Orientador: Selma Maria de Almeida-Santos / Resumo: O conjunto de características manifestadas por uma espécie que levam ao sucesso reprodutivo pode ser chamado de estratégias reprodutivas. Isto inclui, por exemplo, o período reprodutivo, o modo reprodutivo, a sincronia reprodutiva entre machos e fêmeas, o tempo de fertilização e a frequência reprodutiva. O gênero Lachesis é composto de quatro espécies ovíparas restritas às florestas úmidas da América Central e América do Sul. Popularmente conhecidas como surucucu, são um grupo de importância médica, tal como Bothrops e Crotalus. Lachesis muta é a espécie com a maior distribuição, e ocorre na América no Sul, na Mata Atlântica e na Floresta Amazônica. Ao contrário de Bothrops e Crotalus, estudos sobre biologia reprodutiva para o gênero Lachesis são, em sua maioria, desenvolvidos em cativeiro e com foco no comportamento reprodutivo. Propomos, então, descrever em dois capítulos aspectos da biologia reprodutiva da espécie Lachesis muta investigados a partir da anatomia e morfologia de seus órgãos reprodutivos ao longo das estações do ano. No Capítulo 1, investigamos a biologia reprodutiva de L. muta da Floresta Amazônica e da Mata Atlântica brasileira a partir de dados de biometria e histologia de animais preservados em coleções biológicas e provenientes da natureza. No Capítulo 2, descrevemos o oviduto feminino em diferentes estágios do ciclo reprodutivo. Comparando tanto as duas populações quanto a espécie com outros crotalíneos neotropicais, nosso objetivo foi investigar possív... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The set of characteristics manifested by a species leading to reproductive success can be called reproductive strategies. These include, for example, the reproductive period, the reproductive mode, the reproductive synchrony between males and females, the time of fertilization, and the reproductive frequency. The genus Lachesis comprises four oviparous species restricted to Central and South American rainforests and popularly known as bushmasters, and is a group of medical importance, as are Bothrops and Crotalus. Lachesis muta is the most widely distributed and occurs in South America, at the Atlantic Forest and the Amazon Rainforest.Unlike Bothrops and Crotalus, reproductive biology studies for the genus Lachesis weremostly developed in captivity, focusing mainly on reproductive behavior. We propose to describe in two chapters aspects of the reproductive biology of the Lachesis muta species investigated from the anatomy and morphology of its reproductive organs over the seasons of the year. In Chapter 1, we investigate the reproductive biology of L. muta by biometric and histology data from biological collections, focusing on individuals collected in the wild. In Chapter 2, we describe the female oviduct at different stages of the reproductive cycle. Comparing both populations and species with the other neotropical crotalines, we aim to investigate possibles patterns for the Viperidae group, as well as the hypothesis of the influence of abiotic factors on the snake reproduc... (Complete abstract click electronic access below) / Mestre

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

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Sexual selection can influence the mating system of an organism through multiple mechanisms. These mechanisms result in variation in reproductive success among individuals, and include scramble competition, endurance rivalries, contests, mate choice and cryptic choice, and sperm competition. Understanding the mating system of a species requires the identification of which processes are occurring, and to what degree. In this thesis, I explored the influence of mate choice mechanisms on the mating system of the terrestrial red-legged salamander, Plethodon shermani. I also documented the potential for post-copulatory processes (such as sperm competition and cryptic choice) to influence mating system dynamics. The evolution of mate choice requires (among other factors) variation in the reproductive value of potential mates. This variation is made apparent to choosy individuals through cues. Most animals use multiples cues incorporating many modalities to assess the reproductive quality of potential mates. In Chapter 2, I tested the contribution of two cues (chemical and visual) to mate choice by female P. shermani. I found that a male visual cue ("foot-dancing") increased courtship success. In contrast, delivery of non-volatile pheromones during courtship did not influence courtship success in the laboratory setting, but did affect the duration of one of the courtship stages. In Chapter 4, I identified a tactile cue that was significantly correlated with male reproductive success. Thus, P. shermani females could use at least three modalities to assess the reproductive quality of potential mates. Mate choice can also evolve in males. In Chapter 3, I tested this possibility in P. shermani. I found that males vary the reproductive effort they invest in a particular courtship based on the reproductive value of their partner, indicating male mate choice is occurring. A male invested most when paired with a female with large, well developed ova, and invested less with females that were non-gravid or had small ova. In addition to documenting male mate choice, I showed that the male visual display ("foot-dancing") that affected female mate choice was correlated with male condition, implying foot-dancing may be an honest indicator of male quality. Finally, in Chapter 5, I explored the potential for post-copulatory processes to influence the P. shermani mating system. The opportunity for sperm from multiple males to overlap in the female reproductive tract (i.e., the opportunity for females to mate multiply) is necessary for post-copulatory processes such as cryptic choice and sperm competition. The capacity for long-term sperm storage by females can increase the likelihood that this overlap in sperm from multiple males will occur. I found that females can store viable sperm for at least 9 months and in some cases beyond oviposition. In addition, I documented one female with sperm in her sperm storage organ from a mating that occurred 17 months earlier. Such lengthy sperm storage allows the possibility of sperm from one breeding season to interact with sperm from a subsequent season. Thus, the potential for post-copulatory sexual selection within this salamander system is high. / Graduation date: 2012

Chez les espèces aviaires, le stockage des spermatozoïdes s’étend sur plusieurs semaines principalement au niveau du réservoir de la jonction utéro-vaginale, contenant les tubules de stockage des spermatozoïdes (SST). Les mécanismes impliqués dans ce processus restent indéterminés. L’effet de l’insémination artificielle (IA) a été évalué sur le protéome du fluide utérin (FU), des protéines cibles et des glycanes dans les SST, provenant de poules possédant une longue (F+) ou courte (F-) durée de stockage. La longue durée de stockage est associée à une abondance relative dans le FU après IA des protéines exosomales (ANXA4, ANXA5), des protéoglycanes (TSKU), des protéines liant les protéoglycanes (HAPLN3, FN1, VTN), des transporteurs de lipides (VTG1, VTG2, APOA1, APOA4, APOH), et des protéines matricielles de la coquille (OCX32). Au contraire, la faible capacité de stockage est associée à la régulation après IA des protéines immunitaires (PIGR, immunoglobulines) ou pro-inflammatoire (LTA4H), des protéases (XPNPEP1), des chaperones (HSPA8), des mucines (MUC5AC, MUC5B), et de l’ovalbumine (OVALY). Au niveau des SST, les protéines matricielles de la coquille (OC-116, OCX36, OC-17) ont été identifiées dans l’épithélium et la lumière. La longue durée de stockage est associée à la sécrétion luminale de résidus Glc/GlcNAc, à la mobilisation apicale de protéines exosomales (ANXA4), et la non-activation des voies métaboliques impliquant les protéines PIGR, HSPA8, et ANXA5 dans les SST. En conclusion, la composition protéique du FU et des SST requièrent des régulations spécifiques après IA certainement pour garantir le stockage des spermatozoïdes. / In avian species, the sperm storage mainly takes place in uterovaginal sperm storage tubules (SST) during several weeks. Mechanisms implied in this process are not fully understood. The effect of artificial insemination (AI) has been evaluated on the uterine fluid (UF) proteomic composition, and on SST candidate proteins, from hens exhibiting long (F+) or short (F-) sperm storage duration. Long sperm storage duration was associated with the relative abundance in UF after AI of proteoglycans (TSKU), proteoglycan binding proteins (HAPLN3, FN1, VTN), lipid transporters (VTG1, VTG2, APOA1, APOA4, APOH), and eggshell matrix proteins (OCX32). In contrast, poor sperm storage ability was associated with the regulation of immune factors (PIGR, immunoglobulins), pro-inflammatory factors (LTA4H), proteases (XPNPEP1), chaperone (HSPA8), mucins (MUC5AC, MUC5B), and ovalbumin related protein Y (OVALY). At the level of SST, eggshell matrix proteins (OC-116, OCX36, OC-17) were identified in SST cells and lumen. Long sperm storage duration was associated in SST with the luminal secretion of Glc/GlcNAc residues, ANXA4 apical mobilization, and non-activation of metabolic pathway implying PIGR, HSPA8, and ANXA5. In conclusion, the proteomic composition of UF and SST require specific regulation after insemination, most probably to guarantee the success of sperm storage process.

Information on the biosynthetic origin and functional advantage of marine mollusc natural products is not only essential to our understanding of chemical ecology, but to the development and responsible production of therapeutic agents. As demonstrating in situ activity is methodologically hindered, functions inferred by in vitro activity have been assumed for many secondary metabolites. The anatomical and ontogenetic distribution of natural products can not only provide information on the biosynthesis and storage of metabolites, but identify selective pressures likely to affect survivorship at a specific life stage. Thus, dissection and chemical analysis of distinct tissues, in combination with histochemistry may offer a valuable approach. Marine gastropods of the Muricidae are renowned for the ancient dye Tyrian purple, which evolves from choline esters of bromoindoxyl sulphate in the hypobranchial gland through a series of enzymatic and photo-oxidative reactions. Prochromogen hydrolysis by arylsulphatase liberates neuromuscular active choline esters and cytotoxic bromoindole precursors, which also occur in muricid egg masses. Although visual accounts of dye pigments in the muricid gonoduct suggest precursors may be incorporated into egg masses from a maternal source, their biosynthetic origin and the evolutionary significance of the hypobranchial gland is unknown. Thus, the Muricidae, and in particular Dicathais orbita upon which most previous research has been focused, is an ideal model for this novel approach to natural product research. To confirm observations of dye pigments in muricid gonoducts and gain an understanding of their anatomical distribution, a liquid chromatography-mass spectrometry (LC-MS) method was developed to simultaneously quantify pigments, precursors and the prochromogen, tyrindoxyl sulfate. The prochromogen was not only detected in albumen and capsule gland extracts, but bioactive intermediates and the dye 6,6’-dibromoindigo were also present in the latter. These findings provided preliminary evidence for the maternal provision of prochromogens in egg masses of D. orbita and identified regions within which to conduct histochemical investigations. Tyrindoxyl sulphate was also detected in male prostate gland extracts, along with the dibromoindigo isomer, 6,6’-dibromoindirubin and its oxidative precursor, 6-bromoisatin. This not only implies physiological differences exist between male and female gonoducts, but that these secondary metabolites are not solely intended for egg masses and may hold significance throughout the life cycle. Histomorphological inspection of the pallial gonoduct-hypobranchial gland complex was conducted over the annual cycle to determine a mechanism for precursor transfer between these structures. Although an anatomical connection was not detected, the secretions of two hypobranchial cell types thought to be involved in Tyrian purple synthesis were of remarkable biochemical similarity to those of various capsule and albumen gland lobes. Together these findings implied the potential for natural product synthesis within the pallial gonoduct of D. orbita. To establish the role of these glandular lobes in the incorporation of intracapsular fluid and capsule laminae, identical histochemical techniques were applied to transverse capsule wall sections. Biochemical correlations not only provided a simple method of deciphering the complex process of encapsulation in neogastropods, but effectively identified the destination of gonoduct secretions in egg capsules of D. orbita. Comparisons of capsule and gonoduct biochemistry revealed that the intracapsular fluid and inner capsule wall are secreted by the posterior capsule gland lobe, the middle lamina by the lateral lobes and the outer layers by the dorsal lobe, albumen and pedal glands. Investigation into the location of regulatory enzymes and precursors was conducted to establish the biosynthetic origin of Tyrian purple prochromogens and mechanisms governing bioactive precursor synthesis. Novel histochemical techniques for the localization of bromoperoxidase, the enzyme thought to facilitate prochromogen bromination, and tyrindoxyl sulphate were developed and applied to gonoduct, hypobranchial gland, and encapsulated larvae sections. Standard staining reactions for the indole precursor, tryptophan, and arylsulphatase were also applied. The histochemical approach adopted revealed that tyrindoxyl sulphate is de novo biosynthesized through the post-translational bromination of dietary derived tryptophan. Two biosynthetic sites were identified, one related to hypobranchial secondary metabolism and the second of significance to the presence of bioactive precursors in muricid egg masses. Tryptophan is stored within secretory cells of the lateral hypobranchial epithelium and once exocytosed, is united with bromoperoxidase from supportive cells to form tyrindoxyl sulphate. Prochromogen synthesis also occurs in the subepithelial vascular sinus for storage and secretion by medial hypobranchial secretory cells. Bioactive precursor synthesis on the epithelial surface is regulated by the liberation of arylsulphatase from adjacent supportive cells. These findings not only provide evidence for de novo biosynthesis of Tyrian purple precursors, but are first account of natural product biosynthesis within the gastropod hypobranchial gland. Together these findings imply a naturally selected function for the synthesis of bioactive indoles in hypobranchial gland secretions of the Muricidae and Gastropoda. Tyrindoxyl sulphate is also transported within the vascular sinus to lateral and dorsal capsule gland lobes where bromoperoxidase and arylsulphatase also occur. Arylsulphatase was also detected within the albumen gland, which along with the posterior capsule gland lobe, acts as a storage site for dietary tryptophan. Thus, tyrindoxyl sulphate and the constituents for prochromogen and precursor biosynthesis are introduced to intracapsular fluid and capsule laminae by the capsule gland. Histochemistry in combination with LC-MS revealed an identical biosynthetic profile within larval vitellus, which is elaborated during oogenesis and may also receive secretions from the albumen gland. Due to the absence of a hypobranchial gland in veligers, it appears that pelagic larvae rely on vitelline natural products until settlement and metamorphous. These findings together with the in situ antimicrobial activity of bromoindoles suggest Tyrian purple precursors are incorporated into muricid egg masses as a maternal investment in larval defence against pathogens. The results of this investigation clearly highlight the benefits of adopting a histochemical approach to natural product research. This novel alternative to radioisotopes and in situ demonstration of bioactivity, can not only aid in the elucidation of secondary metabolic pathways and chemically mediated interactions, but identify mechanisms of metabolite regulation and differentiate between biosynthetic and storage tissues. Apart from providing insight into the ecological significance of muricid secondary metabolites, the biosynthetic information provided is valuable to our understanding of chemical phylogeny and biosynthetic enzyme sequencing for the environmentally sound development of natural products as biomedical agents.