Sperm function is pivotal to successful sexual reproduction. The phenotype of sperm is defined by the male’s genotype and by the environment sperm encounter during their travel to the oocyte. During their functional lifespan, sperm encounter a variety of environments: After manufacture in the testis, they are stored in males before they are ejaculated along with seminal fluids and transferred to and stored in females for hours up to years before getting a chance to fertilise an egg. The sperm environment in male and female reproductive tract will be determined by male and female genotype, but also by environmental factors that affect sperm directly or indirectly by altering male and female condition. Like somatic cells, sperm age and decline in function over time due to the accumulation of cellular damage. Reactive oxygen species (ROS) that emerge as a by-product of aerobic metabolism and environmental stress are believed to be the main cause of cell senescence. Spermatozoa are in particular susceptible to ROSinduced damage because they have only limited defence and repair mechanism. As it contains many polyunsaturated fatty acids, the sperm membrane is especially prone to peroxidation by ROS and can consequently become leaky. The condition of the sperm membrane can hence be used to assess sperm age. Sperm quality has frequently been measured as sperm viability even though this approach has several biological and technical pitfalls. I developed an osmotic sperm stress test to assess sperm quality and predict future sperm performance that circumvents several of these pitfalls. Further, using osmotic stress to challenge the sperm membrane and observing sperm viability in a longitudinal approach is probably more meaningful in predicting future sperm performance than sperm viability per se. An essential abiotic factor that affects sperm directly and indirectly during storage in males and females is temperature. Ectotherms that inhabit different climates like D. melanogaster can be expected to be locally adapted to temperatures, particularly in fitness-relevant reproductive traits. I assessed the joint and isolated effects of thermal adaptation, of rearing and of ambient temperature on sperm quality by measuring sperm osmotic stress resistance, ejaculate effects on the induction of egg-laying in females, male fertility as well as female fertility and sperm storage in two D. melanogaster strains from Zambia (warm-adapted) and two from Sweden (cold-adapted). I found complex G x E interactions on male and female reproductive traits. Sperm quality was generally higher in the cold-adapted strains and showed negative carry-over effects of hot-rearing, demonstrating the important role of male genotype and developmental temperature on sperm quality. In contrast, there were positive carry-over effects of hot-rearing on male fecundity and male and female fertilisation rate in the hot-adapted strains, supporting local adaptation to heat stress. To investigate direct effects of the female reproductive tract environment on stored sperm, I genetically manipulated female D. melanogaster with a spermathecalspecific GAL4 driver line and hoped to test proposed candidate genes associated with female sperm storage. My results suggested sperm storage defects in the driver line that may either be an unexpected side-effect of the insertion of the GAL4 driver into a spermathecal serine endopeptidase or of the genetic background of the driver line.:Chapter 1 General introduction 1 Sperm phenotype 1 Basic sperm morphology 2 Sperm ageing 3 Environmental effects on sperm 5 Male sperm storage 6 Female sperm storage (FSS) 8 Phase 1: Recruitment of sperm into storage 10 Phase 2: Sperm maintenance 11 Phase 3: Release of sperm from storage 16 References 18 Chapter 2 More pitfalls with sperm viability staining and a viability-based stress test to characterize sperm quality 29 Abstract 29 Author contributions 30 Introduction 31 Methods 33 Methods of sperm viability staining in ecology and evolution 33 Empirical study 33 Statistical analysis 38 Results 38 Methods of sperm viability staining in ecology and evolution 38 Sperm survival examined with cross-sectional vs. longitudinal sampling 43 Sperm viability in the bedbug 43 Sperm viability in the fruitfly 45 Discussion 47 SV heterogeneity 48 Protocol standardization and recommendations 49 Sperm viability vs. sperm quality 50 Sperm stratification 51 Conclusion 52 References 52 Chapter 3 Effects of temperature and thermal adaptation on sperm stored in male and female Drosophila melanogaster 56 Abstract 56 Introduction 57 Material and Methods 61 Fly populations and culture 61 Temperature treatments 62 Wing length 63 Quality of sperm stored in males 63 Male effects on female fertility 64 Female effects on sperm 64 Statistical analysis 66 Results 67 Wing length at 19° and 29°C 67 Sperm viability under osmotic stress 68 Male effects on female fertility 70 Female effects on stored sperm 73 Fertilisation rate 75 Sperm storage 77 Does the decrease in sperm predict female fecundity? 80 Discussion 82 Phenotypic plasticity vs. local adaptation 82 Sperm effects, seminal fluid effects and female effects on fertility 83 Supernumerary spermathecae 87 Conclusion 87 References 89 Chapter 4 Effect of spermathecal proteins on female fertility and sperm storage in Drosophila melanogaster 94 Abstract 94 Introduction 95 Material and Methods 99 Fly Stocks 99 UAS/GAL4 crosses and controls 101 General experimental procedure 103 Experimental fly strains 103 Parameters used to assess sperm storage capability 103 Mating procedure 104 Oviposition and progeny development 104 Part I. RNAi screen (Experiments 1 and 2) 109 Material and methods 109 Experiment 1: Preliminary RNAi screen 109 Experiment 2: RNAi screen 109 Results 110 Experiment 1: Preliminary RNAi screen 110 Experiment 2: RNAi screen 114 Part II: Spermathecal secretory function (Experiments 3 to 5) 121 Material and Methods 121 Experiment 3: Survey of fertility effects of the spermathecal secretory function 121 Experiment 4: Verifying experiment 1 122 Experiment 5: Verifying experiment 2 122 Results 122 Experiment 3: Survey of fertility effects of the spermathecal secretory function 122 Experiment 4: Verifying experiment 1 127 Experiment 5: Verifying experiment 2 128 Part III. Trpa1 and temperature effects 130 Material and Methods 130 Results 131 Sperm number and fertilisation rate 140 Discussion 151 Effect of knock-down of genes with putative role in sperm storage in SSC on fertility 151 Effect of impaired spermathecal secretory function on fertility 153 Effects of enhanced secretory function of the SSC on fertility and sperm storage 154 Wildtype variation in fertility (and sperm storage) with temperature 155 Conclusion 157 References 157 Chapter 5 General discussion 164 Measuring sperm quality in male and female storage organs 164 Environmental effects on sperm stored in males and females 166 Effect of the direct environment on sperm stored in females 168 Conclusion 169 References 170 Acknowledgements 175 Supplementary Material 176 Composition of corn/yeast food 176 Composition of yeast food 176 Chapter 2 176 Chapter 3 177 Full models 177 Survival and mating rate 179 Interaction plots males 181 Fertility of focal males 184 Sex ratio of the progeny of focal males 185 Female fertility 185 Chill coma recovery assay 187 Results 187 Chapter 4 189 Experiment 3 189 Experiment 6 189