School of BioSciences - Theses
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ItemComparative Genetics of Invertebrate Moulting( 2022)The development of molecular phylogenetics in the 1990s led, among many other discoveries, to the finding of a monophyletic clade of moulting invertebrates dubbed Ecdysozoa. Including arthropods and nematodes (alongside numerous smaller groups) Ecdysozoa is unrivalled among living groups for the number of species it contains and for the diversity of these species; moulting is the only non–molecular trait known to be common to all modern ecdysozoans. However, our understanding of how moulting is regulated draws on only a very small number of species, with the vast majority of these being insects of economic importance. Naturally, the sheer size of Ecdysozoa precludes a comprehensive investigation of moulting regulation across this clade; furthermore, many ecdysozoan groups (for instance, aquatic taxa) would be far less suited to laboratory culture and manipulation than the average insect. However, ecdysozoan genome sequencing, once largely restricted to genetic model organisms, pest species and disease vectors, has begun to extend across a far broader range of taxa. This thesis reports my exploration of molecular data in the context of the evolution of moulting. The Ecdysozoa hypothesis was accepted quickly by most workers in arthropod taxonomics, but this should not lead us to underestimate just how significant a change the embrace of Ecdysozoa represented. The better part of a century of investigations into the molecular basis of moulting had been conducted under the assumption that the non–moulting annelid worms were the sister taxon to arthropods, and thus that moulting had either evolved repeatedly or had been lost repeatedly among invertebrates. In order to explain this historical context, I have produced a review of the history of arthropod taxonomy in the Western tradition. This account runs from Aristotle to the present day, but has a particular focus on the development of the Ecdysozoa hypothesis. My experimental work on the genetics of moulting regulation began with an examination of the cytochrome P450 oxidase family CYP307 in the fruit fly genus Drosophila. Although the precise catalytic role of these enzymes has proved elusive, they are required for the synthesis of ecdysteroids, the master moulting regulators of arthropods. It has been known for around fifteen years that multiple duplications in the CYP307 family have occurred in Drosophila, but the functional significance of these duplications has been unclear. I have attempted to address this question by transgenic substitution of CYP307 paralogs, finding that even enzymes which are closely related at the level of amino acid sequence are not functionally interchangeable. The CYP307 family is only one of many acting in the synthesis of ecdysteroids from dietary sterols; almost all of these were first identified in D. melanogaster, and although comparative analyses have generally suggested good conservation of these enzymes across arthropods, exceptions are known. Building on the CYP307 investigation and its suggestion of rapid functional differentiation following duplications in this family, I sought to identify all duplications and losses of ecdysteroid synthesis genes across all available arthropod genomes (at the time I ceased collecting data, 923 genera were represented by at least one genome assembly). In some cases, I was able to connect observed copy number changes to ecological factors (e.g. whether the predominant dietary sterol was of animal, plant or fungal origin), but many duplications and deletions were entirely unexpected and suggestive of additional changes which require further investigation (e.g. replacement of one enzyme with another having similar activity). In some cases, I went on to analyse selective pressures acting on relevant components of the ecdysteroid synthesis and signalling pathways to determine the effects of loss or duplication of ecdysteroidogenic genes. Despite the numerous copy number changes I observed, my findings were generally consistent with the strong conservation of ecdysteroid synthesis genes described by earlier researchers. As a consequence of this conservation, arthropods provide limited evidence concerning the early evolution of ecdysteroid synthesis. This limitation makes the recent release of genome assemblies from lineages closely related to Arthropoda (namely velvet worms and tardigrades) particularly important for understanding ecdysteroid evolution. I used a transgenic approach similar to that previously applied to Drosophila CYP307 enzymes to investigate the function of four cytochrome P450s from the tardigrade Hypsibius exemplaris which showed strong similarity to ecdysteroidogenic enzymes at the amino acid level. I found that H. exemplaris CYP315A1 does not rescue D. melanogaster Cyp315a1 nulls (despite their clear orthology) and that H. exemplaris CYP18K1 overexpression produces a phenotype quite distinct from that associated with D. melanogaster CYP18A1 overexpression. Tardigrades are unlikely to synthesise ecdysteroids, but they, along with representatives of other ecdysozoan taxa such as the priapulids (penis worms), may provide insights into the diverse steroid metabolic pathways of Ecdysozoa. My research has focused on the potential for integration of large–scale molecular analysis with experimentation in tractable model organisms as a means of understanding ancient evolutionary events. While my cross–arthropod screen revealed many changes to the ecdysteroid synthesis pathway which remain to be fully examined, the results of my transgenic experiments demonstrate the use of bioinformatic approaches in identifying promising targets for more direct examination. The moulting process is one of considerable interest both because of its practical importance (disruption of moulting is potentially a potent insecticidal technique) and because of the intrinsic fascination of the morphological changes it enables. However, the combined bioinformatic and transgenic approach I have used could be applied to any process occurring in a taxon from which a model organism has been established.
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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.