Stalk-eyed flies (Diptera: Diopsidae) have emerged as an important model organism in the study of sexual selection. They are characterised by their eyes being located on elongated stalks that protrude from their heads. In many species, males have larger eyespans than females as a result of female mate preference and malemale competition. In this thesis, I investigated several different aspects of stalk-eyed fly biology. First, I provide a comprehensive review that asks whether the literature supports the hypothesis that male sexual traits suffer from heightend inbreeding depression relative to non-sexual traits. This is followed by an empirical study that examines how inbreeding affects a sexual trait (male eyespan) relative to non-sexual traits (female eyespan and male wing length) in the stalk-eyed fly Diasemopsis meigenii. Sexual selection theory predicts that sexual traits should suffer greater inbreeding depression than non-sexual traits. Full-sibling matings were used to generate highly inbred lines of D. meigenii. Over 11 generations of inbreeding, male eyespan suffered more from inbreeding depression than female eyespan and male wing length. After crossing inbred lines, male eyespan was restored more than male wing length, but not female eyespan. Next, I used a P2 mating design to test male offence and defence roles in sperm competition. Female D. meigenii were mated once by a ‘focal male’ and n times by a ‘competing male’ (where n = 1, 3 or 5). Male defence and male offence ability was assessed by mating the focal male either before or after the competing male. I found no evidence of sperm precedence in D. meigenii and suggest that the most likely mechanism of sperm competition is via mixing of sperm from rival males. Finally, I examine how adult sexual size dimorphism in the stalk-eyed fly Teleopsis dalmanni is determined and regulated during larval development.

Sexual selection is responsible for the evolution of formidable adaptations across the tree of life. Traditionally, sexual selection research has focused on male sexual displays and female choice. However, this approach ignores important social and environmental factors that can influence the operation of sexual selection. In this thesis, I aim to contribute to our knowledge of the effects of social and environmental factors, particularly those related to nutrition, on sexual selection. I investigate how the sexual behaviour of females and the nutritional and social environments of both sexes during larval and adult stages modulate sexual selection in the fruit fly Drosophila melanogaster. First, I assess how female promiscuity ("polyandry") affects the strength of sexual selection and patterns of assortative mating in freely interacting populations. There is no current consensus for the conflicting results of previous studies showing that polyandry can have positive, negative or have negligible effect on the opportunity for sexual selection. Using a genetic knockout that increases female sexual receptivity, I show that increasing polyandry reduces the opportunity for sexual selection and shifts the relative role of sexual selection from pre- to post-copulatory in males without affecting assortative mating patterns in freely interacting populations. Next, I consider how plastic responses to nutritionally poor larval environments and adult social environments modulate sexual selection, an area that has been considerably ignored. I find that although nutritionally poor larval environments reduce individuals' mating and reproductive success, plastic responses to social environments might mitigate against these disadvantages. Moreover, I show that plastic responses to larval and social environments influence the relative role of pre- and post-copulatory sexual selection in males, regulate offspring traits through trans-generational effects and determine population fate. Finally, I consider whether male reproductive traits have distinct macronutrient (protein and carbohydrate) requirements, and whether males can regulate their feeding to attain a diet that satisfies the requirements for these traits. I find that both a high short-term rate of offspring production when males mate with virgin females, and a high total number of offspring sired when males mate with previously mated females, require carbohydrate-rich diets, whereas male attractiveness requires a balanced (1:1) macronutrient diet. Furthermore, I show that male protein intake can negatively affect female long-term reproduction when males mate with virgins, but not with previously mated females, revealing a novel intersexual effect of male nutrition. Thus, this thesis deepens our understanding of key evolutionary processes by revealing the negative effects of high polyandry and nutritionally poor larval environments on the operation of sexual selection as well as uncovering male nutritional compromise in the expression of reproductive traits.

The Drosophila adult male accessory gland (AG), which secretes many components of seminal fluid and modifies many behaviours of mated females, is a surprisingly understudied organ compared to many other fly tissues. This has been partly because of a lack of genetic tools allowing manipulation of the cell types that make up the AG epithelium. I have defined a set of transcriptional GAL4 drivers that display specific expression in a subset of AG epithelial cells known as secondary cells (SCs), and used these to investigate SC cell biology and whether there are biological parallels between SCs and the secretory cells of the human prostate. I show that these cells grow in an age-and mating-dependent manner relative to the rest of the epithelium. Furthermore, I have observed that these cells display the remarkable ability to delaminate apically from the epithelium after mating, migrate proximally within the gland in a directional manner, and can even be transferred intact to females during mating. A small-scale genetic screen was performed which established that BMP signalling plays a crucial role in these interesting features of SC biology. Specifically, BMP signalling activity is required for normal SC growth and migratory activity. Hyper-activation of the BMP signalling pathway drives SC overgrowth and promotes spontaneous delamination and migration at a high frequency. Intriguingly, I have also shown that ecdysone receptor (EcR) signalling, a steroid hormone signalling pathway, is an important positive regulator of SC growth. This is of particular interest given an analogous role that the androgen receptor (AR), also a steroid hormone receptor, plays as a potent driver of cell growth in the prostate epithelium. I also show that BMP signalling activity increases EcR expression levels in a cell type-specific fashion, via a potential protein stabilisation mechanism. A surprising discovery has been the observation that the cell cycle regulators, Rbf and E2F1, also genetically interact with EcR and promote SC growth. E2F1 activation drives normal SC growth and positively regulates EcR expression and activity. Many of these findings are reminiscent of biology observed in the human prostate and prostate cancer, where for example, AR expression and activity is driven by E2F1 activity. This mechanism is postulated to be of importance in the progression of prostate cancer to a disease that no longer responds to androgen deprivation therapy. In summary, my research in the Drosophila AG highlights several novel features of adult SC biology, including growth and migration, which are regulated by BMP and steroid hormone signalling activity. Much of this biology appears to closely parallel equivalent cell physiological phenomena in the prostate epithelium and in prostate cancer. In addition, as a consequence of this research, the Drosophila AG is emerging as an important model to study a number of conserved aspects of cell biology, particularly membrane trafficking, relevant to human diseases, including cancer.

Genetic models of the handicap theory of sexual selection propose that ornaments signal heritable male quality, so females mating with the most ornamented males acquire fitness benefits for their offspring. Male ornaments are predicted to have coevolved with female preference to be larger, and so more costly. The key prediction made by the handicap hypothesis is that male sexual traits have evolved heightened condition dependence, a result of the higher differential cost of ornaments relative to other traits. I investigated evidence for condition-dependent sexual ornaments and found little support from well-designed experiments. Most studies had neglected to 1) compare condition dependence in sexual traits with suitable non-sexual controls, 2) adequately account for body size variation, and 3) assess individuals under a range of stresses representative of those experienced in nature. There was also a dearth of experimental studies exploring the genetic basis of condition dependence. I used experiments with stalk-eyed flies to examine predictions made by condition-dependent handicap models of sexual selection. Cyrtodiopsis dalmanni is highly sexually dimorphic for eyespan, and females exhibit strong mating preferences for males with large eyespans. Condition was varied experimentally by manipulating larval food availability. I found that male eyespan was more sensitive to changes in condition than female eyespan and other non-sexual traits. Male eyespan also showed a great increase in standardized phenotypic variance under stress, unlike non-sexual traits. These patterns persisted before and after controlling for body size. In contrast, there was no heightened condition dependence of male eyespan in Sphyracephala beccarri, a species without female mate choice for exaggerated male eyespan and only minor sex differences in eyespan. The genetic basis of ornament condition dependence was investigated in C. dalmanni by comparing the performance of distinct genotypes (inbred lines) along a gradient of environmental stress. Lines that produced a large ornament in one environment tended to do so in others. Stress also amplified these differences between genotypes leading to an increase in the genetic variance of the male ornament. Such patterns were less marked in non-sexual traits, and persisted after controlling for size. I looked for positive correlations between ornaments and viability by assessing the genetic correlations between male eyespan expression and four components of fitness (male fertility, female fecundity, and male and female longevity). I found no evidence that females obtain genetic benefits, other than male attractiveness, for their offspring by mating with well-ornamented males. However, body size-corrected male eyespan was negatively correlated with female longevity. This was unexpected and does not provide support for "good genes" benefits of sexual selection. Possible reasons for such findings (or lack thereof) are discussed.

There has been a continuous production of high quality reports focussing on fruitless as the genetic switch for male sexual behaviour in Drosophila melanogaster, and on fruitless’s contributions to creating a male-specific neural circuit within the CNS. However it has become increasingly clear that fruitless is not sufficient in itself to specify the full complement of male-specific behavioural repertoires. One obvious genetic candidate that contributes to the male neural circuit is doublesex. doublesex has long been known to be pivotal to the specification of the sexually dimorphic adult soma but it’s function in specifying sex-specific neural substrates has, up till now, been largely unexplored. While fruitless has so far shown to be found only in insects, doublesex is a more ancient gene and, as member of the Dmrt family of genes, is both structurally and functionally conserved throughout the animal kingdom. Thus the study of doublesex offers great potential for understanding the neuronal, developmental and physiological logic underlying innate and species-specific behaviours, in not one but both sexes, in organisms throughout the animal kingdom. Using the novel dsxGAL4 transgenic tool, generated by ends-in homologous recombination at the doublesex locus, I have been able to perform a systematic temporal and spatial survey of doublesex expression both within, and outwith, the nervous system. Excitingly, as doublesex is endogenously expressed in both males and females, this has uncovered profound dimorphic differences in male and female neural substrates. In the male this circuit is shared with fruitless (whose expression is restricted to adult males) and has allowed myself, and my colleagues in the Goodwin lab, through functional behavioural analyses, to gain greater understanding into how male-specific behavioural outputs may be generated. Further though, functional analyses impinging on the novel doublesex female circuitry has allowed us to gain new insight into the (largely unstudied) role that females play in the courtship ritual. The dsx GAL4 transgenic tool, and the insights gained in this study, are also of import in relation to dissecting out mechanisms involved in the post-mating physiological and behavioural changes the female undergoes after successful copulation with a male. As well as this, as doublesex is known to play a pivotal role in establishing the dimorphic morphology of the fly, this tool has begun to allow us an understanding of how the assembly of these dimorphic neural circuits is coordinated with the development, and maintenance, of a sex-specific anatomy and physiology to produce the complete male or female ‘state’; Integrating both mind (fly brain) and body (fly soma).

Sexual conflict can occur whenever the evolutionary interests of males and females differ, and when sexually antagonistic selection acts upon traits shared between the sexes, one or both sexes can be constrained from reaching their phenotypic optima. This intralocus sexual conflict can be characterised by a tug-of-war of allelic replacement until it is resolved, but examples of well-characterised sexually antagonistic loci are rare. This thesis investigates the basis and dynamics of intralocus sexual conflict over insecticide resistance at the Cyp6g1 locus in Drosophila melanogaster, and wing colouration in Drosophila simulans. In D. melanogaster, the Cyp6g1 locus is the site of a series of insecticide resistance alleles, one of which is sexually antagonistic when back-crossed to the old isogenic lab strain Canton-S. I investigated the presence of sexual conflict over this same allele in a recently collected and genetically heterogeneous population. I found evidence of balancing selection on resistance (Ch. 2) that could not be explained by overdominance or sex-specific dominance (Ch. 3). However, balancing selection could be explained by resistance conferring increased fecundity to females (Ch. 2-4), and decreased reproductive success to males (Ch. 4). This male cost can in turn be explained by a negative genetic correlation between reproductive success and Cyp6g1 expression (Ch. 4), possibly influencing levels of reproductive investment (Ch. 2). Additionally, I explored the dynamics of the sex-specific fitness effects of resistance across three Cyp6g1 alleles back-crossed to a single genetic background. I found no evidence of sexual antagonism, but revealed that the cost of resistance increased with more derived alleles, and that all alleles were more costly to females (Ch. 5). After decades of strong selection imposed by insecticide use an unresolved sexual conflict persists at the Cyp6g1 locus despite sexual dimorphism in resistance, and it does not appear that more derived Cyp6g1 alleles are necessarily involved in mediating this conflict. Wing interference patterns (WIPs) are a newly discovered trait subject to female mate choice in Drosophila. I explored the potential for intralocus sexual conflict over WIPs by measuring WIP traits from males and females from populations of D. simulans evolved under relaxed or elevated sexual selection. In response to sexual selection male WIPs evolved to be brighter, higher contrast, and shifted to longer wavelengths of light, but there was no associated response to selection in females (Ch. 6). While WIPs did not appear to be constrained from detectably responding to selection by acute intralocus sexual conflict, male WIPs from the relaxed selection regime were similar to female WIPs, suggesting a cost to sexually selected WIPs that may be indicative of sexually antagonistic selection. IASC is pervasive and can influence a wide range of fundamental evolutionary processes including sexual selection, speciation, and extinction. The research presented in this thesis adds to a body of evidence that sexual dimorphism does not necessarily resolve IASC, and documents the first evidence that WIPs do not appear to be subject to acute IASC and can evolve in response to sexual selection.

In Drosophila, the functionally conserved transcription factor, doublesex (dsx), is pivotal to the specification of sexual identity in both males and females. One of its key dedicated roles involves regulating the development of a sexually dimorphic nervous system (NS) that underlies both male and female reproductive behaviours. Specific inhibition of the function of dsx-expressing neurons in males and females results in a global disruption of these sex-specific behavioural outputs. However, little is known about the functional organisation of this dsx circuit that encodes the potential to display these behaviours. Such investigations require the generation of a novel transgenic tool, capable of separating the function of dsx in the NS from that of the body. To achieve this, I generated a novel split-GAL4 dsx^GAL4-DBD hemidriver by ends in homologous recombination. Coupling the novel tool with the pan-neuronal elav^VP16-AD hemidriver, revealed spatial restriction of dsx^GAL4-DBD/elav^VP16-AD expression to dsx neurons only; enabling the realisation of novel patterns of dsx-expression in the peripheral NS. Next, the ability to elicit male-specific behavioural outputs upon activation of all dsx neurons formed the basis of a large behavioural screen aimed at parsing dsx circuitry into functionally distinct clusters. I utilised the novel dsx^GAL4-DBD hemidriver to screen a large collection of extant enhancer trap lines (ET^VP16-AD), for the elicitation of distinct sub-behaviours of male courtship. Here, I show that the activity of dsx-expressing clusters in: i) the brain (dsx-pC1, -pC2 and -pC3 collectively) regulate the early steps of male courtship (initiation, orientation and wing extension), ii) the pro- and mesothoracic ganglia (dsx-TN1 and -TN2) regulate the middle steps of male courtship (wing extension and possibly courtship song) and iii) the abdominal ganglia (dsx-Abg) regulate the late steps of male courtship (abdominal curling, attempted copulation and copulation). These data establish functional correlations between dsx clusters in distinct neuroanatomical foci and specific sub-behaviours of the courtship repertoire. Furthermore, the novel intersectional tool primed a collaborative study on female post-copulatory behaviours. We identified key sensory neurons in the female reproductive tract involved in initiating post-mating behaviours. Subsequent functional interrogations of dsx circuitry in the central NS revealed a subset of dsx-expressing neurons in the Abg that mediate changes in the female behavioural repertoire after mating. Characterisation of this relatively simple neural circuitry sheds light on the organisation of the fly brain. Ultimately, future studies will define principles of neural circuit operation, which may be similarly conserved in the nervous systems of higher animals.

Membrane-bound vesicle secretion provides a novel intercellular communication mechanism, whose roles and regulation remain poorly characterised, particularly in vivo. I have identified two classes of lipid-containing, vesicle-like structures secreted into seminal fluid by epithelial cells of the Drosophila male accessory gland (AG). Exosomes, one class of membrane-bound vesicle formed inside late endosomal multivesicular bodies, are specifically secreted by secondary cells (SCs). The unusual cell biology of SCs allowed me to develop a powerful new high resolution in vivo system to characterise the mechanisms underlying intracellular membrane trafficking events underlying exosome biogenesis using real-time live imaging. I characterise how specific ESCRTs (endosomal sorting complexes required for transport) control SC exosome biogenesis, and identify a novel role for BMP signalling in regulating endolysosomal trafficking events necessary for exosome secretion. I also identify roles for epidermal growth factor receptor (EGFR) and phosphatidylinositide 3-kinase (PI3K) signalling in exosome biogenesis. Importantly, SC exosomes are transferred to females during mating. Here, they fuse with sperm, mirroring in vitro interactions between human prostate exosomes and sperm, and interact with the female reproductive tract epithelium. Blocking SC exosome production specifically suppresses post-mating effects on female receptivity to remating, demonstrating that exosomes have an important reproductive signalling function in vivo, directly or indirectly reprogramming female cells. Finally, I show that main cells, the major epithelial AG cell type, shed lipid-containing microvesicle-like structures from their apical surface. Remarkably, these vesicles carry the seminal peptide, sex peptide, into females during mating and also contribute to the anterior mating plug. In summary, my data reveal previously unsuspected roles for exosomes and microvesicles in Drosophila reproduction that may be evolutionarily conserved. Since these vesicles mediate physiological processes previously thought to involve soluble peptides, my work suggests that current models explaining male reprogramming of female behaviours in flies and higher organisms need substantial revision.

The robust behavioural courtship ritual displayed by Drosophila melanogaster males is governed by their underlying nervous system (NS). Two key genes of the Sex Determination Hierarchy, fruitless (fru) and doublesex (dsx), determine most neuronal substrates for sexual behaviour. In this study we aim to better understand the role fru plays in determining these neural substrates, as a means of better understanding the relationships between brain, behaviour and genes, and thus how the development of neuronal networks shape innate and species-specific behaviours. Fru has two major functions: control of male sexual behaviour, and viability in both sexes. Alternative splicing of fru produces transcription factors driven by four promoters: P1 transcripts are sex-specifically spliced (only viable in the male), and P2-4 transcripts are crucial to both sexes survival. The resulting proteins contain a BTB protein-protein interaction domain at the N-terminus, and one of four C-terminal zinc-finger (ZnF) DNA binding domains. Male-specific proteins (FruM) contain an additional 101 amino acid N-terminal domain, and one of three alternative C2H2 ZnF domains (FruMA, FruMB, FruMC). These male-specific isoforms are expressed in the central nervous system (CNS) beginning in the late stage larvae (L3), peaking during pupation and on into adulthood. Little is known, however, about the roles of the individual isoforms, and no clear transcriptional targets have been identified. The central aims of this thesis are to document the wild-type expression patterns of Fru isoforms throughout development in the CNS, create and characterise isoform-specific mutants, and to identify and evaluate putative transcriptional targets of Fru. Combining these findings will lead to a better understanding of the underlying molecular functions of individual FruM isoforms, as a means to understanding their roles in sexual behaviour. Expression analysis of FruM isoforms throughout development in the NS is described. To further characterise the role of individual FruM isoforms, isoform-specific mutants in fruA and fruB exons were generated using site-specific homologous recombination (HR). These novel mutants were validated by PCR and Fru isoform-specific antibody stainings. Mutants were analysed in fruM- and fru-null genetic backgrounds, to distinguish the roles of sex-specific vs. common isoforms. These analyses included: fertility, viability and morphology. FruA was found to have a role in wing extension with possible repercussions for song production. FruB was found to be developmentally lethal, in addition to having defects in male courtship behaviour. To understand the role of fru in the NS, downstream transcriptional targets of FruM isoforms were identified. DNA adenine methyltransferase identification (DamID) was used to identify putative transcription targets of FruM isoforms. The Dam protein methylates DNA in Drosophila in a sequence-specific manner allowing targets of Fru to be isolated. Candidate genes were identified using computational analysis (including gene ontology, peak analysis and motif analysis) along with a biologically significant connection with fru. The relationship between fru and six candidate genes were characterised using RNAi. The results of these studies advance our knowledge of how FruM isoforms influence the development and physiology of the NS underlying male sexual behaviour in Drosophila.

Elaborate sexual ornaments evolve because mate choice exerts strong sexual selection favouring individuals with high levels of ornament expression. Consequently, even at evolutionary equilibrium, life history theory predicts that ornamental traits should be under directional sexual selection that opposes contrasting selection to reduce the costs associated with their maintenance. Otherwise, the resources used to maintain ornaments should be used to improve other life history functions. Elaborate female ornaments have only evolved in a few species, despite females commonly experiencing strong sexual selection. One explanation for this rarity is that male preferences for female ornaments may be self-limiting: females with higher mating success become less attractive because of the lower paternity share they provide to mates with every additional sperm competitor. The unusual species in which female ornaments do occur can provide rare insight into how selection can favour the expression of expensive characters in females despite their costs. The main goal of my thesis was to determine how sexual selection acts on exaggerated sexual ornaments, and give new insight into how these ornaments may have evolved, in spite of the self-limiting nature of selection on male preferences. To determine the strength of sexual selection acting on female ornamentation in dance flies, we developed new microsatellite markers to assess polyandry rates by genotyping stored sperm in wild female dance flies. We first used polyandry rates to determine whether ornament expression was associated with higher mating success in female Rhamphomyia longicauda, a species that has evolved two distinct and exaggerated female ornaments. Contrary to our predictions, we found no evidence that females with larger ornaments enjoy higher mating success. We then compared polyandry rates in R. longicauda to those of two other species of dance fly, one (Empis aestiva) that has i independently evolved female ornaments on its legs, and another (E. tessellata) that does not possess any discernable female ornaments. We also estimated the opportunity for sexual selection, which we found to be similar and relatively low in all three species. Moreover, the standardized sexual selection gradients for ornaments were weak and non-significant in all three species. Females with more elaborate ornaments, in both within- and cross-species comparisons, therefore did not enjoy higher mating success. Overall, these results suggested that sexual selection operates rather differently in females compared to males, potentially explaining the general rarity of female ornaments. Our amplifications of stored sperm were able to reveal more than just mate numbers. We developed new methods to study patterns of sperm storage in wild female dance flies. We investigated how the skew in sperm genotypes from mixed sperm stores changed with varying levels of polyandry. Our data suggested that sperm stores were dominated by a single male in R. longicauda, and that the proportion of sperm contributed by this dominant male was largely independent of the number of rival males’ sperm present in the spermatheca. These results were consistent with the expectation of males using sperm ‘offence strategies’ in sperm competition and that the most successful male is likely to be the female’s last partner before oviposition. As a whole, my thesis contributed new molecular resources for an understudied and fascinating group of organisms. It exploited these new resources to provide the first estimates of lifetime mating success in several related species, and suggested that the general prediction that ornament expression should covary with sexual selection intensity does not seem to hold in this group. Instead, both the unusual prevalence of ii ornaments and the inconsistent evidence for sexual selection that sustains them in dance flies may owe their existence to the confluence of two important factors. First, the conditions under which sperm competition occurs: as last male precedence is likely, males are selected to prefer the most gravid females to secure a high fraction of her offspring’s paternity as they are unlikely to mate again before oviposition. Second, potent sexually antagonistic coevolution between hungry females and discerning males: females have evolved ornaments to disguise their stage of egg maturity to receive the benefits of nuptial gifts, while males face the challenge of distinguishing between gravidity and ornamentation in females.