School of BioSciences - Theses
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ItemThe evolutionary and functional characterisation of the ecdysteroid kinase-like (EcKL) gene family in insects( 2020)Many thousands of gene families across the tree of life still lack robust functional characterisation, and thousands more may be under-characterised, with additional unknown functions not represented in official annotations. Here, I aim to characterise the evolution and functions of the poorly characterised ecdysteroid kinase-like (EcKL) gene family, which has a peculiar taxonomic distribution and is largely known for containing an ecdysteroid 22-kinase gene in the silkworm, Bombyx mori. I hypothesised that EcKLs may also be responsible for insect-specific ‘detoxification-by-phosphorylation’, as well as ecdysteroid hormone metabolism. My first approach was to explore the evolution of the EcKLs in the genus Drosophila (Diptera: Drosophilidae), which contains the well-studied model insect Drosophila melanogaster. Drosophila EcKLs have evolutionary and transcriptional similarities to the cytochrome P450s, a classical detoxification family, and an integrative ‘detoxification score’, benchmarked against the known functions of P450 genes, predicted nearly half of D. melanogaster EcKLs are candidate detoxification genes. A targeted PheWAS approach in D. melanogaster also identified novel toxic stress phenotypes associated with genomic and transcriptomic variation in EcKL and P450 genes. These results suggest many Drosophila EcKLs function in detoxification, or at least have key functions in the metabolism of xenobiotics, and additionally identify a number of novel P450 detoxification candidate genes in D. melanogaster. I then broadened the phylogenomic analysis of EcKLs to a manually annotated dataset containing an additional 128 insect genomes and three other arthropod genomes, as well as a number of transcriptome assemblies. Phylogenetic inference suggested insect EcKLs can be grouped into 13 subfamilies that are differentially conserved between insect lineages, and order-specific analyses for Diptera, Lepidoptera and Hymenoptera revealed both highly conserved and highly variable EcKL clades within these taxa. Using phylogenetic comparative methods, EcKL gene family size was found to vary with detoxification-related traits, such as the sizes of classical detoxification gene families, insect diet, and two estimations of ‘detoxification breadth’ (DB), one qualitative and one quantitative. Additionally, the rate of EcKL duplication was found to be low in lineages with small DB—bees and tsetse flies. These results suggest the EcKL gene family functions in detoxification across insects. Building on my previous ‘detoxification score’ analysis, I used the powerful genetic toolkit in D. melanogaster and developmental toxicology assays to test the hypothesis that EcKL genes in the highly dynamic Dro5 clade are involved in the detoxification of selected plant and fungal toxins. Knockout or misexpression of Dro5 genes, particularly CG13659 (Dro5-7), modulated susceptibility to the methylxanthine alkaloid caffeine, and Dro5 knockout also increased susceptibility to kojic acid, a fungal secondary metabolite. These results validate my evolutionary and integrative analyses, and provide the first experimental evidence for the involvement of EcKLs in detoxification processes. Finally, I aimed to find genes encoding ecdysteroid kinases in D. melanogaster, focusing on Wallflower (Wall/CG13813) and Pinkman (pkm/CG1561), orthologs of a known ecdysteroid 22-kinase gene. Wall and pkm null mutant animals developed normally, but misexpression of Wall caused tissue-specific developmental defects, albeit not those consistent with inactivation of the main ecdysteroid hormones, ecdysone and 20-hydroxyecdysone. In addition, my hypothesis that Wall encodes an ecdysteroid 26-kinase was not supported by hypostasis experiments with a loss-of-function allele of the ecdysteroid 26-hydroxylase/carboxylase gene Cyp18a1. Combined with existing expression and regulatory data, these results suggest Wall encodes an ecdysteroid kinase with an unknown substrate, and hint at previously unknown complexity in ecdysteroid signalling and metabolism in D. melanogaster. Overall, this thesis provides a detailed exploration of the functions of the EcKL gene family in insects, showing that these genes comprise a novel detoxification gene family in multiple taxa, and that they may also contribute to understudied aspects of ecdysteroid metabolism in a model insect. This work also demonstrates the power and potential of integrating evolutionary, genomic, transcriptomic and experimental data when characterising genes of unknown function.
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ItemSystemic impacts of low dose insecticide exposures in Drosophila: a mechanism centred on oxidative stress( 2020)The plight of insect populations around the world has gained increasing attention. A recent meta-analysis published in Science reported an average decline of terrestrial insect abundance by average 9% per decade since 1925 (van Klink et al. 2020). While this is a lower rate of decline than reported in earlier meta-analyses (Sanchez-Bayo and Wyckhuys 2019) it still suggests that many terrestrial insect species are under threat. The extinction of terrestrial insect species would severely affect agriculture and ecosystems due to the vital role that many species play in pollination, the recycling of organic matter, pest control and other ecosystem services. Insecticide exposure has been proposed to be one of the significant contributing factors for population declines of non-pest species. Insecticide contamination in biomes, resulting from intensive usage on agricultural crops, is likely to lead to exposures for many non-pest insect species. Low doses of insecticides are known to impact the fitness and behaviour of various insect species, but the underlying molecular, cellular, and physiological impacts of such doses in insects are not well defined. The absence of a mechanism that explains how low doses affect insects is an obstacle to ascertaining the extent to which insecticides may contribute to the demise of populations. The aim of this study was to scrutinize the impacts of low insecticide doses on the metabolism and physiology of the model organism Drosophila melanogaster in order to propose a mechanism to explain the impact of such doses on insect biology. Two insecticides were investigated in detail. The first of these is the synthetic neonicotinoid imidacloprid. Having been banned in the EU due to some evidence of a role in collapse in honeybee colonies, imidacloprid remains one of the most widely used insecticides in the world. The second insecticide is spinosad. Composed of two structurally similar natural fermentation products from the soil bacterium Saccharopolyspora spinosa, this insecticide is classified as organic and considered to be less harmful to beneficial insects. Both insecticides target evolutionarily conserved nicotinic acetylcholine receptors (nAChRs) in the Central Nervous System (CNS) of insects. nAChRs are pentameric ligand gated ion channels. Activation by the natural ligand, acetylcholine, leads to a flux of calcium, potassium or sodium ions into neurons, regulating a myriad of responses in the insect brain. The Drosophila genome encodes 10 nAChRs subunits (Dalpha1 to Dalpha7 and Dbeta1 to Dbeta3), meaning that there is a vast number of subunit combinations that could assemble to form functionally distinct receptor subtypes. Imidacloprid targets the Dalpha1, Dalpha2, Dbeta1 and Dbeta2, subunits, whilst spinosad targets the Dalpha6 subunit. Acute exposure to imidacloprid, at doses that do not kill Drosophila larvae, rapidly increased in the levels of reactive oxygen species (ROS) in the brain, most likely due to the sustained calcium flux into neurons caused by the interaction between the insecticide and its nAChR targets. This led to oxidative stress marked by mitochondrial dysfunction that in turn led to a significant decrease in energy (ATP) levels. While this process was initiated in the brain, lipid storage in the metabolic tissues (fat body, Malpighian tubules, and midgut) was affected. Transcriptomic analysis of the larval brain and fat body revealed a significant perturbation in the expression of genes involved in metabolism, oxidative stress, and immune response. Using genetic manipulations to elevate ROS levels exclusively in the brain, lipid storage was shown to be perturbed in the metabolic tissues, indicating that a ROS signal initiated in the brain radiates to other tissues. Severe damage to glial cells and neurons (i.e. neurodegeneration) was observed in the visual system of adults subjected to chronic low-dose exposure to imidacloprid. This precipitated a progressive loss of vision. Spinosad showed a different mode of action, blocking nAChRs and preventing calcium influx. The blocked receptors were shown to be recycled from the neuronal membranes through endocytosis. This mechanism led to an increase in the number and size of lysosomes in the CNS, characteristic of lysosomal storage diseases, which precipitates elevated generation of ROS by impairing mitochondrial activity and neurodegeneration. The high levels of ROS measured in the CNS after spinosad exposure, were associated with a cascade of phenotypes in metabolic tissues similar to the ones observed after imidacloprid exposure. Experiments examining the lipid environment in Dalpha6 knockout mutants (resistant to spinosad) indicated that impacts observed in the metabolic tissues of spinosad-exposed larvae are due to the interaction between Dalpha6 and spinosad. These data corroborate the hypothesis that impairments observed in metabolic tissues are triggered by a chemical signal from the brain, suggested to be a peroxidized lipid. Although there were some differences in the responses observed for the two insecticides (e.g. in transcriptomes and lipidomes), a similar cascade of processes was observed to be initiated following the elevation of ROS levels in the brain. A potent antioxidant, N-Acetylcysteine amide, strongly suppressed a range of phenotypes observed in both larvae and adults, indicating a causal role for ROS and oxidative stress. As the nAChR targets of these insecticides are conserved among insects, it is likely that similar impacts would be precipitated by exposures in other non-pest species, albeit at different doses. As insecticides from a wide range of chemical classes create markers of oxidative damage, the low dose mechanism of action observed for imidacloprid and spinosad may apply more broadly. This requires investigation. Considered together, the low dose impacts of imidacloprid and spinosad severely impair insect biology, without necessarily killing. These impairments could render insect species more vulnerable to the other major threats proposed to contribute to the decline of populations: climate change, habitat loss, pathogens, and parasites.
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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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ItemWolbachia fitness benefits and symbiont interactions in Drosophila( 2017)Wolbachia are Alphaproteobacteria that inhabit vacuoles within cells of insects and other arthropod hosts. In adapting to this nutrient-rich environment they have lost many metabolic functions possessed by free-living bacteria, and rely on maternal transmission via ova produced by host females for lineage continuity. Nevertheless, around one-third to one-half of insect species are estimated to harbour Wolbachia infection, making them likely the most abundant endosymbionts on earth. Although infection prevalence varies considerably across host species, Wolbachia often profoundly influence host biology, and are notorious for a diverse range of abilities to promote their own transmission by manipulating host reproduction. The most common such ability, known as cytoplasmic incompatibility (CI), entails a toxin-antitoxin type mechanism whereby Wolbachia present in male gonads secrete a factor that modifies developing sperm. If the same Wolbachia strain is present in eggs fertilized by such sperm, a second Wolbachia-derived factor can affect rescue of the modification. This disadvantages females lacking Wolbachia, because if they mate by chance with an infected male, their offspring (or a large proportion) will not be viable, whereas offspring from other mating types are expected to be unharmed. CI can drive the spread of Wolbachia infection through a host population, but only in a frequency-dependent fashion. Where infected males are initially rare, the population-wide effect of CI may be quite small and in itself insufficient to outweigh occasional maternal transmission loss, or reduced fitness of infected individuals if, as expected, presence of Wolbachia imposes some metabolic cost to host resources. This leads to a prediction of bistable dynamics with a threshold infection frequency—above which infection tends to spread, but below which infection tends to be lost from the population over time. Spatial spread of bistable infection is predicted to be relatively slow and easily stopped by geographic barriers or variations in host population density. Importantly, bistability does not readily explain how Wolbachia infection can spread in new host populations from arbitrarily low initial frequencies. Recently, several Wolbachia strains have also been shown to have antiviral effects in their native hosts, as well as when transinfected into novel hosts. This phenomenon has tremendous potential for applied use to combat spread of insect-vectored disease; in particular the wMel strain of Wolbachia, native to Drosophila melanogaster, has been shown to effectively block replication of dengue virus—the most significant arbovirus of humans—when transinfected into the principal dengue vector, Aedes aegypti. Field releases of wMel-infected Ae. aegypti are currently underway. The wMel infection induces strong CI in this novel host, but is otherwise somewhat costly to fitness and therefore predicted (and observed) to exhibit bistable dynamics. By contrast, increasing evidence suggests that naturally-occurring Wolbachia infections routinely enhance host fitness independent of CI. This thesis examines effects of native Wolbachia infections on host fitness in the model organisms, Drosophila melanogaster and D. simulans. Chapter 1 introduces Wolbachia and the phenomenon of endosymbiosis generally. Chapter 2 provides some of the clearest evidence yet of rapid frequency-independent spread of Wolbachia through natural host populations from what are likely to have been point sources of introduction. The magnitude of positive fitness effects for two Wolbachia variants in eastern Australian D. simulans are estimated. Findings here have led to a reinterpretation of Wolbachia infection dynamics previously observed in California. Chapter 3 documents the persistence over at least 20 years of a cline in wMel infection frequency in D. melanogaster in eastern Australia, and examines fitness effects of harbouring this infection during periods of reproductive dormancy as a possible causative mechanism. Chapter 4 seeks to recapitulate dynamics of wAu and wRi infection in D. simulans field populations inferred from chapter 2 within large experimental cages, and finds fitness enhancement for both infections of even greater magnitude in the experimental population context. This work further explores the hypothesis that inhibition of common viral pathogens is the principal mechanism of Wolbachia enhancement of host fitness. Taken together, these findings add to our understanding of Wolbachia infection dynamics that may have important implications for future applied programs. Finally, chapter 5 discusses potentially useful future directions for this and the broader field of Wolbachia research.
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ItemNetrinA and Frazzled in regulation of wing disc epithelia( 2017)The chemotrophic factor Netrin and its receptors play a critical role during axon outgrowth, organogenesis and cancer progression. In my PhD I have found that Drosophila NetrinA and its receptor Frazzled also control epithelial-mesenchymal transition (EMT), a key process of embryogenesis, regeneration and tumor metastasis. Using wing imaginal disc eversion as a genetic model, I have demonstrated that both loss of netrinA and elevation of frazzled expression in the peripodial epithelium suppress zonula adherens dissociation and alter cytoskeleton organization, but do not affect basement membrane degradation. Loss-of-function and overexpression analysis of frazzled in the disc proper epithelium shown that Frazzled controls localisation of junctional proteins (E-Cadherin and β-catenin), F-actin polymerization and cellular contractility. A key question is whether the Frazzled signaling pathways that regulate cell motility are distinct from those that control epithelial phenotypes. To address this I have conducted a structure function analysis to determine which intracellular domains of Frazzled are required for inhibiting wing eversion, and which are required for regulating E-Cadherin, F-Actin and cell shape. I shown that P1 domain is responsible for eversion disruption, E-Cadherin delocalization and cellular contractility, but not for formation of the F-actin protrusions. P3 domain is required for cell motility. Finally, I performed an RNAi screen to identify components acting downstream of NetrinA/Frazzled signaling. I have found that the apical polarity protein Par6 is required for inhibiting EMT and partly for E-Cadherin delocalization. The results establish a new role for Netrin signaling in a developmental EMT and highlight the complexity of NetrinA/Frazzled pathway regulation of epithelial cells.
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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.