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Condensins are multi-protein complexes that have key functions such as chromosome compaction, regulation of gene expression and 3D chromatin conformations. Despite the wealth of knowledge on condensins in dividing cells, their role in post-mitotic, specialised cells remains largely unknown. In this thesis I explore a novel role for condensin I subunit Cap-G in Drosophila neurons. In vivo evidence suggests that condensin I subunits Cap-G and Barren are expressed and localised in neurons. The generation of an exclusively post-mitotic, neuron-specific knockdown of Cap-G revealed an exciting role for condensin in maintaining neuronal identity and health. Removal of Cap-G from post-mitotic neurons resulted in severe developmental arrest, behavioural defects and an overall disruption of gene expression. Furthermore, knockdown animals exhibited a downregulation of neuron-specific genes and an upregulation of ectopic, non-neuron specific genes, suggesting loss of neuronal identity. Cap-G DNA binding profiles in neuronal stem cells and neurons reveal dynamic, cell-specific binding of Cap-G across cell types. Moreover, genes bound by Cap-G significantly overlap with the disrupted genes detected in knockdown animals. Overall, the evidence suggests that Cap-G is necessary for healthy neuronal development and maturation and has a neuron-specific, gene regulatory role in Drosophila. The work in this thesis provides the first evidence that condensin is necessary in post-mitotic neurons for transcriptome stability and maintenance of neuronal identity.

Centrosomes are the main microtubule-organising centres in many animal cells. They consist of a pair of centrioles surrounded by a dense mass of protein known as the pericentriolar material. As the cell enters mitosis, this pericentriolar material expands dramatically in a process called centrosome maturation. In flies, the protein Centrosomin (Cnn) assembles into a network that acts as a scaffold for the recruitment of the hundreds of other proteins making up a mature centrosome. Cnn network formation is known to depend on phosphorylation of a central region within Cnn. However, it is unclear how Cnn assembles into a network and how this process is controlled. In this thesis it is shown that Cnn network formation in vitro depends on an interaction between the conserved C-terminus of Cnn and the phospho-regulated central region. The crystal structure of part of this central region is solved, revealing that it forms a dimer of α-helical hairpins that are sterically incompatible with binding of the Cnn C-terminus. The structure is therefore proposed to constitute an “inactive” state. Furthermore, the structure suggests a mechanism wherein phosphorylation acts by disrupting the hairpin fold, thereby exposing the Cnn C-terminus binding site and hence allowing formation of an “assembly-competent” state where the Cnn C-terminus is bound. In addition, the calcium-binding protein Calmodulin is shown to crosslink the Cnn C-terminus in crystallo. However, Calmodulin inhibits network assembly of Cnn constructs in solution, possibly by preventing the assembly-competent state from forming a network. Taken together, this thesis draws on structural and functional data to provide important atomic-level insights into the mechanism of mitotic centrosome assembly in flies.

The nervous system of most animals is a highly complex network that transmits signals throughout the organism. The complexity of the human nervous system can, at times, appear inscrutable; therefore, it is practical to break it down into fewer components and to study a simplified, yet still complex analogue. This thesis utilises the fruit fly Drosophila melanogaster as a model to forward our understanding on neuronal development and maintenance. Neurons are the most modular of all cells, comprising of a cell body, axon and dendrites. The development and function of each compartment relies heavily on the cytoskeleton, a major component of which are microtubules, which are required for intracellular transport, as well as establishing neuronal morphology. In this thesis, I identify microtubule regulators that are necessary for the dendritic development of Drosophila sensory dendritic arborisation neurons, where I show that the minus-end targeting protein, Patronin, prevents unregulated minus-end growth within dendrites. Unlike in mammalian interphase cells, Drosophila Patronin does not function at Golgi within da neurons. Depletion of Patronin from class I da neurons results in dendrite over-branching and excessive growth of microtubules away from the soma. Given that most microtubules have their minus ends facing away from the soma in the dendrites of these neurons, I propose that the absence of Patronin leads to the unregulated growth of minus-ends away from the soma that invade terminal dendrites and prevent their retraction. I also find that that the microtubule depolymerising enzyme, Klp10A does not function in class I dendrites, and that overexpression is sufficient to rescue anterograde minus-end polymerisation that results from Patronin depletion. These findings are the first to show a role for Patronin in microtubule minus-end growth restriction in vivo, and further increase our understanding of microtubule regulation in the nervous system.

Operant conditioning is the process by which animals learn to associate their own behaviour with positive or negative outcomes, biasing future action selection in order to maximise reward and avoid punishment. It is an important strategy to ensure survival in an ever-changing environment. Although operant conditioning has been observed across vertebrate and invertebrate species, the underlying neural mechanisms are still not fully understood. The Drosophila larva is an excellent model system to study neural circuits, since it is genetically tractable, with a variety of tools available. Although it is quite small, it is capable of a diverse range of behaviours and can achieve complex learning tasks. However, while the mechanisms underlying classical conditioning, where animals learn about the appetitive or aversive qualities of an external sensory cue, have been extensively studied in larvae, it has remained an open question whether they are capable of operant conditioning. This is in part due to the challenges which arise during the training process: in order to train an animal to associate its own actions with their outcomes, the experimenter needs to be able to deliver rewarding or punishing stimuli directly in response to behaviour. In this thesis, I introduce a novel high-throughput tracker suitable for training up to 16 larvae simultaneously. I have developed a customised software for real-time detection of various actions that larvae perform: left and right bend, forward crawl, roll and back-up. Light and heat stimuli can be administered at individual animals with minimal delay, enabling optogenetic or thermogenetic activation of circuits encoding reward or punishment in response to behaviour. Using this system, I show that Drosophila larvae are capable of operant conditioning. Pairing bends to one direction, e.g. the left, with optogenetic activation of a large group of reward-encoding dopaminergic and serotonergic neurons is sufficient to induce a learned preference for bending towards this side after training. I explore whether there are other types of actions which larvae can learn to associate with valence, and introduce a second operant conditioning paradigm, in which larvae modify their behaviour following pairing of the stimulus with forward crawls. To identify new candidate neurons signalling valence in a learning context, I also conduct a classical conditioning screen, in which I pair an odour with optogenetic activation of distinct neuron types covered by different driver lines. While activation of many types of gustatory sensory neurons paired with the odour was insufficient for memory formation, I find that the serotonergic neurons of the brain and the subesophageal zone (SEZ) can induce strong appetitive learning. Finally, I show that activity of serotonergic rather than dopaminergic neurons is sufficient for memory formation in the operant bend direction paradigm, and that operant conditioning is impaired when restricting activation to the serotonergic neurons of the brain and the SEZ. My results suggest a novel role of serotonergic neurons for learning in insects as well as the existence of learning circuits outside of the mushroom body. Different subsets of serotonergic neurons mediate classical and operant conditioning. This works lays a foundation for future studies of the function of serotonin and the mechanisms underlying operant conditioning at both circuit level and cellular level.

Novel vector control measures such as the release of Wolbachia-infected Aedes aegypti offer a promising new pathway for dengue control. However, to realistically model the likely impact of these measures, improved mathematical models of Aedes aegypti population dynamics are needed, with the final goal being spatially explicit models of Aedes aegypti population dynamics, calibrated against high quality entomological data. Here we begin to address the challenge of developing such models by (i) examining the role of spatial structure in shaping the dynamics of Aedes aegypti populations at fine spatial scales and (ii) using advanced inferential methods to fit a dynamical model of Aedes aegypti population dynamics to entomological field data, while allowing for the highly variable nature of mosquito trapping data. We explore the effects of larval breeding habitat fragmentation on fine-scale Aedes aegypti population dynamics for a variety of different landscapes, examining how features of the underlying landscape and the dispersal behaviour of the mosquito affect the dynamics observed. In addition, by modelling the same population at different levels of spatial granularity, we investigate the appropriate level of spatial granularity for models to adopt to represent the fine-scale dynamics of Aedes aegypti populations. We examine the results of a small-scale field trial testing the use of Wolbachia as a tool for Aedes aegypti population suppression in Singapore by calibrating a stochastic model of Aedes aegypti population dynamics against the detailed entomological data collected during the trial. We model both the underlying population dynamics and the trapping process, thereby accounting for variability in mosquito trapping data when estimating the impact of the trial on local Aedes aegypti populations. Thus, the work presented in this thesis represents an important step forward in the challenge of developing models needed to realistically assess the likely impact of novel vector control measures.

The goal of this project is to address the phylogeny and evolutionary biology for one of the most diverse and species-rich genera of flower flies (Diptera: Syrphidae), Platycheirus LePeletier & Serville. Although studied taxonomically for a long time, the phylogenetic relationships of its species groups, subgroups and species are not well understood; this is especially the case for large genera of hoverflies. The COI gene is widely used to clarify phylogenetic relationships among taxa. A fragment of 658 base pairs of COI was analysed for 74 species, 72 Platycheirus and two outgroups (both Melanostoma spp.). The monophyly of the genus Platycheirus was well supported. However, phylogenetic relationships of the Platycheirus species groups and subgroups was still not completely clarified. We used phylogenetic comparative methods to study the evolution of quantitative traits of Platycheirus. First, we estimate the level of phylogenetic signal in both reproductive and morphological traits using Pagel's λ and Blomberg's K. We found that male reproductive traits tend to evolve rapidly and divergently with low phylogenetic signal relative to morphological traits, while most female reproductive traits do not. Second, we quantify the rate of evolution of six morphological and seven internal reproductive traits of species within seven other genera of Syrphidae using their estimated phylogenetic trees. Using a likelihood-based approach, the evolutionary rate estimates show clearly that the reproductive traits evolve about 110 times more rapidly than all morphological traits except proboscis length; the latter exhibits a faster rate of evolution than other morphological traits, faster than some reproductive traits. These results provide quantitative evidence that male reproductive traits are less constrained phylogenetically, perhaps because they are under strong sexual selection. Third, the activity rhythms and foraging behaviours of Platycheirus species in relation to environmental conditions was studied in an evolutionary context. The foraging behaviour for some of the Platycheirus and Melanostoma species seen visiting flowers for nectar or pollen was recorded. Although tested with too few data, the results for both Pagel's and Blomberg's methods suggest that this behavioural trait is not constrained phylogenetically. Further, there was detectable phylogenetic signal in neither Pagel's λ nor Blomberg's K in indices of the responses of species to temperature. This indicates the greater lability of behavioural traits in contrast to morphological and reproductive characters. Finally, we used phylogenetic multivariate techniques to elucidate the main patterns of morphological evolution and their association with the evolution of reproductive traits among species of Platycheirus. Major evolved differences between species appear to lie primarily in relative proboscis length, and secondarily in relative wing size. The number of mature eggs stored in one ovary was positively correlated with relative proboscis length. There was a trade-off between ovariole number and egg size in females, but this was not associated with morphology except the width of the abdomen. Male traits showed a trade-off between testis length and the size of the accessory glands, but again this was not associated with morphology.

Cell competition is the process whereby less fit cells termed "losers" are selectively eliminated from a tissue by their fitter neighbors - or " winners." This sacrifice of aberrant cells is thought to have evolved at the advent of multicellularity to enforce cell co-operativity and ensure the fitness of the host organism. Accumulating evidence over the last 40 years has suggested key roles for cell competition during development, adult tissue homeostasis and at the onset and during the progression of diseases including cancer. However, if we are to exploit competition in the treatment of human pathologies and in tissue regeneration, we still have a lot to learn about the underlying mechanisms that ultimately instruct the elimination of loser cells. The main goal of this work was to identify the key molecular events that are responsible for initiating the loser status of Minute heterozygous cells. As many Minute mutations affect ribosomal genes, it has long been assumed that the loser status is closely linked to their associated slow growth phenotype, which occurs a consequence of reduced protein synthesis. Surprisingly, I have found that the loser status is independent of rates of translation. Instead, the activity of a single transcription factor, Nrf2, that typically co-ordinates an oxidative stress response, is sufficient to instruct the elimination of cells by their neighbors. Given the importance of Nrf2, I have sought to identify events occurring both upstream and downstream of the pathway in the loser context. Here, I have shown how multiple loser cell types are suffering from an underlying proteotoxic stress, as a result of an imbalance in proteostasis and their accumulation of toxic protein aggregates. In addition, I have developed a screening strategy to identify key molecules downstream of Nrf2 that could be involved in loser cell recognition. These findings not only provide new insights into the mechanisms of cell competition, but broaden the implications of the process to age-related diseases including those that result in neurodegeneration.

Drosophila suzukii (Matsamura) is an invasive dipteran pest of soft fruit crops. Native to Japan and SE Asia it was first detected in the Mediterranean growing regions of Europe and the western states of the USA in 2008. Since then it has been expanding its range across both continents causing huge economic damage to the horticultural industries there. Current control measures are heavily dependent on broad spectrum insecticides and labour intensive cultural control. Therefore, there is a large incentive to investigate alternative, more environmentally benign, control methods such as biological control or biopesticides. The viruses of D. suzukii offer a potential source of pathogens suitable for the development of such a biopesticide. Chapter 2 explores the diversity of viruses found naturally associating with D. suzukii in both its native and naturalised ranges. In it, I describe 18 new RNA viruses belonging to a variety of virus clades. Although none of these viruses belong to those clades traditionally used as biological control agents, we suggest further work for the development of a viral control agent based on our data. Not only are the viruses of D. suzukii of direct applied interest to the horticultural industry, they also offer a powerful model system for the study of virus host dynamics in the wild. The ecosystems recently invaded by this pest contain many other species of Drosophila which harbour their own raft of viral pathogens. In chapter 3 I explore the extent to which these viruses are shared between species and how virus prevalence changes over time. Understanding the patterns of virus 'host-shifts' after host range change could help us better predict the success of particular biological invasion events and further informs our understanding of emerging viral diseases in both humans and livestock. The ability of a virus to shift host ultimately comes down to its ability to overcome its host's immune system. In chapter 4 I investigate the comparative genome-wide transcriptomal immune responses of D. suzukii and its congener D. melanogaster after treatment with two highly divergent viruses. The relative responses of these flies was shown to be highly dissimilar as was the response of males and females of the same species. Few model species allow comparative expression studies of this depth granting us unprecedented insights into the evolution of insect innate immune systems.

Drosophila Protein Inhibitor of Activated STAT (PIAS) is required for chromatin organisation. PIAS proteins are SUMO E3 ligases that provide target specificity for the addition of small ubiquitin like modifiers (SUMO) to proteins to alter their function. Previous experiments in the Heun laboratory, which used a PIAS overexpression system, identified a relationship between PIAS and XNP (X-linked nuclear protein). Both proteins play a role in silencing transposable elements and likely interact. XNP and ADD1 (ADD (ATRX-DNMT3-DNMT3L) domain-containing protein 1) together make up the two halves of their mammalian homologue, the chromatin remodeller ATRX. XNP and ADD1 have been shown by us and others to be SUMOylated. We hypothesise that the SUMOylation of XNP by PIAS could be essential for its role in chromatin organisation and aimed to study this interaction within a Drosophila model using S2 tissue culture cells and embryos. In this thesis, I defined the interactomes of PIAS, ADD1 and XNP using immunoprecipitation-mass spectrometry (IP-MS) in the presence and absence of SUMOylation. Using CRISPR/Cas9 epitope tagged PIAS in S2 cell lines and primary antibodies against endogenous PIAS, I could confirm an interaction between PIAS and XNP in a SUMO dependent manner. Interestingly, I found PIAS interacts with ADD1 and heterochromatin protein 1 (HP1) in a SUMO independent manner. These interactomes support the hypothesis that SUMOylation of XNP by PIAS is involved in the association of XNP with ADD1 to promote heterochromatin organisation.

It is increasingly evident that mechanical forces play a vital role in regulating tissue development and homeostasis. During development, epithelia undergo complex morphogenesis, shaping organs by generating and responding to forces. The shape, organisation and numbers of epithelial cells are essential for overall tissue morphology. Furthermore, mature epithelia are continually subjected to stresses that may disrupt organisation of an organ. These epithelia must therefore respond to mechanical forces to maintain tissue integrity. I have used the pseudostratified epithelium (PSE) of the Drosophila wing disc to study the role of mechanical forces in regulating growth and form. PSE are found in organ precursors of many organisms due to their ability to confine the cell density required for morphogenesis. PSE tightly regulate the movement of nuclei to the apical surface prior to mitosis. This process is termed interkinetic nuclear migration (IKNM) and is essential for tissue organisation. In this thesis, I present potential mechanical regulation of IKNM dynamics through developmental increases in cell density. Such regulation may be important for regulating tissue growth. I find a role for the force generating machinery and lateral cell-cell adhesions in driving nuclear movement. These molecular effectors have differential effects on IKNM through development, suggesting that the dependency on these effectors changes in line with tissue architecture. In the second part of this thesis, I characterise a stretch-sensitive polarisation of Myosin that is downstream of Rho-mediated actin polymerisation, via Diaphanous. Cortical actomyosin is enriched at stretched junctions, in order to resist cell shape deformations and maintain epithelial integrity. I find that upon sustained stretch, actin remodelling is vital to stabilise new cell shapes. This likely enables cells to endure forces generated during morphogenesis, and subsequently stabilise new tissue structures. Overall, these findings exemplify the ability of epithelia to respond to the mechanical environment to regulate organ development.

In this thesis I investigate the role of dopamine modulated plasticity during olfactory learning in the fruit fly Drosophila Melanogaster. Although previous plasticity models explore many qualitative aspects of how dopamine orchestrates plasticity that also apply to the fruit fly, no model explains recent relief learning and extinction experiments. In relief learning if a shock precedes an odour, the fly will learn to approach the relief signalling odour. Extinction is the disappearance of a previously learned behaviour when the behaviour is not reinforced. I address this problem by building a model that is based on the known anatomy of the fruit fly's olfactory circuit, as well as recent behavioural and imaging data in four results chapters. In chapter 2 I introduce a three-layer spiking model based on the first layers of the olfactory circuit to study the transformations of dense spatio-temporal odour patterns into sparse representations in the fly. In chapter 3 I introduce a model of the olfactory associative memory centre to study olfactory aversive learning and extinction. I show that a learning rule that doesn't require postsynaptic spikes, like in traditional models of plasticity, is sufficient to reproduce learning experiments in the fruit fly. In chapter 4 I explore appetitive learning and extinction. I hypothesize that dopaminergic neurons can inhibit each other and that suppressing the activity of a select group of dopaminergic neurons during odour exposure forms an aversive memory. In chapter 5 I investigate the role of potentiation in two experiments: relief learning and aversive re-learning. The aversive re-learning experiment shows that a fly can learn to avoid an odour, reduce its avoidance and re-learn to avoid the odour in a flexible manner depending on the absence or presence of shock in consecutive trials. I show that with potentiation re-extinction is also possible in the fly. My results provide a new perspective on dopamine-modulated learning in the fruit fly and suggest that dopamine orchestrates both potentiation and depression during plasticity.

The work presented in this thesis explores insecticide resistance using NMR metabolomics in pupa, early pupa and early adult mosquitoes of both sexes. Firstly, sex differences were investigated in early pupa and early adult strains of Anopheles gambiae and Aedes aegypti. Secondly, Cyp4g16 and Cyp4g17 knock-down strains of An. gambiae were studied to further understand the cuticular hydrocarbon (CHC) biosynthesis mechanism. Lastly, wild type susceptible and resistant strains of An. gambiae and Ae. aegypti were studied to understand metabolic differences in resistance. A common theme in this work is the utilisation of NMR metabolomics, focusing on polar metabolic profiles and its application in insecticide resistant mosquitoes. Improvements on the analyses of the NMR data to make the identification of metabolites from the NMR spectra more robust was also undertaken in the course of this project. The vast majority of mosquito studies focus on females since only females bite. This means that there is often a great amount of information on male mosquitoes that is not utilised. In order to investigate sex differences in wild type An. gambiae (and knock-down strains) and Ae. aegypti, a metabolomics protocol was established. This study found that early female pupae and adults have higher levels of lactate and glucose compared to males, whereas, their pyruvate and propionate levels were found to be lower. Cuticular resistance is a relatively newly discovered mechanism, with much about it that is still unknown, Electron microscopy studies have shown that insecticide resistant mosquitoes of An. gambiae possess a thicker cuticular layer. Furthermore, the decarbonylases, Cyp4g16 and Cyp4g17, have been found to be critical in CHC biosynthesis and are highly expressed in insecticide resistant strains. Although not fully characterised, these enzymes are thought to catalyse a decarbonylation step in CHC biosynthesis. The study here showed evidence that both Cyp4g16 and Cyp4g17 take part in the biosynthesis of methyl branched-alkanes. Additionally, these enzymes have temporal activity where Cyp4g16 activity can be observed in both pupa and adult stages, whereas Cyp4g17 activity is only observable in the adult stage. Using resistant and susceptible strains of both An. gambiae and Ae. aegypti, metabolic differences were observed. Trehalose was found consistently higher in all resistant strains IIand so it has been proposed as a biomarker candidate. Furthermore, systems using trehalose were suggested as potential targets in insecticide development. In conclusion, the work in this thesis shows that it is possible to use NMR metabolomics to study the metabolic responses found in insect pupae and adults, to distinguish sex differences and the obtained development stage- dependent temporal information on the activity of specific enzymes. Furthermore, through the identification of elevation in trehalose in resistant mosquitoes, this study has proposed a potential biomarker for identification of resistant mosquito species.

Virus-host interactions determine virus transmissibility and virulence, and underlie coevolution that shapes interesting biological phenomena such as the genetic architecture of host resistance and host range. Characterization of the virus factors that exert selective pressure on the host, and the host genes which underlie resistance and adaptation against viruses will help to define the mechanistic pathways embroiled in host-virus coevolution. In this thesis, I describe the viral causes and host consequences of host-virus coevolution. These include genomic signatures consistent with antagonistic coevolution in antiviral RNA interference pathway genes such as high rates of positive selection and polymorphism, loci that underlie genetic variation in resistance to virus infection, and apparent conflict between NF-κB signalling and DNA virus infection. The RNA interference (RNAi) pathway is the most general innate immune pathway in insects, underlined by the observation that many viruses encode suppressors of RNAi (VSRs). The relationship between RNAi and VSRs has garnered attention as a plausible battleground for host-virus antagonistic coevolution, and genomic patterns in Drosophila support this hypothesis. However, genomic patterns in the N-terminal domain of the key RNAi effector gene, Argonaute-2, have not been described. In Chapter 2, I sequence the Argonaute-2 N-terminal domain using PacBio long-read sequencing technology to describe variation within and across Drosophila species, and test whether this variation is associated with resistance to Drosophila C Virus. The RNAi pathway evolves adaptively in Drosophila, but this has not been formally extended across invertebrate species. In Chapter 3, I quantify rates of adaptive protein evolution and describe evidence for selective sweeps in RNAi pathway genes using population genomic data from 8 insect and nematode species. These analyses indicate that RNAi genes involved in suppression of transposable elements and defence against viruses evolve rapidly across invertebrates, and I identify genes with signatures of elevated adaptation in multiple insect species. Host genes that underlie host-virus interactions have been described in RNA virus infection of Drosophila, however substantially less attention has focussed on the host response to DNA viruses, primarily because no DNA viruses have been isolated from Drosophila. In Chapter 4, I describe the isolation of Kallithea virus, a Drosophila dsDNA nudivirus, and characterise the host response to infection and genetic variation in resistance. I find that Kallithea virus infection causes early male-specific lethality, a cessation of oogenesis, and induction of undescribed virus-responsive genes. Further, I describe genetic variation in resistance and tolerance to Kallithea virus infection, and identify a potential causal variant for virus-induced mortality in Cip4. Insect viruses commonly encode viral suppressors of RNAi, however there are a multitude of antiviral immune mechanisms besides RNAi which may select for viral-encoded inhibitors. In Chapter 5, I describe the requirement for RNAi and NF-κB in immunity against Kallithea virus, and map gp83 as a virus-encoded inhibitor of NF-κB signalling. I find that gp83 inhibits Toll signalling at the level of, or downstream of NF-κB transcription factors, and that this immunosuppressive function is conserved in other nudiviruses.

Meiotic drive genes are a class of segregation distorter that gain a transmission advantage in heterozygous males by causing degeneration of non-carrier sperm. This advantage must be balanced by fertility or viability costs if drive is to remain at stable frequencies in a population. A reduction in male fertility due to sperm destruction reduces the fitness of the rest of the genome, accordingly mechanisms to circumvent the effects of drive may evolve. Such adaptations will have implications for how likely it is that drive will persist. The primary theme of this thesis has been examining fertility consequences of meiotic drive in a Malaysian stalk-eyed fly, Teleopsis dalmanni. I demonstrate that drive carrier males are not sperm limited, despite the destruction of half their sperm. They produce ejaculates with sperm numbers equivalent to wildtype male ejaculates. Furthermore, drive males achieve this with greatly enlarged testes. However, resources are not unlimited; drive males also have reduced body size, and reduced accessory glands and eyespan for their body size. Accessory gland size limits male mating frequency, and male eyespan is a sexually selected trait used in female choice and male-male competition. I discuss how these patters fit with theoretical models that predict males should invest in producing an optimal ejaculate according to levels of expected sperm competition, even if they are low-fertility males. A second interrelated theme of this thesis has been to examine the benefits of polyandry, female mating with multiple males, using wild-caught individuals. Polyandry is widespread across many taxa and almost ubiquitous in insects. However, there is much debate around its proximate and ultimate causes. There are many costs associated with mating and so polyandry requires an adaptive explanation. I utilise data on wild-caught T. dalmanni to explore how natural variation amongst females and males influences fertility gains for females.

During animal development, cells undergo various behaviours, such as migration and shape change, which need to be coordinated. How this coordination is achieved is still elusive. During morphogenesis of the adult abdominal epidermis of Drosophila, the larval epithelial cells (LECs) are replaced by the adult histoblasts. The LECs migrate directedly and, subsequently, cease migration, constrict apically and die. Here, I use in vivo 4D microscopy to study the spatial and temporal organisation of the actomyosin cytoskeleton of LECs as a potential mechanism to coordinate migration and apical constriction. The analysis of LECs apical actomyosin network shows that are planar polarized during migration, undergoing pulsed contractions in the back of the cell, while protruding at the front. During constriction, the cytoskeleton of LECs displays radial polarity with contractions localising in the centre of the cell. Behavioural change, thus, involves a change in the polarity of the contractile network. The properties of the contractile network are further studied manipulating actomyosin contractility by interfering with Rho kinase and Myosin phosphatase. The results show that the regulation of Myosin activation not only impacts on the contractility of the network but also on the network's dynamics and the cell's behaviour. A loss-of-function analysis of several Rho-GEFs, the activators of the Rho GTPases, is performed to study the molecular mechanisms that underlie the behavioural change. The depletion of individual RhoGEFs identifies 5 genes involved in the regulation of specific aspects of cell migration and the apical constriction of LECs. Altogether, the results suggest that cytoskeletal architecture and autonomous network dynamics underlie the behaviour of the contractile network. The results furthermore suggest that pulsed contractions, along with the cortical actomyosin network, underlie behavioural change, being one of the force generation mechanisms that orchestrate abdominal morphogenesis.

Insect flight is a complex behaviour that goes beyond pure mechanism. Integrating genetics, morphology, physiology, sensorimotor control, kinematics and aerodynamics, researching the multiform nature of insect flight is of great scientific interest and also a challenge. Consequently, exploring different strategies for reducing the dimensionality of large flight datasets to uncover meaningful relationships is critical to furthering our understanding of insect flight biomechanics. The aim of my DPhil is to explore different strategies for analysing large datasets of free-flight kinematics in three Dipteran species: Drosophila melanogaster (fruit fly), Calliphora vicina (blowfly) and Eristalis tenax (drone fly). In Chapter 2, I critically evaluate the k-means clustering algorithm as a means of partitioning dipteran body kinematics, and show that it is an unsuitable target for body kinematic data that shifts continuously. In Chapter 3, I show that sexual dimorphism in fruit flies affects their wing beat kinematics beyond size dimorphism. In Chapter 4, I used the k-nearest neighbour clustering algorithm to develop a way of predicting the gear stops that blowflies engage in based on their wing beat kinematics. In Chapter 5, I contrast the wing beat kinematics and aerodynamic profile of all three species. Overall, my thesis shows firstly, that fruit flies, blowflies and drone flies occupy distinct kinematic morphospaces, and secondly, that careful consideration should be given to the choice of technique used to reduce the dimensionality of the data.

Dipteran insects include some of the most aerobatic animals on the planet, including species such as drone flies (Eristalis tenax) which are able to hold a steady hover in the face of environmental perturbations before executing rapid and precise manoeuvres. A deep understanding of the mechanisms of wing kinematic control that afford such abilities is vital if we are to emulate these capabilities in our own bio-inspired machines. This thesis makes use of cutting-edge techniques to visualise these mechanisms in unparalleled detail and determine how they function to control the way in which the wings move. My work here begins by documenting the mechanical interaction of two exoskeletal components around the wing hinge that forms the gear change mechanism of dipteran wing kinematic control. I show its effects on the movement of the wings and explain how the mechanism acts to bring about these changes in Calliphora vicina and E. tenax. I then move on to study another enigmatic structure near the wing hinge, the Pterale C, and argue against a role of this component in mechanically altering wing movement. Instead, I conclude that the Pterale C may be a sensory structure which informs the insect's central nervous system of the position and speed of the wing during the latter half of the downstroke. Finally, I utilise time-resolved synchrotron X-ray computed microtomography to visualise the internal musculature of C. vicina during flight, where I assert that several muscles influence wing movements by restricting the motion of wing hinge components. Throughout this thesis, the inhibition of wing motion and energy absorption pervades each mechanism. Through the application of our understanding of dipteran flight, it may be possible to design bio-inspired machines which begin to approach the aerial capabilities of these insects.

Many epithelial cell types, particularly those in glands, employ distinct regulated and constitutive secretory pathways to perform their functions. Understanding how membrane trafficking and secretion are coupled to other cellular functions like growth, which is also thought to be linked to secretory and endocytic functions, is fundamental to cell biology and to certain pathologies such as diabetes and cancer. During my DPhil, I have been developing the secondary cells (SCs), which reside in the male Drosophila accessory gland, as a new in vivo system to dissect out the genetic regulation of membrane trafficking and its link to secretion and cell growth. SCs are epithelial cells located at the distal tip of each accessory gland. They contain a number of large intracellular compartments (>5 μm diameter) allowing membrane trafficking to be easily visualised. This in turn makes scoring specific trafficking phenotypes in normal and genetically manipulated cells very reliable. Using imaging of both live and fixed cells, I show that SC compartments have specific Rab signatures characteristic of both secretory and endolysosomal compartments. The late endosomal and lysosomal marker, Rab7, is predominantly found on acidic compartments whilst Rab11, a slow recycling endosomal marker, is localised on the membranes of about ten non-acidic compartments containing dense-core granules (DCGs) and the protease angiotensin-converting enzyme (ANCE). I show that Rab GTPases regulate the maturation and number of compartments, the secretion of intraluminal vesicles, called exosomes, and the growth of SCs. Rab11 and Rab7 appear to control the balance between large non-acidic and acidic compartments in SCs, while Rab35, which coats smaller fast-recycling endosomal compartments (~1 μm), also controls the number of these large compartments. BMP signalling regulates SC growth and membrane trafficking. Using knockdown experiments, I show that all components associated with BMP signalling are involved in this process. Interestingly, I demonstrate that as well as ANCE, Dpp, the fly homologue of mammalian BMP 2 and 4, is localised inside DCGs of SCs. Dpp is the predominant ligand controlling SC growth and exosome secretion via autocrine and paracrine mechanisms. Interestingly, mating elicits the secretion of DCG compartments (DCGs), a process dependent on the Synaptosomal-associated protein 24kDa, SNAP24. BMP pathway activation is also increased in a Dpp-dependent fashion, presumably as a result of Dpp secretion, and it accelerates the rate of DCG biogenesis. I propose a model in which Dpp regulates the replenishment of DCGs in response to release via an autocrine feedback loop.

The mechanism that controls adult stem cell commitment is not fully elucidated, although it is known that the transcriptional control plays a major role in this process. Since the discovery of stem cells in the Drosophila melanogaster posterior midgut, many transcription factors have been identified to control whether intestinal stem cells (ISC) remain in the stem compartment, commit into the absorptive fate (enterocytes, EC) or differentiate into enteroendocrine (EE) cells. However, the molecular mechanisms governing these cell fate decisions remain to be fully elucidated. We have identified a network of basic helix-loop-helix (bHLH) transcription factors to be key for ISC fate and the maintenance of the tissue homeostasis. The class I bHLH factor Daughterless (Da) impedes terminal differentiation by forming Da:Da homodimers, accumulating cells as ISCs or as absorptive committed cells (enteroblasts, EB). However, the class V HLH protein Extramacrochaete (Emc) inhibits the formation of Da:Da by forming dimers which lack transcriptional activity. We confirmed that emc is expressed in the posterior midgut and we showed that emc over-expression produces terminal differentiation of ISCs into ECs. Moreover, we found that Emc function is downstream of the Notch-Delta pathway. Importantly, we identified that Emc is also important in EBs to block de-differentiation of EBs into ISCs. This blocking is due to Emc inhibition over a bHLH class II, Scute (Sc). We showed that Sc has a dual role; it drives the expression of stem cells genes, such as Dl, while when being expressed at high levels, Sc initiates secretory differentiation. Finally, we studied the function of a bHLH-leucine zipper called Cropped (Crp), whose expression produces the complete arrest of cell division and differentiation in ISCs. Our results indicate that a simple Sc/Da/Emc network of bHLH factors act as a three-position toggle switch, choosing between the stem, secretory and absorptive fates by swapping dimerization partners. This is the first time that such a mechanism can account for all cell fate transitions in the fly gut, and it has direct implications for the maintenance of the mammalian intestine.

The aim of this study is to establish the habitat preferences of the mosquito Culex modestus in the UK so that its likely distribution can rapidly be determined in areas most vulnerable to West Nile Virus (WNV) becoming established in the UK. Culex modestus was first found in the North Kent marshes in 2010. It is of particular public health interest due to its importance as a bridge vector of WNV from the amplifying host, birds, into humans and equines. Currently little is known about Cx modestus habitat preferences and population dynamics. Larval sampling was conducted in the North Kent marshes between June and November 2015 and 2016. Initially, four habitat areas in the Hoo peninsula were sampled, collecting larvae from wetland drainage ditches along with information on both abiotic and biotic factors at each Sample Point. Culex modestus larvae were present from early July until late October at all four Locations, with populations peaking between late August and September. In 2016, an additional six Locations were sampled along the southeast coast of the UK. The key findings were that Cx modestus is most likely to be found in narrow, shallow ditches, containing sedge vegetation and aquatic vegetation. They were more common in warmer waters with a neutral to alkaline pH and a high level of total dissolved solids (TDS). In 2016, Cx modestus was found to occupy two new Locations outside of Kent, in Essex along the Blackwater Estuary. A Habitat Suitability Index (HSI) was developed, based on the abiotic and biotic factors that were shown to be statistically significantly associated with its presence in the North Kent sites. The HSI showed encouraging results, particularly in its ability to predict which of the additional Locations were most suitable for Cx modestus.

Cell number plasticity drives organismal growth, and is coupled in the CNS to the emergence of neural circuits, ensuring appropriate function. In mammals, neurotrophins promote cell survival via Trk and p75\(^{NTR}\) receptors or induce cell death via p75\(^{NTR}\) and Sortilin. In \(Drosophila\), DNTs bind Toll receptors promoting cell survival, but whether they regulate cell death within the CNS remains unknown. I show Toll receptors have distinct and overlapping spatial and temporal expression and functions. Driving RNAi knockdown and overexpression of each Toll, I show that different Toll receptors are required in glia for adult locomotion; in neurons for the regulation of VNC size; and to induce cell survival or death in distinct contexts. I focused on the signalling mechanisms downstream of Toll-6. My data show DNT-Toll-6 signalling switches between promoting cell survival or death via NFkB, ERK, or JNK signalling. These outcomes depend on the cleavage state of the DNT, time and available downstream adaptors. Toll-6 induces cell survival via MyD88 and cell death via dSarm, and these alternative outcomes depend on Weckle. Altogether, my data contribute to showing that the Toll receptors, DNTs and downstream signalling adaptors constitute a novel mechanism of cell number plasticity within the CNS.

Apoptosis-induced Proliferation (AiP) refers to an evolutionary conserved process that stress-induced apoptotic cells stimulate neighbouring cells to undergo extra proliferation to compensate for the loss of apoptotic cells. It is therefore involved in tissue regeneration in multi-cellular organisms. I found that dAtg1 (Drosophila autophagy-related gene 1), a gene best known in activating autophagy, is required for AiP. dAtg1 is transcriptionally induced by the JNK pathway, a stress response signalling pathway, during AiP and it regulates AiP by acting upstream of growth signals e.g. Wg/Wnt. Surprisingly, other Atg genes involved in autophagy are not required for AiP. Therefore, dAtg1 regulates AiP independent of its canonical roles in mediating autophagy. In parallel, I investigated the non-apoptotic cell death induced by Eiger (Egr), the Drosophila tumour necrosis factors (TNFs). In mammals, TNF induces apoptosis and, when apoptosis is blocked, necrosis. In Drosophila, the type of cell death induced by Egr remains elusive. I found that expression of egr in the developing Drosophila eye primarily induces apoptosis through the canonical apoptosis pathway. Intriguingly, when apoptosis is blocked by inactivation of effector caspases DrICE and Dcp-1, Egr induces necrosis instead. Therefore, mechanisms underlying TNF-induced cell death are more conserved in Drosophila than previously thought.

Group B Sox genes play a critical developmental role in both vertebrates and insects. Within the model species Drosophila melanogaster, two SoxB genes, Dichaete and SoxNeuro, have been shown to act as ‘master regulators’ in the early development of the central nervous system. SoxB genes have only been characterised in a handful of arthropod species thus far, with most work to date focusing on drosophilids. The purpose of this investigation was twofold. First, I set out to resolve the phylogenetic origins of arthropod SoxB genes, as mutually exclusive models explaining their emergence are still contested. I have identified and annotated the SoxB of several invertebrate taxa. In total, my investigation includes 24 different metazoan taxa, and represents the largest investigation of arthropod SoxB phylogeny to date. In light of this research, I have proposed a new model of SoxB evolution which resolves the conflicting elements of the two primary competing models. Second, to study the evolution of SoxB in terms of functional conservation/divergence, I selected the emerging model organism Tribolium castaneum to draw a comparative analysis with Drosophila melanogaster. I first began by characterising the spatiotemporal expression patterns of SoxNeuro mRNA in early Tribolium embryos using whole mount in situ hybridisation, and examined published Dichaete expression patterns in the context of central nervous system development in T. castaneum. Using these data, I draw a comparison to the expression profiles of Dichaete and SoxNeuro orthologues in Drosophila melanogaster and other species. I have found that both Dichaete and SoxNeuro expression patterns in the developing central nervous system are remarkably well-conserved across species. I also attempted to characterise genome-wide binding for both Dichaete and SoxNeuro proteins in Tribolium in what would have represented the first genomic investigation of its kind in this emerging species. Using a tethered DNA adenine methyltransferase (Dam) enzyme for both SoxNeuro and Dichaete, I hoped to characterise the genomic loci with which each protein interacts within the beetle genome (a technique known as DamID). Unfortunately, these last set of experiments have proved unsuccessful, despite several attempts which have made use of different promoters, different DNA enrichment methodologies, and tackling unforeseen DNA contamination issues. Nevertheless, the troubleshooting experiments that I have carried out will pave the way for further genomic experiments in Tribolium, easing the establishment of genomic research in this emerging organism.

The ability to adhere to the extracellular matrix (ECM) is critical for numerous cell types and tissues including epithelia and muscle. Cell-ECM adhesion is primarily mediated by integrins which provide a direct link between the ECM and the actin cytoskeleton. Integrin adhesions are frequently associated with a core of 60 different proteins (integrin-associated proteins, IAPs). Integrins are required for muscle attachment and in Drosophila, loss of integrins and several IAPs results in embryonic lethality and muscle detachment. However, the IAPs FAK, RSU1, tensin, vinculin and zyxin are not required for viability or embryonic muscle attachment. Furthermore, FAK, RSU1, tensin and vinculin have been observed to localise to muscle attachment sites in Drosophila, indicating that they have some function in muscle attachment. Unlike FAK, RSU1, tensin and vinculin, it was not previously known whether zyxin is expressed in Drosophila muscles. To test this, I generated a genomic zyxin-GFP construct that should contain most of the endogenous zyxin promotor. The genomic zyxin-GFP construct was not observed at muscle attachment sites, suggesting that it is not normally expressed in muscle. I wished to know whether FAK, RSU1, tensin and vinculin are required for muscle function. Various behavioural assays were employed to test for muscle function in larvae and adult flies. The results suggest that larval muscle function was normal in flies lacking these IAPs, but that adult muscle function might be impaired, although it proved difficult to demonstrate a clear functional defect. I then tested whether the IAPs FAK, RSU1, tensin and vinculin are required for normal morphology of adult muscles, focusing on the adult indirect flight muscles (IFMs). The IFMs are fibrillar muscles which attach to the cuticle via specialised epithelial cells known as tendon cells. At the end of the myofibril, where the myofibril attaches to the tendon cell, is a dense region of actin and IAPs known as the modified terminal Z-band (MTZ). I have found that the MTZ is not a homogenous zone of proteins, but is instead organised into at least three distinct layers. Because of the similarity between the structure of the MTZ with that of a hand, I refer to the layers as ‘fingers’, ‘palm’ and ‘wrist’. I discovered that the IAPs FAK, RSU1, tensin and vinculin are each required for the proper structure of the MTZ in unique ways. The fingers were elongated in IFMs lacking FAK, RSU1, tensin or vinculin, while the palm was disrupted in IFMs lacking RSU1, tensin or vinculin. Finally, I was intrigued by the enrichment of the actin-binding protein filamin/Cheerio in the palm and wished to know if it is required for palm function. Deletion of the C-terminus of filamin/Cheerio resulted in a reduction in palm length. Filamin/Cheerio is a mechanosensitive protein which exists in a closed and open conformation. I found that filamin/Cheerio must be open in order to help form a normal palm. Furthermore, vinculin is required to convert filamin/Cheerio from and closed to an open filamin/Cheerio state so that it can perform its function in the palm.

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.

Nutrition is a vital determinant of lifespan, reproduction, health and ageing. Much has been done to investigate the lifespan consequences of short-term (proximate) nutritional manipulation, but much less is known about long-term (evolutionary) nutritional manipulation and nutritional mismatches. In this thesis I addressed this important omission, using the fruit fly, Drosophila melanogaster, model system. I empirically tested two evolutionary theories: the Thrifty Phenotype and Thrifty Genotype hypotheses, which predict the general life history consequences of nutritional mismatches over the lifetime or over evolutionary time, respectively. I also tested how the latter interacted with long-term nutritional selection regimes. Contrary to predictions, I showed that the costs of nutritional mismatches between developmental and adult diets were not universal, but instead dependent on the nature of the mismatch, sex and the components of life history measured (Chapter 2). Similarly, the costs of mismatches between evolved and proximate nutrition were dependent on evolved feeding regime, sex, life history component measured and proximate diet (Chapter 3). I discovered that there was enhanced sexual dimorphism for lifespan in nutritionally selected lines, which was associated with sex-specific life history patterns and a partial resolution of sexual conflict (Chapter 4). Transcriptome-wide analysis of these nutritionally selected lines revealed differential expression in genes with functions related to lifespan, post-mating responses, regulation and epigenetic modification (Chapter 5). Finally, I found that manipulation of another important component of altered lifestyles, activity level, had no effect on lifespan or reproduction (Chapter 6). Overall, my results make a novel contribution to the study of nutritional mismatches and long-term nutritional selection. The results also highlight the importance of simultaneously studying both sexes and several age-specific components of life history, in different proximate environments, to fully elucidate the fitness consequences of nutritional manipulation.

The growth and development of all animals involves transitions between different physiological states. The key developmental transition of critical weight (CW) in the fruit fly Drosophila melanogaster dramatically changes the growing larva’s response to nutrient restriction (NR). Developmental progression is arrested by NR before CW whereas it proceeds without delay when NR occurs after CW. It is known that the time of onset of CW and other developmental transitions are regulated by the steroid hormone ecdysone but questions remain concerning the nature of the physiological changes at CW and how they might confer NR-resistant developmental progression. To begin to answer these questions, I have analysed how the larval metabolome changes when nutrition is altered either side of the CW transition. The larval metabolome was recorded via nuclear magnetic resonance (NMR) spectroscopy and fitting reference spectra to recorded peaks enabled identification of the metabolites. Absolute metabolite concentrations could then be back-calculated from these spectra using the volume determination with two standards (VDTS) technique (Ragan, et al. 2013), which was further adapted to measure metabolite concentrations from the volume released from homogenisation of solid whole larval and adult samples. Through use of these techniques, I found that progression past CW correlates with the ability of fed and NR larvae to sustain a substantial increase in the concentration of tyrosine. An interesting interplay between tyrosine and a possible storage form of the metabolite: o-phosophotyrosine (OPT), suggests a process regulating the conversion between the two that may indirectly affect the biosynthesis of ecdysone. Dietary and genetic manipulations have been undertaken to draw a molecular mechanism for how varying tyrosine levels affected by CW attainment can effect time to pupariation (larval maturation). These results highlight how the field of NMR-metabolomics can be used to direct subsequent experiments to address biological questions.

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.

Pollination is an ecosystem service critical to both crop production and the functioning of many ecosystems. Hoverflies (Syrphidae) are a key group of non-hymenopteran pollinators. Using a combination of field and molecular techniques, this thesis explored hoverfly communities in grasslands, investigating how they are structured, and their possible role in pollination. In addition, it also created a library of hoverfly DNA barcodes that has applications for monitoring. Hoverfly abundance and species richness was not related to grassland plant community, but was influenced by flower abundance. Distinctive hoverfly communities were also associated with agriculturally unimproved grassland communities. Focussing on species-rich marshy grasslands, a four year study showed that hoverfly abundance and species richness was influenced by weather conditions. Hoverfly abundance was at a maximum at a daily mean temperature of 15°C and at moderate rainfall, declining with both high and low temperature and rainfall. Species-richness declined with increasing daily temperature. Sequences of the standard animal barcode COI were obtained from 82 hoverfly species caught in Britain. This was added to world-wide publicly available sequences for British species to create a library of over 70% of the British hoverfly fauna. These barcodes were effective at distinguishing hoverfly species, although discrimination was poor in a small number of genera. By DNA metabarcoding pollen derived from hoverflies, the structure of hoverfly pollen transport networks in marshy grasslands was investigated. In Eristalis species, networks were generalised at the whole network and species level, but showed some specialisation by individual hoverflies. Networks of 11 hoverfly species showed that although pollen carried by hoverflies came from a few common plant taxa, different hoverfly species carried distinctive pollen loads derived from at least 40% of the entomophilous plants present. Collectively, these results increase our understanding of hoverfly community function, and their role in grassland ecosystems.

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.

Over the past three decades, nitric oxide (NO) has been recognised as one of the most versatile and important players in many aspects of physiology, including immune responses. More recently, S-nitrosylation, the incorporation of a NO moiety into a protein thiol group, has emerged as a major post-translational modification (PTM) during pathophysiological responses in plants and animals. The main goal of this work was to investigate the role of S-nitrosylation in physiology and innate immunity of animals using the genetic reference system, Drosophila melanogaster. The S-nitrosylated derivative of glutathione (GSH), S-nitrosoglutathione (GSNO), is the main non-protein S-nitrosothiol (SNO) in the cell and extracellular fluids. GSNO can trans-S-nitrosylate other thiols and is considered a reservoir of NO bioactivity. The levels of GSNO and total S-nitrosylation have been shown to be controlled by S-nitrosoglutathione reductase (GSNOR) in yeast, plants and mammals. By employing an overlapping deletion technique to knock-out gsnor, a role for S-nitrosylation in the immune response of D. melanogaster is proposed. Compared to wild type flies, gsnor overlapping deletion flies presented lower expression of antimicrobial peptides in response to infections, and succumbed more rapidly to both Gram-positive bacterial and fungal pathogens. As the Toll pathway mediates responses against these pathogens, key components of this network were tested for their propensity to being S-nitrosylated. Two CLIP-domain serine proteases of the Toll signalling pathway, Persephone (PSH) and Spätzle-Processing Enzyme (SPE), were shown to be S-nitrosylated both in vitro and in vivo and this process seemed to control the quaternary structure of these proteins and interfere with the immune response of D. melanogaster. At least for PSH, S-nitrosylation at C254 has an immune significance as the expression of non-Snitrosylable PSHC254S in gsnor knock-out flies partially recovered the resistance of these animals to infections with the entomopathogenic fungus Beauveria bassiana. These findings might represent a novel mechanism by which NO and S-nitrosylation regulate immunity. Further results presented in this thesis reveal an interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) in D. melanogaster physiology and immunity. Similarly to what has been reported in Arabidopsis thaliana, gsnor knock-out flies presented higher tolerance to the herbicide paraquat, an inducer of superoxide (O2 -) production. Moreover, additional mutations in Catalase (Cat), a hydrogen peroxide (H2O2) scavenger enzyme, partially restored the immunodeficiency phenotypes of gsnor knock-out flies. These findings suggest an inter-relation between the levels of ROS and RNS during stress responses of plants and animals. In addition, CRISPR/Cas9 technology was employed to generate gsnor knock-outs in the genome of D. melanogaster. These flies were shown to have no GSNOR activity, presented lower tolerance to pharmacological-induced nitrosative stress and succumbed faster to infections with B. bassiana compared to wild type flies. These results support the role played by GSNOR in regulating NO homeostasis and immunity in D. melanogaster.

In many species females have been shown to preferentially mate with males that exhibit the most elaborate sexual ornaments. The handicap hypothesis is a major theory proposed to explain the evolution of such exaggerated traits. It postulates that the ornament is costly and handicaps the bearer such that only high quality males are able to produce the most exaggerated ornamentation. In this thesis I examined questions about male ornament evolution and the handicap hypothesis in the stalk-eyed fly, Teleopsis dalmanni. I examined how meiotic drive, a selfish genetic element that produces female-biased broods, associated with male eyespan (the sexually selected trait in T. dalmanni) in natural populations. I demonstrated a link between meiotic drive and ornament size, whereby small eyespan males were more likely to carry the meiotic drive X chromosome. I then examined how meiotic drive affected the condition-dependent expression of male eyespan. I found that although the mean eyespan of meiotic drive males was smaller, the overall degree of condition dependence was unaffected. Next, I explicitly tested whether there was empirical evidence for the handicap hypothesis in T. dalmanni. In wild populations, I found that under high experimental stress, survival was strongly correlated with male eyespan. In contrast, there was no relationship between eyespan and survival when flies were under low experimental stress. These results provide strong support for the handicap hypothesis. Laboratory experiments yielded similar results. I then explored how environmental quality influenced key components of sexual selection (lek structure and behaviour). I found that under low environmental quality, mean and variance in harem size and the strength of mate choice declined, suggesting reduced sexual selection in poor environments. Finally, I describe for the first time the existence of male mate choice in T. dalmanni, a species that had previously been invoked to exemplify a traditional model of female choice.

Flying insects, like many other animals that rely on their sense of vision to guide behaviour, have a tendency to maintain a default orientation of their eyes relative to the environment. During flight, reflexes act to keep the head level and minimise retinal image shifts resulting from rotational steering manoeuvres, or from external perturbations such as wind gusts and turbulent air flow. Gaze stabilisation serves a number of functions, which include: i) simplifying the estimation of translational self-motion, ii) aligning the head-based sensory systems with the inertial vector which facilitates the transformation of sensory signals into motor commands, iii) supporting the tracking of moving targets, and iv) reducing motion blur in the visual input. This thesis reports studies on species-specific adaptations and general principles underlying multisensory gaze stabilisation in a number of different Dipteran flies. A variety of stimulation methods were explored, along with their suitability for a linear systems analysis of the gaze stabilisation system across species. Using results obtained from the well-characterised blowfly for comparison, novel experimental work was performed on the gaze stabilisation behaviour of robberflies, hoverflies and horseflies. Species from each family were shown to stabilise their heads in compensation for body rotations around the roll axis. The performance of the reflex was found to be species-specific and dependent on the sensory modalities involved. Experimental evidence suggests that in contrast to the other families, hoverflies appear to make use of the inertia of the head to maintain a level gaze, a novel finding that has previously been reported only for dragonflies. Finally, the integration of signals in the context of gaze stabilisation obtained by the two visual systems in blowflies - the ocelli and compound eyes - were explored in both behavioural and electrophysiological experiments. This research opens new lines of investigation by identifying behaviours that demonstrate different control strategies employed by the nervous systems of flying insects.

Peptides and biogenic amines are important regulators of muscle activity in reproductive tissues, and therefore can play important roles in the reproductive success of insects. The role of neuropeptides was investigated in regulating contractions of the male accessory gland (MAG) and the ejaculatory duct (ED). The FlyAtlas tissue expression database indicated that DMS, dromyosuppressin (DMS) pro-hormone, is highly expressed in the MAGs of D. melanogaster. However, DMS, could not be found using HPLC and mass spectrometry, but was detected by immunohistochemistry which revealed extensive staining of neuronal processes on the surface of MAGs and ED of Drosophila melanogaster, D. yakuba, D. erecta, D. virilis, D. simulans, and the pest species D. suzukii. The GAL4-UAS expression system revealed DMS neuronal fibres descending from the abdominal ganglion onto the surface of the MAG, ED, and seminal vesicles. A pair of rectal cells and a single cell, that we have called the ejaculatory duct cell because of its projections to the ED, also contain DMS. The rectal and ED cells were isolated and subjected to MALDI-TOF mass spectrometry. Molecular ions of several neuropeptides were detected, and the identity of DMS and sNPF4-11 were confirmed by fragmentation sequencing. Both DMS and sNPF 4-11 reduced the frequency of contractions of the MAG and ED, suggesting that these peptides are involved in regulating ejaculation of seminal fluid and spermatozoa. Furthermore, the finding of DMS-R1 and DMSR2 expression in the epitracheal cells suggested that both receptors might be involved in adult ecdysis. The silencing of DMS expression using nSyb-GAL4/UASdicr2 > DMS-RNAi (108760), shows abnormal abdomen phenotype and mortality at day 5 post-emergence. Inhibitors of neuropeptide metabolism were shown to be larvicidal when fed to two species of mosquitoes (Anopheles gambiae and Aedes aegypti) identifying an enzyme target for the development of novel control chemicals.

In this thesis I use Drosophila melanogaster as a model organism to study the possible cognitive mechanisms controlling plastic behavioural responses to sperm competition. This plastic behaviour involves a male D. melanogaster responding to the presence of a rival male by increasing mating duration when housed with a female. I provide a general context to the work (Chapter 1) before examining my model in more temporal detail by investigating how the length of time males were exposed to a high sperm competition environment affected maintenance time of the plastic behaviour. I show that for males to accurately portray the sperm competition environment in their behaviour over a useful timescale they must possess accurate sensory systems. Without these, behaviour is still fully plastic, but change occurs at a slower speed than males with full sensory ability (Chapter 3). I then show that extended mating duration is controlled by a suite of well-known learning and memory genes highlighting the need for specific memory pathways to reflect ecological change (Chapter 4). However, those same genes do not change in their expression due to increased sperm competition, potentially pointing to some other mechanism of temporal change underlying the behavioural change (Chapter 5). Due to this reliance on learning and memory, I show that an increase in sperm competition can affect cognitive ability, and increase expression of synaptic genes over a longer time period (Chapter 6). Finally, I summarise my thesis findings and discuss how future research can build on the research presented to develop the field (Chapter 7). My research shows that learning and memory is paramount for males to react to changes in the sperm competition environment on a relevant timescale where behaviour and the environment have not become mismatched. In addition, I show that sperm competition pressures can cause an increase in male individual cognitive ability, posing the question of whether competition is one of the main drivers of non-mammalian cognitive ability.

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.

Phlebotomus argentipes (Diptera: Psychodidae) is an important vector responsible for the transmission of Leishmania donovani that causes visceral leishmaniasis (VL) or kala-azar, in the sub-continent of India. The aims of this study were to investigate the semiochemicals that mediate oviposition and mating behaviour and also the courtship behaviours in P. argentipes. The result of ovipositional behaviour bioassays shows gravid P. argentipes females preferred to oviposit their eggs in the present of conspecific eggs and also eggs extract. This suggests the presence of an oviposition pheromone on the surface of the eggs which can be removed by washing with an organic solvent and transferred to an alternative surface. A Y-tube olfactometer was used to test an upwind anemotactic response of virgin females to male headspace volatiles and male extract, in the presence or absence of host odour. The results strongly suggest that a volatile maleproduced sex pheromone is present in P. argentipes. The results also suggest that under certain circumstances of the age of males and females and the presence of host odour, the females are attracted to live male and male extract of P. argentipes. Thus, presence of host odour might have a synergistic effect on the male-produced sex pheromone. Quantitative description and detailed of courtship behaviour(s) in both males and females of P. argentipes were observed. The results show that male behaviours during courtship are vital for the success of the mating. These predictor behaviours include approach wingflapping, abdomen bending and copulation attempt by male P. argentipes. Understanding of the biology, ecology and chemical mediated behaviour in P. argentipes will enhance and widen the knowledge leading to the improved of the efficiency and efficacy of the current sand fly control programmes.

"One of the deadliest animals in the world", is the description given to the mosquito by the World Health Organisation. Collectively mosquitoes are a vector for a range of pathogens to humans. Recent developments in genetic engineering have demonstrated that the transgenic self-limiting technology developed by Oxitec Ltd can be deployed to reduce populations of Aedes aegypti. The technology uses a transgene that, when inherited, causes lethality if the mosquito does not have access to tetracycline during development. Recent advancements at Oxitec Ltd involve targeting the self-limiting system specifically to females. This allows for males (which are required for a release) to be readily separated from females without the need for costly mechanical separation. The work presented in this thesis aims primarily to further development of the Oxitec Ltd technology in Ae. aegypti and Aedes albopictus. In Ae. aegypti this is achieved through the development of paternal effect, a male specific construct which render the sperm inviable if reared in the absence of tetracycline. This paternal effect is subsequently combined with a genetic sexing strain in an 'all in one' construct, such that when reared in the absence of tetracycline only sterile males are produced. Development in Ae. albopictus comes through characterisation of certain life history traits of the wild type strains currently maintained at Oxitec Ltd. It is important to develop self-limiting insects in a background strain with certain traits, such as the ability to be produced under mass rearing conditions, and sensitivity to insecticides. In conclusion the work presented here represents a major advancement of Oxitec Ltd's self-limiting technology in Ae. aegypti, through production of a genetic sexing strain capable of producing sterile males. In Ae. albopictus wild type characterisation has laid the foundations for future transgenic development in a suitable background strain.

A major facet of nervous system development entails the projection of axons from neuronal cell bodies towards synaptic partners, such as other neurons or muscles. This process, referred to as “axon guidance,” relies upon receptors on axons detecting secreted or membrane-associated guidance cues within the developing organism, which attract or repel axons. While many cues and receptors have been identified over the last few decades, many within studies of the relatively simple fly embryonic nervous system, bioinformatic analyses of the Drosophila proteome suggest numerous uncharacterised proteins might be implicated in directing axonal growth. Such proteins are predicted to be expressed at the cell membrane, to harbour domains common to established axon guidance proteins, and to be expressed in the nervous system while axons are extending. The current study focuses on three genes predicted to encode proteins with the above characteristics: CG7565, CG31814, and otk2. An examination of a line with a P-element in the coding sequence of CG7565 revealed aberrations throughout the embryonic motor neurons. Abnormalities in these nerves were also observed in a line with a deficiency spanning CG7565, as well as in embryos misexpressing CG7565 in motor neurons or somatic muscle. Motor neuron projections were absent in embryos harbouring a P-element in the 5’ UTR of CG31814, which were rescued by the precise excision of the transposon. In null otk2 mutant embryos, various motor axons were absent or abnormal, and aberrations in the same branches were apparent when misexpressing otk2 in motor neurons or somatic muscle. Moreover, analyses of transheterozygous embryos provide evidence that otk2 genetically interacts with the established axon guidance genes, otk, sema-1a, and fz2, and that otk interacts with fz2. Analysis of a line with a deficiency that removes otk and otk2 revealed more severe phenotypes than were observed in single mutants, providing further support for cooperation between the off-tracks. Thus, taken together these observations implicate three largely uncharacterised genes in axon guidance and reveal novel insights into the signalling pathways in which these and established axon guidance genes participate.

Genome-wide binding and expression studies in Drosophila melanogaster have revealed widespread roles for Dichaete and SoxNeuro, two group B Sox proteins, during fly development. Although they have distinct target genes, these two transcription factors bind in very similar patterns across the genome and can partially compensate for each other's loss, both phenotypically and at the level of DNA binding. However, the inherent noise in genome-wide binding studies as well as the high affinity of transcription factors for DNA and the potential for non-specific binding makes it difficult to identify true functional binding events. Additionally, externalfactors such as chromatin accessibility are known to play a role in determining binding patterns in Drosophila. A comparative approach to transcription factor binding facilitates the use of evolutionary conservation to identify functional features of binding patterns. In order to discover highly conserved features of group B Sox binding, I performed DamID-seq for SoxNeuro and Dichaete in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. I also performed FAIRE-seq in D. pseudoobscura embryos to compare the chromatin accessibility landscape between two fly species and to examine the relationship between open chromatin and group B Sox binding. I found that, although the sequences, expression patterns and overall transcriptional regulatory targets of Dichaete and SoxNeuro are highly conserved across the drosophilids, both binding site turnover and rates of quantitative binding divergence between species increase with phylogenetic distance. Elevated rates of binding conservation can be found at bound genomic intervals overlapping functional sites, including known enhancers, direct targets of Dichaete and SoxNeuro, and core binding intervals identified in previous genome-wide studies. Sox motifs identified in intervals that show binding conservation are also more highly conserved than those in intervals that are only bound in one species. Notably, regions that are bound in common by SoxNeuro and Dichaete are more likely to be conserved between species than those bound by one protein alone. However, by examining binding intervals that are uniquely bound by one protein and conserved, I was able to identify distinctive features of the targets of each transcription factor that point to unique aspects of their functions. My comparative analysis of group B Sox binding suggests that sites that are commonly bound by Dichaete and SoxNeuro, primarily at targets in the developing nervous system, are highlyconstrained by natural selection. Uniquely bound targets have different tissue expression profiles, leading me to propose a model whereby the unique functions of Dichaete and SoxNeuro may arise from a combination of differences in their own expression patterns and the broader nuclear environment, including tissue-specific cofactors and patterns of accessible chromatin. These results shed light on the evolutionary forces that have maintained conservation of the complex functional relationships between group B Sox proteins from insects to mammals.

One of the greatest problems for sub-Saharan Africa is shortage of epidemiological data to support planning for provision of adequate public and animal health services. The overriding challenge is to provide the necessary resources to facilitate the process of regular data collection in support of disease surveillance and vector monitoring across target regions. Due to such circumstances, there is currently an increasing interest towards devising cheaper but yet significantly reliable means for availing the needed epidemiological and vector data for planning purpose. This study comes as a contribution towards solving such challenges. The study has three research components starting with a review of past Uganda national tsetse and trypanosomiasis control efforts as a means towards appreciating the dynamics of controlling the vector and disease. This is an analysis of what was applied, what worked, what didn't, and why it didn’t as linked to the broader vector and disease control system. Secondly through the use of remote sensing, geographical information systems and global positioning technologies tsetse species were sampled within Lake Victoria Basin. Only two species of tsetse were trapped, G. f. fG. f. fuscipes which was widely distributed across the surveyed area, and G. Pallidipes which was detected in a few isolated locations close to the border with Kenya in Eastern Uganda. The analysis of land cover with tsetse findings showed an important association between G. f. fuscipes and particular vegetation mosaics. Unfortunately, while the results are highly informative, approaches for data collection such as this one are costly and unlikely to be sustained by the already over-burdened health systems in the low developed countries of Africa. The third and main part of this study investigates, demonstrates and delivers the possibilities of applying spatial epidemiological modelling techniques to produce both tsetse distribution and abundance maps. Four spatial and non-spatial regression models (Logistic, Autologistic, Negative binomial and Auto-negative binomial), were constructed and used to predict tsetse fly presence and tsetse fly abundance for the study area. The product is an improved understanding of association between environmental variables and tsetse fly distribution/abundance and maps providing continuous representations of the probability of tsetse occurrence and predicted tsetse abundance across the study area. The results indicate that tsetse presence and abundance are influenced differently. Tsetse abundance is highly determined by river systems while tsetse presence is majorly influenced by forested landscapes. Therefore, efforts to control trypanosomiasis through vector control in the Lake Victoria basin will call for delineation of such clearly identified high tsetse accumulation zones for targeted tsetse control operations. This will ensure optimum utilization of the scarce resources and above all contribute to the protection of humans and animals against trypanosomiasis infection.

A fast and powerful gaze stabilization reflex supports self-motion es- timation and flight control in flies. Changes in body posture are conveyed by a variety of sensory modalities and compensated for by fast and accurate head movements. This thesis aims to further our understanding of the behavioural basis of compensatory head move- ments, and presents a first foray into the in vivo imaging of the motor systems that actuate these control reflexes. Major sensors that contribute to gaze stabilisation are the visual ocelli and compound eyes on the head, and the mechanosensory halteres on the thorax. The integration of visual feedback and mechanosensory feedforward control gives rise to a two-degree-of-freedom controller, a design which is extensively used in engineering applications. I per- formed a linear systems analysis of compensatory head roll in response to forced thorax oscillations in the fly. The feedforward pathway ex- hibited a high bandwidth and constant gain and reduced the response delay of the reflex. Large stability margins in the feedback pathway supplied by the compound eyes guaranteed stable behaviour in the face of response variability. The occlusion of the ocelli did not change the gain of the feedback pathway, but significantly reduced the la- tency. I investigated the use of iodine-enhanced computed x-ray microto- mography (microCT) to perform fast three-dimensional imaging of the neck and flight motor systems. Virtual dissections of major func- tional units illustrate the possibilities and limitations of microCT. To observe the configuration of motor systems in behaving flies I per- formed gated microtomography using hard x-rays at the TOMCAT beamline of the Swiss Light Source, a third generation synchrotron. While 3D tomograms of the neck motor system proved elusive, this thesis presents the first in vivo tomograms of the flight motor and wing hinge during tethered flight.

Many higher animals have evolved the ability to use the Earth’s magnetic field, particularly for orientation. However, the biophysical mechanism by which magnetoreception is achieved remains elusive. One theoretical model (the radical pair mechanism - RPM) proposes that the geomagnetic field is perceived by chemical reactions involving the blue-light photoreceptor Cryptochrome (CRY). Recent evidence supports the RPM in Drosophila melanogaster and reveals a mechanistic link with the circadian clock. Here I have confirmed, albeit with rather different results, that a low frequency electromagnetic field (AC-EMF) along with a Static Field (SF) exposure does affect circadian and activity behaviour in the fruit fly. Furthermore, I have developed two new assays to investigate the effects of EMF in Drosophila melanogaster, negative geotaxis and an additional light wavelength preference assay, revealing a net CRY-dependent response. My data support the idea of CRY mediated magnetoreception, thereby indirectly supporting the RPM. Furthermore, I provide some striking new results that challenge our view that only the canonical clock neurons contribute to behavioural rhythms in Drosophila melanogaster.

Insects are highly successful and their large numbers lead to economic loss through crop damage and disease transmission. Insecticides provide a valuable tool for control of insect populations. However, as resistance is increasing to existing products, new modes of action are required for the development of novel products. Understanding of the biological mechanisms underlying stress resistance in insects may provide insight into new potential insecticide targets. Malpighian tubules are critical for epithelial fluid transport and xenobiotic tolerance in insects. The function of Malpighian tubules in desiccation stress tolerance was explored by examining changes in gene expression, protein levels, fluid transport rates, and metabolism following stress exposure. The results indicate a reduction in secretion rate during desiccation that is reflected in accumulation of metabolites that are normally processed and excreted by the tubules. Moreover, the involvement of Drosophila melanogaster diuretic hormones corticotrophin releasing factor-like (DH44) and leucokinin (LK) were examined using genetic manipulations based on the GAL4-UAS system. Highly selective manipulation of the DH44-producing neurons via knockdown of DH44 and neuronal ablation indicates that suppression of DH44 signalling contributes to desiccation tolerance. This result is supported by the finding that knockdown of DH44 receptor 2 in the Malpighian tubule principal cells improves survival during desiccation stress. Previous work suggests the possibility of interaction between LK and DH44 signalling as LK receptor (LKR) is colocalised to the DH44 neurons. This hypothesis is supported by the results of this study as selective knockdown of LKR and DH44 in the DH44 neurons produced opposing effects on desiccation tolerance. Moreover, knockdown of DH44 in the DH44 neurons or ablation of these neurons resulted in significantly decreased LKR expression in the Malpighian tubules. Finally, a novel role for the Malpighian tubules in starvation tolerance was uncovered by the study, with LKR gene expression increasing significantly following starvation. Knockdowns of either DH44-R2 or LKR in the Malpighian tubules significantly impaired starvation tolerance. Here, a mechanism for this role of renal epithelia in starvation tolerance is proposed.

In this thesis I investigated the genetic basis of several behaviours to answer questions surrounding the evolution and mechanistic basis of behaviour. Firstly, I took a single-gene approach to investigate the influence of fruitless (fru) on the courtship behaviour of Drosophila. fru is an alternatively-spliced transcription factor that is necessary for the production of male sexual behaviours, and has also been implicated in producing species-specific differences in courtship song. I investigated the patterns of selection acting on fru at the sequence level and found that positive selection was restricted to the alternatively spliced exons of fru. From this I hypothesised that the positively selected changes in fru would contribute to species-specific differences in courtship song. To test this I examined how isoform-specific fru loss-of-function mutants influence courtship song, and generated “species-swapped” flies whereby regions of fru that showed evidence for positive selection were transferred from four species of Drosophila, into D. melanogaster. Contrary to prediction, I found flies that lacked isoforms containing positively selected regions did not show any differences in courtship song. Unfortunately “species-swapped” flies were not generated in time to examine phenotypes and neuroanatomy as intended. Next, I examined the genetic basis of cold acclimation in two species of Drosophila using a transcriptomic approach. I found that the genes differentially expressed in response to cold acclimation were largely different in each of the species; however, the biological processes they were involved in were broadly similar. Finally, I investigated the transcriptomic changes associated with parental care in the burying beetle Nicrophorus vespilloides to determine if males and females alter the genes they express when parenting alone versus with a partner. I found that males greatly reduced their transcriptional response when parenting with a partner, suggesting they reduce the care they provide when present with a female.

The development of behavioural resistance in Anopheles mosquitoes is an important threat to the sustainability of current vector interventions, which selectively target mosquitoes that are found indoors. Changes in the following behaviour have been reported in several Anopheles vectors: biting time, host preference, biting and resting behaviour. However, a critical review of the published literature reveals that behavioural resistance has not been demonstrated clearly in Anopheles, highlighting important limitations in using entomological data to investigate the evolution of behaviour. The availability of next generation sequencing and low-cost custom genotyping technologies makes it possible to investigate behavioural resistance using genetic information as an alternative approach. Using such technologies, this thesis aims to investigate whether Anopheles vectors are evolving towards outdoor-biting behaviour ('exophagy') by examining the genetic difference between the indoor- and -outdoor population of the main African malaria vectors, An. coluzzii and An. gambiae from The Gambia and Uganda, respectively. We also investigated the role of olfactory-related genes in driving the potential genetic difference between the two populations. We found limited divergence between the two populations overall, however close inspection identified SNPs potentially associated with outdoor-biting behaviour as well as genomic regions displaying elevated levels of differentiation between the two populations. Most notably, gustatory receptors, Gr11 and Gr12 and the odour receptor, OR1 were identified as well as, genes related to transcription, signal transduction and catalysis. We discuss the potential role of these genes in determining biting behaviour and suggest that further investigation of these genomic regions is required to confirm the early-stage divergence of indoor- and outdoor-biting Anopheles populations.

Traditional studies of simple perceptual choice tasks in vertebrates identified behavioural characteristics of deliberate decision-making that guided the development of general mathematical models, and the search for neurophysiological correlates. Current experimental and modelling efforts aim to uncover biophysical and circuit level mechanisms of decision-making processes. However, genetic manipulability constraints and lack of high-throughput assays make further progress in vertebrate studies a steep endeavour. In this thesis I studied decision-making in Drosophila melanogaster in trained two-alternative forced-choice olfactory tasks with varying stimulus contrast, using a high-resolution single fly behavioural assay. Analysing accuracy and reaction time as a function of task difficulty (i.e., stimulus contrast) showed that flies display behavioural characteristics of evidence accumulation processes, a signature of vertebrate decision-making: reaction times increased and perceptual accuracy declined as stimulus contrast decreased. Mutants for the gene encoding the transcription factor FoxP took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult tasks. Using the putative FoxP promoter to ascertain FoxP expression identified subsets of mushroom body intrinsic Kenyon cells, in αβ core and γ neurons, as potential sites of FoxP action. Disrupting FoxP expression or decreasing neuronal excitability specifically in αβ core neurons mimicked the phenotype observed in FoxP mutants. FoxP expression therefore affects the development or function of αβ core neurons in the progression of a decision process towards commitment. To identify molecular processes involved in evidence integration regulated by FoxP I further screened 2nd and 3rd chromosome deficiency lines in a sensitised FoxP mutant background, uncovering genomic regions of interest for further study. Finally, analysing naive performance in tasks of increasing difficulty showed that naive discriminations are faster and less accurate than trained ones, pointing to the existence of two decision-making systems. FoxP mutants appear to engage the slower, more accurate decision making system and the mushroom body seems to be involved in naive discriminations. The molecular and neuronal players involved in olfactory decision making in Drosophila melanogaster uncovered in this thesis will allow researching decision making systems to an unprecedented level of detail.

Centrosomes are the major microtubule (MT) organising centres in most animal cells, and they play important roles in many cellular processes. Centrosomes comprise a pair of centrioles surrounded by a matrix of proteins known as the pericentriolar material (PCM). Centrosomes assemble around the mother centriole when the PCM is recruited to nucleate and organise the MTs. For decades, the PCM has been viewed as an amorphous cloud of proteins. Although there are hundreds of PCM proteins, a small number of these are thought to assemble an underlying PCM scaffold that extends around the mitotic centriole and subsequently recruits other mitotic PCM proteins. In flies, Centrosomin (Cnn) and Spindle-defective protein 2 (Spd-2) are such essential proteins, and Asterless (Asl) helps recruit these proteins to the mother centriole. However, it is unclear how these proteins assemble into a scaffold at the centrosome, and none of these core PCM assembly proteins have been characterised at a structural level. In this thesis, I have identified and recombinantly expressed functional domains of Cnn, Spd-2 and Asl in order to characterise them in vitro. In collaboration with colleagues in the laboratory, the biological significance of these domains has been investigated in vivo. I define a Phospho-Regulated Multimerisation (PReM) domain within Cnn that is phosphorylated by Polo kinase in vitro, that dimerises via a leucine zipper, and that can form higher order oligomers when phospho-mimicking mutations are incorporated into the protein in vitro. In vivo, forms of Cnn that cannot be phosphorylated in the PReM domain cannot efficiently assemble a Cnn scaffold, while phospho-mimicking forms can spontaneously assemble into scaffold structures. I show that the Centrosomin-Motif-2 (CM2) domain of Cnn is also required to promote Cnn scaffold assembly. The crystal structure of CM2 reveals an unusual asymmetric dimer that further assembles into a tetramer in crystallo. Strikingly, Cnn-CM2 demonstrates a zinc-binding activity that stabilises the dimer, and preliminary in vivo studies indicate that the tetramer interface is essential for Cnn scaffold assembly in vivo. Finally, I show that Cnn-CM2 exhibits a weak interaction with the first of three ASPM, Spd-2, Hydin (ASH) domains in Spd-2, and that this domain facilitates Spd-2 expansion into the PCM region. The structure of two of the Spd-2 ASH domains has been investigated using X-ray crystallography and Nuclear Magnetic Resonance spectroscopy, revealing an Immunoglobulin (Ig)-like protein fold. These studies provide the first atomic insights into the molecular mechanism underlying mitotic PCM assembly, and reveal how the assembly of a Cnn scaffold may be controlled to ensure robust centrosome assembly in vivo.

Elaborate morphological ornaments can evolve if they increase the reproductive success of the bearer during competition for mates. However, ornament evolution is incredibly rare in females, and the type and intensity of selection required to develop female-specific ornamentation is poorly understood. The main goals of my thesis are to clarify the relationship between the type and intensity of sexual selection that drives the evolution of female ornamentation, and investigate alternative hypotheses that might be limiting or contributing to the development of female ornaments. I investigated the ecology and evolution of female-specific ornaments within and between species of dance flies from the subfamily Empidinae (Diptera: Empididae). The dance flies display incredible mating system diversity including those with elaborate female-specific ornaments, lek-like mating swarms, aerial copulation and nuptial gift giving. To elucidate the form of sexual selection involved in female-ornament evolution, I experimentally investigated the role of sexual conflict in the evolution of multiple female- specific ornaments in the species Rhamphomyia longicauda. Through manipulative field experiments, I found that variation in the attractiveness of two ornaments displayed by females indicates that sexual conflict, causing a coevolutionary arms race, is an important force in the evolution of multiple extravagant female ornaments. Using R. longicauda again, I tested for a role of functional load-lifting constraints on the aerial mating ability of males who paired with females displaying multiple large ornaments. I found no evidence of functional constraints influencing the mating opportunities of elaborately ornate females, but instead discovered a relationship consistent with positive assortative mating for mass. Biased sex ratios are predicted to increase the intensity of sexual selection in a population, which in turn, is predicted to influence the evolution of ornamentation. I measured the incidence and prevalence of vertically transmitted symbiotic bacteria that has been observed to distort the sex ratio in other Dipteran hosts. While my survey revealed that symbionts occur at high incidence and variable prevalence across dance fly hosts, I found no effect of symbiont infection levels on population sex ratios, or female- specific ornament evolution. Further investigation into the relationship between sex ratios and female-ornament evolution using the comparative method revealed that the operational sex ratio (OSR) of a population did not predict continuous measures of female ornamentation across species. However, female-ornament evolution did predict male relative testis investment across species indicating that female ornaments likely indicate increased levels of polyandry. My thesis reveals that sexual selection theory developed to describe male-specific ornament evolution cannot readily be translated to apply to females. I show that male mate choice, rather than functional constraints or ecological associations with bacteria, is likely driving the evolution of female-specific ornaments. I also identify sexual conflict as an important selective force in the evolution of female-specific ornaments.