narrowed-down to potentially include the prothoracic gland cells, or the neurons innervating the prothoracic gland. In insecticide exposure assays, loss of Da5 was shown to not confer altered response to spinosad, suggesting that Da5 may not be contributing to the primary spinosad target. In Chapter Three, loss of the Db1 subunit is shown to result in pleiotropic consequences, including severely shortened longevity, reduced male courtship, limited locomotion, and unsuccessful wing expansion. Since wing expansion is controlled by a well-characterised hormone (bursicon) in a small and well-defined subset of neurons (the CCAP neurons), this phenotype was examined further. Removal of Db1 specifically from CCAP neurons using somatic CRISPR was sufficient to disrupt wing expansion and loss of Db1 was shown to cause loss of the hormone bursicon. Together, these experiments identify CCAP neurons as a specific subset requiring Db1 for normal function. Chapter Four extends findings from the previous chapter, by testing whether alternative Db1 alleles, and a non-Drosophila b1 subunit orthologue, can rescue the Db1 loss of function phenotypes. Db1 cDNA rescue constructs containing the amino acid replacements R81T (found in resistant populations of aphids), and R81Q (naturally occurring in insensitive arachnids), were both found to rescue all elements of the Db1 phenotype, but only R81Q was found to confer high levels of imidacloprid resistance. The aphid subunit Mpb1 was also able to rescue loss of Db1, and fully restored sensitivity to imidacloprid, revealing substantial functional conservation between the b1 subunits in these two species. This work provides a platform for studying resistance-conferring amino acid replacements in pest nAChR subunits within the Drosophila model. Understanding the endogenous roles of nAChR subunits will be essential for characterising the function of every pathway in the insect brain. By characterising the roles of Da5 and Db1, this thesis provides great insight into the fitness costs insects may endure when evolving insecticide resistance. It also reveals the developmental and behavioural pathways that may be affected when pest and non-pest species are chronically exposed to the low doses of insecticides that contaminate the environment.
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ItemIn vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster( 2018)Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
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ItemPopulation genomics and transcriptomics in the cotton bollworm, Helicoverpa armigera( 2018)Helicoverpa armigera is an agricultural pest that causes billions of dollars' worth of damage each year. As H. armigera has evolved resistance to insecticides, an understanding of resistance genes will provide useful insights into managing this pest. One approach to identify candidate genes is to scan the genome for signs of strong and recent selective sweeps. This extends the search beyond typical candidate genes (detoxifying enzymes and molecular targets) although a limitation of the approach is that the selective agent causing a sweep may not be an insecticide. Another approach is to compare the differences between lab-selected and unselected cohorts. Genes that are differentially expressed are good candidates for further investigation. Here, I present estimates of some baseline parameters such as nucleotide diversity and the extent of linkage disequilibrium to lay a foundation for detecting selective sweeps in H. armigera, and I identify a gene exhibiting the hallmarks of strong and recent selection. I also present some preliminary findings from an analysis of differentially-expressed genes between selected and unselected cohorts of H. armigera in response to a pyrethroid insecticide.
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ItemUnderstanding aphids: transcriptomics, molecular evolution and pest control( 2018)Aphids exhibit fascinating biological features including parthenogenesis, symbiosis, altruism and host-plant preference; all of which would be better understood if genetic tools and molecular biological techniques were applied to them. Aphids are also agricultural pests that vector plant viruses and new approaches to control them are required. This thesis addresses questions motivated by an interest in the biology of aphids and a desire to improve the agricultural impact of aphids. It does so through transcriptomic analyses and RNA interference (RNAi) technology. I examined the ways in which the transcriptome of aphid changes with host-plant, between tissues, within species and between species. The three-aphid species studied (the green peach aphid: Myzus persicae, the mustard aphid: Lipaphis erysimi, and the cabbage aphid: Brevicoryne brassicae) are all pests of economically important brassica crops (such as cabbage, cauliflower, mustard and canola). These data may provide insights into the way different aphid species deal with plant secondary compounds such as glucosinolates. These data also allowed me to examine the structure, function and evolution of myrosinase enzymes that have allowed some aphid species to develop an anti-predator ‘mustard bomb’. RNAi has been suggested as a way to specifically target pest that would be more ‘environmentally friendly’ than conventional insecticides. I experimentally assessed the feasibility of orally-delivered RNAi to control aphids and the potential of this technology to be developed as a functional genomic tool. RNAi was fed to aphids via artificial diets at various concentrations and with various delivery agents and via transgenic Arabidopsis thaliana plants that I created that produced dsRNA’s corresponding to aphid genes. These studies lead me to suggest that more work needs to be done to limit the effects of RNase enzymes of the aphid gut digesting orally delivered RNAi and to more carefully characterize factors that may affect within-species variation in RNAi efficacy.
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ItemIdentifying downstream targets of FILAMENTOUS FLOWER, a YABBY transcription factor that promotes organ polarity and lateral growth in Arabidopsis( 2017)Angiosperm leaves are typically polar structures with a distinct arrangement of cell types along the adaxial-abaxial (upper-lower) axis. Studies of leaf development in the model dicot plant Arabidopsis thaliana have shown that adaxial-abaxial patterning is not only associated with the formation of distinct cell types, but also triggers growth along the lateral axis leading to the formation of the leaf blade or lamina. Adaxial-abaxial patterning factors identified in a variety of plants including Arabidopsis, Antirrhinum and maize are either small regulatory RNAs or transcription factors. Although first categorized as being involved in adaxial-abaxial patterning, the YABBY (YAB) family of transcription factors is now thought to play a pivotal role in coordinating various developmental programs involved in leaf blade formation. As part of an approach to identify targets of the Arabidopsis YAB gene FILAMENTOUS FLOWER (FIL), this study generated transgenic lines with inducible FIL activity. Constitutive activation of FIL resulted in the partial abaxialisation of leaves and reduced blade growth, phenotypes that have previously been attributed to ectopic YAB expression. Further analysis showed that constitutive FIL activity increased the rate of cell cycle progression in specific regions of the developing leaf, as well as increasing sensitivity to exogenously applied auxin. The latter phenotype was inferred from increased activity of an auxin-signalling reporter, suggesting that FIL modulates auxin responses in Arabidopsis. Transcriptional profiling with microarrays was subsequently used to monitor genome-wide changes in gene expression following FIL activation. This analysis identified groups of genes that were either positively or negatively regulated by FIL and extensive testing of a subset of these genes showed that some were direct targets. On the basis of these results it is proposed that FIL functions as both a transcriptional activator as well as repressor during leaf development. Among the positively regulated genes identified as FIL targets, two are well-known abaxial patterning factors, KANADI1 (KAN1) and AUXIN RESPONSE FACTOR4 (ARF4). Given the well-defined role these factors play during leaf development, this study focused on their regulation. Analysis of mutant lines lacking activity of the leaf-expressed YABs revealed a significant reduction in KAN1 expression, but not ARF4 expression. These results confirm that FIL is a regulator of KAN1, but presumably regulates ARF4 in combination with other factors. In conclusion, this study identified direct targets of the bifunctional plant transcription factor FIL. Finding that two of these targets promote abaxial cell identity during leaf development supports the case for YABs being important polarity regulators. Given the expression pattern of the YABs, it is argued that YABs are unlikely to function upstream of KAN1/ARF4. Instead, a model is proposed in which YABs promote adaxial/abaxial patterning through a system of positive feedback loops that ultimately maintain the activity of these early abaxial patterning genes during leaf blade formation.
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ItemThe genetic basis of resistance to the Ryanodine Receptor modulator chlorantraniliprole in Drosophila melanogaster( 2017)The development of synthetic insecticides in the mid 20th century lead to a revolution in pest control. However, issues with environmental toxicity, adverse human exposure and insecticide resistance have meant new safer alternative pest control methods are required. Chlorantraniliprole belongs to a promising new class of insecticides that exert control by targeting the Ryanodine Receptor. As this class, the group 28 synthetic diamides, has a unique chemistry and a mode of action that is distinct from most insecticides, its market share has rapidly increased since it was first introduced in 2007. Here, I use genomic, transcriptomic and phenotypic analyses of the Drosophila Genetic Reference Panel (DGRP) to examine the way chlorantraniliprole interacts with an insect’s biology. This research reveals that a novel muscle- associated gene, Stretchin Myosin Light Kinase, is strongly associated with resistance in the DGRP. In addition, a co-expressed set of detoxification enzymes, under control of the Cap ‘n Collar/Keap1 pathway were found to be constitutively up-regulated in a subset of the DGRP and that their transcriptional abundance was correlated with survivorship on chlorantraniliprole. Transgenic ‘knock up’ of one of these putative detoxification enzymes, Cyp12d1, confers increased resistance to chlorantraniliprole and resistance to the related compound cyantraniliprole. Furthermore, a lab selection experiment based on a large Australian population of D. melanogaster also confirms an association between Cyp12d1 and resistance. This contributes to a growing body of evidence suggesting that cytochrome P450 enzymes play a role in chlorantraniliprole resistance. Through the quantitative genetic approaches employed, it is possible to demonstrate that the genetic architecture underpinning resistance changes with dose. Furthermore, as the DGRP was established before the introduction of chlorantraniliprole this study demonstrates that alleles of large effect are pre- existing in naïve populations and such alleles may increase in frequency as this class of insecticides become more widespread. Finally, this study illustrates the systems genetic approach offers unprecedented power to understand the biology perturbed by insecticides.
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ItemThe significance of low level mosaicism in Fragile X Syndrome.( 2017)Fragile X Syndrome (FXS) is the most common single disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD). FXS is fundamentally caused by a trinucleotide CGG sequence repeat expansion within the 5’ untranslated portion of the FMR1 gene, to more than 200 repeats which is called Full Mutation (FM). This is associated with abnormal methylation of this gene’s promoter and silencing of FMR1 expression. To date, the type and severity of the neurocognitive phenotype has been related more closely with the methylation status than with the number of CGG repeats in the FM range. Smaller CGG expansions are called premutation (PM) (55 and 200 CGGs), grey zone (GZ) (45-54 CGGs) and normal size (<44 CGGs) alleles, and are usually associated with presence of an unmethylated FMR1 promoter, and normal FMR1 expression. There is a proportion of FXS individuals in which two or more population of cells can be found, and who are called mosaic or mosaicism. These individuals possess cells with FM and smaller CGG sizes (e.g. Normal, GZ or PM), which lead to milder forms of the FXS phenotype. This PhD explores the clinical and diagnostic significance of low level mosaicism (LLM) and DNA methylation variation amongst different CpG sites within FMR1. The first aim of this study was to define the lower limit of detection LLM of six FMR1 tests currently used in diagnostic laboratories. This was achieved using three FMR1 PCR commercial kits (AmplideXTM, X-Sense and FastFraXTM) targeting the CGG region and three methylation tests (Methylation Specific- Quantitative Melt Analysis [MS-QMA], Sequenom®EpiTYPER system [MALDI-TOF MS] and mSouthern blot) targeting methylation of Exon1/Intron1 boundary region and specific CpG sites within FMR1 promoter. The second aim was to screen a large number of males with ID and ASD of unknown etiology referred for FXS testing to determine the prevalence of mosaicism containing FM together with a normal or GZ allele (cryptic FMR1-FM) who are not being identified as a part of standard FXS testing. This was achieved using MS-QMA and the positive results were confirmed with the FMR1 tests assessed in Aim 1. The third aim of this study was to examine the association between DNA methylation of FMR1 and mRNA levels and severity of intellectual functioning impairment in individuals with a cryptic FMR1-FM identified in Aim 2 and males and females with typical FXS (only FM and PM/FM alleles). Three significant contributions to the FXS field resulting from this PhD are: i) implementation of a new highly sensitive assay for detection LLM missed by standard testing; ii) determination of the prevalence of cryptic FM-FMR1 in males with idiopathic ID/ASD referred for FXS testing; iii) clinical and molecular characterization of LLM and its methylation characterization amongst tissues and CpG sites; and demonstration that LLM levels are significantly correlated with severity of cognitive impairment and ASD as well as FMR1 mRNA levels.
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ItemA genome-wide analysis of carbon catabolite repression in Schizosaccharomyces pombe( 2018)The process by which eukaryotic microorganisms preferentially utilise glucose as a carbon source is coordinated by a network of sensory and signalling pathways, which converge at the transcriptional level to control the function of a conserved regulatory mechanism known as carbon catabolite repression (CCR). In the fission yeast, Schizosaccharomyces pombe, CCR is mediated by Scr1, a C2H2 zinc finger transcriptional repressor orthologous to Saccharomyces cerevisiae Mig1, and Aspergillus nidulans CreA. In addition, a conserved co-regulatory complex, comprised of Tup11, Tup12 and Ssn6 proteins (hereafter Tup/Ssn6), is required for maximal transcriptional repression. Also implicated in this process is the transcriptional activator, Rst2, which in the absence of glucose, induces the expression of gluconeogenesis and sexual differentiation genes. To date, the molecular mechanism of CCR in S.ipombe has not been characterised in depth, and so there is limited knowledge of the range of genes that are subject to transcriptional repression, or of the functional relationship between Scr1, Tup/Ssn6, Rst2, or other factors that influence the establishment and/or maintenance of CCR. This study combined genetic techniques with a suite of high-throughput sequencing approaches to investigate the process of CCR in S. pombe. RNA-seq and ChIP-seq approaches showed that Scr1 represses approximately 2% of the S. pombe genome in the presence of glucose including hexose uptake, glycolysis, TCA cycle, pentose phosphate pathway, and gluconeogenesis genes. In addition, unexpected roles for Scr1 in the regulation of iron homeostasis and stress-induced meiosis were discovered, integrating Scr1 and CCR more broadly into the regulation of general metabolism and stress responses in S.ipombe. Biochemical pulldown approaches showed that Scr1 physically interacts with the Tup/Ssn6 complex in vitro and further ChIP-seq showed co-localisation of Tup11 with Scr1 at gene promoters in glucose-sufficient conditions. Interestingly, Tup11 was shown to remain at the promoter of certain target genes that were activated in the absence of glucose, suggesting roles for the Tup/Ssn6 complex in gene activation. Additional ChIP-seq analysis of Rst2 in the absence of glucose revealed localisation to gene promoters formerly repressed by Scr1 in glucose-sufficient conditions. Surprisingly, Rst2 was also found to co-localise with Scr1 and Tup11 at certain genes in the presence of glucose suggesting unforeseen regulatory roles for this factor in glucose-sufficient conditions and hinting at a potential competitive or co-operative relationship between Scr1 and Rst2 at these genes. In addition to increasing knowledge of CCR, these findings also have important biotechnological implications. S. pombe is industrially utilised to produce bioethanol, a renewable biofuel of significant environmental and economic importance. In a concurrent approach, an S. pombe isolate used for industrial scale bioethanol production from sugarcane molasses was analysed for modifications to carbon metabolism, CCR or other processes. Whole genome sequencing identified structural variation, including a 100kb duplication of a subtelomeric region in chromosome III, and potential evidence for horizontal gene transfer of coding sequences from Schizosaccharomyces octosporus. Further transcriptomic analysis identified a distinct transcriptional signature of this industrial isolate in both laboratory media and the molasses feedstock. Importantly, regulatory rewiring of central carbon metabolism and stress response pathways was evident. Finally, direct examination of CCR within this strain via Scr1 ChIP-seq, revealed significant plasticity with respect to the number of Scr1 targets, particularly in the molasses feedstock where Scr1 was associated with multiple actively transcribed genes, suggesting alteration of the CCR pathway within the industrial strain genetic background. Overall, this study has shown that CCR in S. pombe forms a core regulatory network that responds to glucose primarily at the transcriptional level to facilitate the regulation of a range of metabolic processes in both laboratory and industrial contexts. Thus, this work significantly improves our understanding of the CCR process in S. pombe and forms an important resource for the study of carbon regulation in eukaryotes. These findings will also be useful for the development of fission yeast strains that possess improved bioethanol production characteristics.
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ItemThe evolution of pathogenicity and isolate variation in Talaromyces marneffei( 2017)The opportunistic fungal pathogen of humans, Talaromyces marneffei, is one of very few pathogens in an order of over a thousand species and the only species that has the capacity to switch between two morphologically distinct growth forms (known as dimorphism). Growth at 25°C results in a saprophytic multicellular, hyphal form while infectious growth in a host occurs as a uninucleate unicellular yeast that resides within phagocytic cells of the immune system. The intracellular niche of T. marneffei differs significantly from the niches of other Talaromycetes. The identification of the mechanisms by which T. marneffei can survive and grow in this intracellular niche is a major aim of this study. Comparisons of the genomes of three closely related non-dimorphic, non-pathogenic species with the T. marneffei genome identified unique features that contribute to niche specific growth and the ability to cause disease. Most significant of these were an overall reduction in genome size and gene number in T. marneffei with substantial gene losses in families responsible for environmental interaction. These and other findings strongly indicate that T. marneffei has adapted to an intracellular host niche distinct from its saprophytic relatives. Against this background of gene loss three gene families were identified that had been significantly expanded in T. marneffei. These expanded gene families showed putative extracellular and cell surface localisation and consisted of cell wall galactomannoproteins (mpl family), aspartyl proteases (pop family) and a family of small proteins with very little functional characterisation in any species (mib family). Genes in the pop, mpl and mib families were over-represented in subtelomeric regions, under positive selection, had copy number variation in T. marneffei isolates and many had high levels of repetitive adjacent sequences including several transposon families. In the host T. marneffei grows as an intracellular pathogen within phagocytes and as such extracellular proteins interact directly with the host. Therefore another aim of this study was to characterise these expanded gene families and their role in pathogenesis. Deletion studies in pop genes revealed roles in yeast cell formation during intracellular growth, while high variability in cell-to-cell protein production for two mib genes suggested a role in cell surface variation when interacting with the host. Understanding the type and degree of variation within the population of a fungal pathogen can reveal its population structure and potential to adapt to stressors such as antifungal compounds. Genome wide variation in the T. marneffei population had yet to be examined therefore an aim of this study was to characterise the degree and type of this variation. To this end several clinical and environmental isolates of T. marneffei were examined for variation in chromosomal structure, which is a common means of generating phenotypic variation in other fungi. While no obvious abnormalities were observed, gene copy number variation in subtelomeric regions was widespread and several strains showed specific small mutations with impacts in antifungal resistance and phenotypic instability. Overall this study has revealed the genomic and genetic changes within T. marneffei and between it and other Talaromycetes. Many of these changes help to explain its unique niche as an intracellular pathogen within an almost entirely non-pathogenic clade. This research also highlights specific genes and gene families with roles in this pathogenesis and identifies potential therapeutic targets and genes involved in host interactions for future investigation.
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ItemProbing insecticide biology using Drosophila melanogaster( 2017)Insecticides are often used to control insect pests, but resistance to these chemicals arises quickly, leading to agricultural losses and public health concerns. Understanding how insects cope with insecticides is necessary when designing rational pest management strategies, but much still remains unknown regarding the fate of insecticides once inside the body. Furthermore, the genetic variation that governs an insects ability to survive insecticide exposures has not been fully described. Here, a 3 pronged approach is applied to study insecticide biology using the model insect Drosophila melanogaster. First, an acute, sub-lethal insecticide response assay was developed, which provided information complementary to that obtained from more common toxicology assays. In particular, behavioural response observed in a hyper-resistant target site mutant suggests additional target sites for the insecticide spinosad. This bioassay was then applied in a forward genetics approach to describe the genetic basis of resistance to the insecticide imidacloprid. This approach identified a variety of neuronal genes and the previously identified drug metabolizing enzyme Cyp6g1, which was explored through genetic manipulation. Finally, a reverse genetics approach was employed in order to study the effect of an ABC transporter protein Mdr65 on insecticide resistance. Removing the gene made the insects more susceptible to a subset of the insecticides tested, and this was confirmed with genetic and chemical complementation tests. These data provide information both on the genetics and kinetics of insecticide biology. Such information will help to better understand insecticide resistance and design rational resistance management strategies.