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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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.