Despite the benefits of sexual reproduction, sharing a genome can put constraints on the evolvability of a species. This is due to sexual conflict, where the interests of each sex is in direct opposition to one another, and the benefit of one sex can be the cost of the other sex (i.e. sexual antagonism). Sex chromosomes have been the focus of much of the research done on sexual conflict due to their unique nature and are particularly interesting in the context of sexually antagonistic variance. In the first experiment (Chapter 2), I used experimental evolution to investigate the standing sexually antagonistic variation on the X-chromosome of the common vinegar fly, Drosophila melanogaster. Unlike most other experimental evolution experiments where selection has been limited to males, I limited the inheritance of the X-chromosome to females only. I used a non-recombining Xchromosome balancer to control the inheritance of the female-limited X-chromosome. Throughout the evolution experiment, I tested different phenotypic traits that have previously been shown to be sexual antagonistic, as well as investigating how the transcriptome changed through female-limited selection (Chapter 3). The results were mixed but indicated that limiting selection of the X-chromosome to females could, to some extent, change the antagonistic variation and move traits towards the female optimum. In the second experiment (Chapter 4), I exchanged sex chromosomes between populations with divergent geographic origins. I used flies with special genetic constructs (e.g. autosomal balancers, fused-X chromosomes) to control the population crosses, so that sex chromosomes were introduced into a new background without any prior interaction. I found that introducing a novel sex chromosome increased male reproductive fitness through improved sperm competition at the cost of offspring viability. 25 generations after introducing the novel sex chromosome (Chapter 5), the increase in male fitness was undetectable and their fitness was again the same as the wild types. Collectively, this indicates an antagonistic coevolution between the sex chromosomes. Together, these two experiments shed new light on sexual conflict and the antagonistic coevolution between the sexes at the genetic level, both between and within the sex chromosomes. These novel insights could help further the understanding of how sex chromosomes may affect speciation.

Although the X chromosome is usually similar to the autosomes in size, gene density and cytogenetic appearance, theoretical models predict that its hemizygosity in males may cause unusual patterns of evolution. The sequencing of several genomes has indeed revealed differences between the X chromosome and the autosomes in the rates of gene divergence, patterns of gene expression and rates of gene movement between chromosomes. In this thesis, I have attempted to investigate some of these patterns and their possible causes. The first two chapters consist of theoretical and empirical work intended to analyse the rates of evolution of coding sequences of X-linked and autosomal loci, with particular emphasis on faster-X evolution, the theory that more effective selection on the X can lead to higher rates of adaptive evolution on this chromosome. By analyzing X-linked and autosomal coding sequence in several species of Drosophila, we found some evidence for more effective selection on the X, particularly evident in the higher levels of codon usage bias detected at X-linked loci. We argue that this could be due to higher levels of recombination on the X chromosome increasing its effective population size (NeX) relative to the autosomal effective population size (NeA). To further investigate this hypothesis, we have modeled the effect of increased NeX/NeA on rates of evolution and confirmed that this can contribute to faster-X evolution. The last two chapters deal with the evolution of sex-biased genes and the possible causes for their differential accumulation on the X. We used EST data to create expression profiles for D. melanogaster male-, female- and unbiased genes. Our results suggest that the expression levels of sex-biased genes are incompatible with the accepted iii model of sex-biased gene evolution. We also show that the deficit of testis-expressed genes that is observed in Drosophila seems to be stronger for highly expressed genes. In fact, for very lowly expressed genes, we observe a small excess of testis-expressed genes on the X. We attempt to discuss this pattern in view of what is currently known about the evolution of sex-biased gene expression.

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound.

Atg8 proteins play a major role in autophagy. Atg8 is involved in the formation of autophagosome and also, serves to recruit selective autophagy receptors, autophagy regulators and autophagy substrates. These receptors, regulators and substrates are characterized by the presence of the LIR motif that mediates Atg8 interaction. Despite the discovery of several Atg8 interactors in human and yeast, knowledge of these interactors in Drosophila is limited. The known Atg8a interactors (Drosophila homolog of Atg8) in Drosophila include Ref(2)P, functions in selective autophagy of ubiquitinated substrates whereas Atg1, functions in autophagy initiation. We searched for novel Atg8a interactors in Drosophila and their role in selective autophagy of ubiquitinated protein aggregates and autophagy regulation. Using a bioinformatics approach, we identified novel putative LIR-containing proteins in Drosophila proteome and characterized three of the promising candidates in vivo using various biochemical and molecular biology techniques. One of the candidates was UbcD4, a ubiquitin ligase containing putative LIR and UBA (involved in ubiquitin binding) domains. Despite this, we could not detect UbcD4 interaction with Atg8a, suggesting that the putative LIR is not functional. Localization and western blot analysis showed that UbcD4 accumulates as aggregates under Atg8a-deficienct conditions and colocalizes with various markers of protein aggregation. Knockdown of UbcD4 indicated that UbcD4 mediates aggregate formation in old flies but not in young flies. Thus, we identified a novel component of ubiquitinated protein aggregates that mediate aggregate formation in Drosophila brains under autophagy-deficient condition. The second candidate was PAR1, a serine/tyrosine kinase with putative LIR and UBA domains. Interaction studies demonstrated that PAR1 interacts with Atg8a. Further, PAR1 is not a major component of protein aggregates formed in response to Atg8-deficiency, demonstrating that PAR1 is not found with ubiquitinated protein aggregates and thus does not participate in the removal of ubiquitinated proteins through ubiquitin-dependent selective autophagy. Further, we showed that overexpression of kinase-dead PAR1 induces mCherry-Atg8a puncta (a marker of autophagy) under fed condition, suggesting a novel role of PAR1 in autophagy regulation. The third candidate was Sequoia, a putative LIR, and ZnF-C2H2 domains (involved in DNA-binding) protein. Sequoia interacts with Atg8a, and it does not accumulate under Atg8a-deficient condition. Knockdown of Sequoia and overexpression of LIR-mutated Sequoia induces autophagy under fed conditions in the larval fat body. Additionally, expression analysis indicated that Sequoia acts as a repressor of Atg7. Thus, we identified a novel Atg8-ineracting protein that negatively regulates autophagy under fed condition. To further the understanding of selective autophagy in Drosophila, we characterized the role of Ref(2)P in mediating the cytotoxicity associated with the expression of mutant huntingtin (Htt), a toxic protein that accumulates in Huntington’s disease (HD) brains. We examined whether overexpression of Ref(2)P ameliorates HD-associated phenotypes in a Drosophila HD model. This HD model faithfully recapitulates HD-associated phenotypes such as Htt aggregation, motor dysfunction, and short lifespan. It was observed that overexpression of Ref(2)P does not rescue the above-mentioned HD-associated phenotypes. These analyses suggest that selective autophagy receptor Ref(2)P does not mediate Htt-induced toxicity in Drosophila. Finally, a label-free shotgun proteomics was employed to identify proteins with increased accumulation in Atg8a-deficient versus wild-type conditions to further the knowledge of autophagy mechanisms. An increase in the accumulation of 69 and 57 proteins was identified in Triton-soluble and Triton-insoluble fraction respectively. These proteins may rely on autophagy for their degradation. These proteins were further searched computationally for the occurrence of the putative LIR motif. The results presented here open the pathway for the discovery of novel Atg8a interactors and autophagy substrates and thus provide insights into novel mechanisms of autophagy in Drosophila.

Drosophila is a valuable experimental organism can be used as a reverse genetics model. Drosophila Malpighian (renal) tubules are important epithelial tissue in which to study transport mechanisms. RNA-seq has been chosen to investigate Drosophila Malpighian (renal) tubules to identify novel genes following a three- way comparison between three popular transcriptome profiling methods. Two types of novel gene have been found in Drosophila tubules, coding genes and noncoding genes. Reverse genetics has been applied to identify novel coding gene function in Drosophila tubules. Three-way analysis of Drosophila expression microarrays, Drosophila tiling micrarrays and Drosophila RNA-seq reveal that most gene expression levels are well correlated between the three technologies. Drosophila expression microarrays and RNA-seq are correlated better than the correlation between Drosophila tiling microarrays and RNA-seq. Drosophila expression arrays and Drosophila tiling arrays all suffered from cross-hybridization, miss target detection and hybridization background noise, and also have low dynamic range for detecting lowly and highly expressed genes. Drosophila tiling microarrays also have a high false-positive detection rate, which may lead to overestimate the transcriptional activities of the genome. RNA-seq has overcome the drawbacks of microarrays and become the leading technology for genome sequencing, transcriptome profiling, novel gene discovery, and novel alternative splicing discovery with wide dynamic range. However, Drosophila expression microarrays and tiling microarrays still remain useful. Three-prime expression microarrays offer a means to measure the differential three-prime end processing, and tiling microarrays can be used for novel gene discovery. In this sense, the three technologies complement each other. Poly(A) selected RNA-seq has been used as a discovery tool for searching novel genes in Drosophila Malpighian tubules in this thesis. A TopHat and Cufflinks pipeline has been used as an analytical pipeline for novel gene discovery and differential gene expression analysis between Drosophila tubules and whole flies in order to find the tubule-enriched genes. Reverse genetics has been applied to Drosophila to achieve a gene knockdown and overexpression by using the unique Gal4/UAS system to achieve the novel gene knockdown or overexpression in specific tissue and cell types. Novel coding gene CG43968 has been discovered. The location of this gene has been confirmed in tubule main segments, principle cell cytoplasm or apical membrane. The function of this gene has been identified as involvement in tubule secretion, which may relate to calcium transport. Reverse genetics has been confirmed as particularly important for the functional study of novel genes.

I have analysed the regulation and function of the Him gene to gain new insights into Drosophila heart development and its controlling factors. My results show that Him is important during the early specification of pericardial cells and cardioblasts. Loss of Him leads to a reduced number in both of these cell types by the end of embryogenesis. Over-expression of Him throughout the heart results in supernumerary pericardial cells. Him is expressed in all embryonic pericardial cells from embryonic stage 12 to approximately stage 15. I have identified an enhancer fragment that reproduces this expression pattern. Phylogenetic footprinting revealed three highly conserved regions within this sequence. I undertook an extensive mutational analysis of this enhancer to identify regulatory elements within it. I identified Tinman as a direct activator of Him expression. My data indicate that Him is activated in a widespread area of the dorsal mesoderm and the amnioserosa and is actively limited to the pericardial cells. A 5 bp mutation within the enhancer sequence allows for expression within the cardioblasts. Both heart cell types develop from the dorsal mesoderm and some share immediate progenitors. By stage 13, Him is pericardial cell specific and Mef2 is cardioblast specific. This is essential for normal heart development. If Him is not excluded from the cardioblasts, expression of the muscle-cell specific differentiation gene myosin is disrupted, similar to what has been described for Mef2 null mutants. If Mef2 is expressed in pericardial cells, the larval development of the pericardial cells is severely disturbed. A possible explanation for these data is that Him is part of a genetic program that prevents the premature differentiation of heart cells and its down-regulation permits the pericardial cells to undergo their correct development.

Organisms face a constant risk of attack from parasites. While classic immunology has revealed numerous physiological and molecular mechanisms that underpin host immunity, the recently developed field of ecological immunology has attempted to understand the ecological and evolutionary causes that explain the diversity of such immune mechanisms. However, progress in the field has been hampered by the complex relationship between immunity and fitness as well as the methodological limitations of our experiments. There is an urgent need for eco-immunological studies that combine life history theory with experimentally tractable but ecologically realistic host and pathogen models. In this thesis, I tackle three novel aspects of host defence against parasites in an established model for insect immunity, the fruit fly Drosophila melanogaster, with the entomopathogenic fungus Metarhizium robertsii, one of the most successful natural insect pathogens. In particular, I show in Chapter 2 that an immune and stress response gene, Turandot M, provides specific immunity against sexually transmitted fungal infections; but, this protective effect comes at a cost to life history in the absence of infection. In Chapter 3, I show that when exposed to the fungal pathogen, the fruit fly alters its temperature preference by seeking out cooler temperatures, which results in a dramatic shift in its life history strategy while simultaneously enhancing antifungal resistance, though not tolerance. Finally, I demonstrate in Chapter 4 that exposure to fungal parasites induces fitness-associated maternal effects on offspring meiotic recombination and life history, both of which have the potential to accelerate adaptive evolution. Taken together, these results demonstrate the benefits of integrating life history theory in eco-immunological research. They show that life history responses are an integral component of host defence against parasites, and that Drosophila-Metarhizium is a promising model system for ecological immunology.

I present a novel method for the unsupervised discovery of behavioural motifs in larval Drosophila melanogaster and Caenorhabditis elegans. Most current approaches to behavioural annotation suffer from the requirement of training data. As a result, automated programs carry the same observational biases as the humans who have annotated the data. The key novel element of my work is that it does not require training data; rather, behavioural motifs are discovered from the data itself. The method is based on an eigenshape representation of posture. Hence, my approach is called the eigenshape annotator (ESA). First, I examine the annotation consistency for a specific behaviour, the Omega turn of C. elegans, and find significant inconsistency in both expert annotation and the various Omega turn detection algorithms. This finding highlights the need for unbiased tools to study behaviour. A behavioural motif is defined as a particular sequence of postures that recurs frequently. In ESA, posture is represented by an eigenshape time series, and motifs are discovered in this representation. To find motifs, the time series is segmented, and the resulting segments are then clustered. The result is a set of self-similar time series segments, i.e. motifs. The advantage of this novel framework over the popular sliding windows approaches is twofold. First, it does not rely on the ‘closest neighbours’ definition of motifs, by which every motif has exactly two instances. Second, it does not require the assumption of exactly equal length for motifs of the same class. Behavioural motifs discovered using the segmentation-clustering framework are used as the basis of the ESA annotator. ESA is fully probabilistic, therefore avoiding rigid threshold values and allowing classification uncertainty to be quantified. I apply eigenshape annotation to both larval Drosophila and C. elegans, and produce a close match to hand annotation of behavioural states. However, many behavioural events cannot be unambiguously classified. By comparing the results to eigenshape annotation of an artificial agent’s behaviour, I argue that the ambiguity is due to greater continuity between behavioural states than is generally assumed for these organisms.

Drosophila melanogaster has been an important model organism for over a century, cumulating in a vast array of mutant and transgenic stocks, the publication of the genome, its subsequent annotation and more recently the production of the online gene expression database, FlyAtlas. Much of what we know about developmental biology was pioneered in Drosophila and it is possibly the most well studied and understood model organism, in terms of development, genetics and physiology. The so-called ‘omics’ era of biology has resulted in a relatively data poor discipline quickly becoming a data rich one. Therefore the need for a good model organism, which offers the balance between genetic power and relevance has never been more important, as scientists begin to evaluate and analysis this data. We will argue that Drosophila melanogaster offers the best opportunity to study the relevance of omics data. FlyAtlas is an online resource, which allows scientists to look at tissue specific gene expression in the fruit fly Drosophila melanogaster. Unexpected expression patterns of previously characterised genes may hint at novel functions, thus helping to close the phenotype gap. To test this hypothesis we looked at the neuronal gene Fasciclin 2 (fas2), which has been exhaustively characterised (over 500 papers), with neural functions ranging from axonal growth in development to synapse stabilization in the adult. Surprisingly FlyAtlas showed fas2 is predominately expressed in the Malpighian tubule (a renal, rather than neural, tissue), hinting at a previously unreported function in this tissue. Results suggest fas2 may play an important role in apical microvilli development and stability in the principal cells of the tubules. We have also shown that Fas2 may be involved in actin localisation. Fas2 shows dynamic localisation in response to cAMP and over expression of the protein results in a significant increase in secretion when tubules are stimulated with cAMP. We also present evidence that Fas2 co-localises with F-Actin bundles in response to cAMP, hinting at a role for the actin cytoskeleton in secretion. Proteomics experiments carried out in order to determine Fas2’s, interacting partners proved problematic. For this reason 2D Blue Native PAGE and sucrose gradient techniques were optimised in order to facilitate this problem. 4 Unfortunately we were unable to isolate Fas2, however we have shown that BN-PAGE offers a robust protocol for the isolation of protein/protein complexes. We can also conclude from these experiments that 2D BN-PAGE offers an ideal comparative data source for transcriptomics data such as FlyAtlas. The second gene tested in this study is the sex determination transcription factor Doublesex (dsx). Dsx has been extensively studied in its role in differentiation of both the soma and to some extent the nervous system in males and females. FlyAtlas results indicate that it is also expressed in the Malpighian tubules, again hinting at previously unknown function in this tissue. Further to this the male and female transcripts of dsx are expressed in a sex specific manner. Our results confirm these observations and dsx was localised to the principal cells of the main and lower segments of the tubules. Male tubules however do not express dsx in the transitional segment whereas females do, suggesting that perhaps this segment of the tubule constitutes a previously unknown sex specific function. We have determined that Tra RNAi is effective at knocking down the female transcript in female tubules, allowing for the study of masculinised tubules in an otherwise female fly. Experiments concluded that although males and females show differential survival in response to bacterial infection, this is not controlled by dsx expression in the tubules. Preliminary results also suggest that two genes CG8719 and YP3 are differentially expressed in male and female tubules and offer ideal candidates to study dsx role in sexually dimorphic gene expression in the tubules. In conclusion this study verifies the use of FlyAtlas to determine novel functions for well-known genes in D.melanogaster. In turn this indicates the importance of omics data, as a staring point for further functional analysis of both genes and proteins.

fruitless (fru) is a pleiotropic gene which produces an array of variant transcription factor isoforms to fulfil a range of developmental and behavioural roles, from the regulation of axonal pathfinding during embryogenesis, all the way to the precise orchestration of individual steps of the Drosophila melanogaster male courtship ritual. Much of the transcriptional differentiation is achieved through the use of multiple promoters, sex-specific splicing and variant C-termini. Alternative splicing at the 3′ end enables generation of transcripts containing one of at least three zinc finger (Zn-F) domains (A, B or C). Due to the close proximity of these Zn-F–encoding exons at the fru locus, almost all extant fru mutants reduce or eliminate expression of all isoforms from a given promoter(s). This has prevented genetic dissection of their individual roles, and limited functional assignment to the zinc finger triumvirate as a whole. Using gene targeting by homologous recombination, this project set out to generate precise null mutations for type-A and -B Fru isoforms in an attempt to determine which aspects of fru function are conferred by these isoforms. Isoform-specifc antibodies were also generated to confirm the loss of individual isoforms within the generated mutants, and to investigate the expression of different isoforms throughout development. Generation of such an antibody to FruB proteins enabled the developmental expression pattern of this isoform to be assessed for the first time, and expression in the male-specific serotonergic neurons of the abdominal ganglion suggested a possible role for male-specifc FruB isoforms in male fertility. Investigation of the developmental expression patterns of FruC revealed a novel immunostaining pattern for this isoform in a group of coalescing cells which appear towards the end of embryonic development. Isolation of specific-isoform mutants was achieved, giving rise to multiple mutant phenotypes, varying in severity between mutant lines. Analysis of mutants lacking type-A and -B Fru isoforms demonstrated the importance male-specific type-A and/or type-B isoforms in establishing male fertility, and suggested essential roles for sex-non-specifc type-A and/or type-B isoforms in the viability and morphology of both sexes.