Rozprawy doktorskie na temat „Sperm competition”

Sperm competition, when sperm from more than one male compete to fertilise the same ova, has driven a diversity of adaptations. Increasingly, molecular techniques have been used to study the effect of post-copulatory sexual selection, including sperm competition, on proteins that are essential to reproduction. Genomic studies have revealed the rapid evolution of ejaculate proteins in polyandrous species. Additionally, there is evidence that gene expression can be altered plastically, in response to cues of sperm competition. Such studies are limited however, as the transcriptome does not always equal the proteome. Here, quantitative proteomics techniques are utilised to explore plasticity in reproduction, at a molecular level, in the house mouse (Mus musculus domesticus) (Chapters 2 – 4). In addition, adaptations to sperm competition are considered within mammalian testes and sperm proteins (Chapters 5 and 6). Contrary to predictions of sperm competition theory, within rodents, sperm production is suppressed in subordinate males. In addition, dominant males develop significantly larger seminal vesicles. Here, quantitative proteomic analysis reveals that the composition of the secretion within the major accessory sex gland in house mice differs according to social status. Subordinate males produce a more concentrated protein secretion, which contains a greater proportion of the protein SVS2. This protein is essential in copulatory plug formation, and increasing the proportion of SVS2 within their seminal fluid may allow subordinate males to produce copulatory plugs of an equivalent size to those produced by dominant. Within mammals, the oviduct is the site of fertilisation and can exert control over sperm storage and movement. As male house mice can plastically alter their ejaculate production and allocation according to local sperm competition risk, it is feasible that females may alter the environment within the oviduct in response to similar cues of multiple mating, to maintain control of paternity and reduce the risk of polyspermy. I found no evidence for plasticity in the oviduct proteins of female house mice following either a high or low level of interactions with male house mice. Indeed, the oviduct proteome was more similar within siblings, balanced across treatments, than within treatment groups. I did find evidence for plasticity in female behaviour, when interacting with a novel male, and in female ano-genital distance. Mothers in populations with high levels of sperm competition may benefit from producing more competitive male offspring. I analysed male offspring produced by female house mice that had encountered either a high or low level of male odour and interactions prior to pregnancy. Quantitative proteomic investigation revealed that ejaculate composition differed according to the level of male interaction their mother had encountered before pregnancy. In contrast, I found no evidence for variation in male mating behaviour, reproductive morphology or ejaculate size. This study reveals the potential for maternally driven, subtle alterations in ejaculate composition. Sperm competition has driven the evolution of testicular architecture. Comparative analyses reveal that relatively larger testes also produce sperm more efficiently. Improved spermatogenesis efficiency is primarily due to a faster rate of each seminiferous epithelial cycle, thereby increasing sperm production rate. This trait is linked to sperm competition, and so sperm competition is selecting for a greater sperm output, primarily by increasing the rate of production. Sexual conflict and post-copulatory sexual selection drive the rapid evolution of genes involved in reproduction. The reproductive proteomes of relatively closely related species may therefore be very different from each other. Comparative proteomic analysis of cauda epididymal samples from two groups of mammals, rodents and ungulates, reveals broad similarities in the sperm proteome. Closer analysis of proteins known to be involved in sperm – egg interactions suggests these proteins are very divergent, and exhibit a low level of sequence homology.

Bitterling are a group of freshwater fishes that lay their eggs on the gills of living freshwater mussels, using the mussel as a protective environment for embryo development and utilising the mussels own respiration to ensure fertilisation. This unusual spawning mechanism, using a spawning site that can be easily manipulated, makes bitterling ideal of reinvestigating sperm competition and making system evolution. Here, using a range of bitterling species, a series of aquarium experiments were conducted, as well as morphological and histological studies of the sperm and testes. It was shown that males were highly sensitive to sperm competition, ejaculating at a higher frequency and subsequently becoming more sperm depleted where sperm competition was high. There were few differences between mating tactics except in relative testis size, where larger males had proportionally larger reproductive apparatus, but ejaculates were of a similar size. The timing of ejaculates was found to be crucial, with a peak in sperm concentration within the mussel mantle cavity 30 seconds after ejaculation. The spatial clustering of fertilisation opportunities and OSR were found to affect ejaculate frequency, ejaculate distribution among mussels, the dominance of gender males and subsequently the opportunity of subordinate males to sneak fertilisations. Significant differences in the spermatogenic strategy and the structure of the reproductive apparatus among species were identified, as well as significant differences between species in the morphology of spermatozoa.

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.

Most species of reptile studied to date have polygynandrous mating systems and possess specialised sperm storage regions. Consequently, there is a high potential for sperm competition in this group. Using comparative analyses, I examined the level of sperm competition in New Zealand geckos and how this has influenced the evolution of their reproductive morphology. Across lizards and snakes, there was more than a 40-fold variation in relative testis size. New Zealand geckos fell in the middle of this range and lacked sexual dimorphism in head size, suggesting that most species have polygynandrous mating systems. I confirmed this for one species, Hoplodactylus maculatus, which is gregarious, lacks territoriality and has a courtship pattern that suggests a high level of promiscuity for both sexes. I found that hemipenis size in New Zealand geckos was positively correlated with relative testis size, suggesting that sperm competition has resulted in the evolution of larger intromittent organs. However, the surface features of the hemipenis were relatively conservative across species. Although there was no relationship between sperm length or putative sperm storage site (SST) morphology and relative testis size, species with fewer SSTs, and thus more intense sperm competition, had longer sperm. H. maculatus males produced two types of sperm which differed not only in length but also in fertilising capacity, the short morph lacking DNA. This is the first known example of such sperm polymorphism in a vertebrate and may have evolved in response to sperm competition, the non-fertilising morph potentially helping to block the sperm of rival males or filling sperm storage sites. The motility of these short sperm was positively correlated with temperature; however, at higher temperatures motility declined with time, suggesting a trade-off between motility and longevity. Such temperature influences on male reproductive physiology have important implications for males of ectothermic species under sperm competition.

Macropods exhibit the second largest sexual dimorphism in body size for any vertebrate, suggesting there is a major role of sexual selection, with the potential for different mating tactics amongst male kangaroos. It is suggested that dominant males invest heavily in large forelimb muscles, which aids in male-male competition, female coercion and increased attractiveness to females, gaining them the majority of matings. A subdominant male may not invest in muscle mass development, but rather rely on other reproductive tactics such as sneaky matings and investment in competitive spermatozoa characteristics; for example more spermatozoa (larger testes) and faster or longer spermatozoa. We examined whether there was a trade-off between investments in forelimb muscle development and sperm competition in the western grey kangaroo (Macropus fuliginosus). Sperm traits, body mass, and forelimb muscle masses were measured for 35 male M. fuliginosus and an index of ‘muscularity’ was calculated using the residuals of individual muscle masses against body mass. There were no significant relationships between forelimb muscle investment and sperm competition traits. Significant relationships were recorded between relative testes mass and relative epididymis mass (r2 = 0.26, p = 0.003), total motility and progressive motility (r2 = 0.31, p = 0.001) and spermatozoon velocity and spermatozoon head (r2 = 0.47, p < 0.001) and tail lengths (r2 = 0.19, p = 0.011) with the longer spermatozoa swimming slower. These results suggest that there is no evidence for a significant relationship between sperm competition and forelimb muscles. There was no evidence for a trade-off between these two measures of sexual selection, suggesting that these traits are independent of each other.

ABSTRACT In the last forty years, the historical notion of monogamous females has been gradually eroded away, and female multiple mating is now look as a common and ubiquitous phenomenon in nature, triggering theoretical and experimental attention to its biological implications and evolutionary consequences. Sexual selection is the evolutionary process that favours the increase in frequency of the genes that confer a reproductive advantage to the individuals carrying them. Polyandry implies that sexual selection may persist even after the copulation up to the point of fertilization, and in some cases beyond. In this scenario, male mating not necessarily results in successful insemination, but depends on the outcome of post-copulatory sexual mechanisms influencing the fertilization success, namely cryptic female choice and sperm competition. Female choice is the possibility for females to non-randomly bias paternity, favouring the ejaculate of the best quality male, in order to maximize their fitness. On the other hand, male-male competition results in sperm competition when the ejaculates of two or more males compete to fertilize the same set of eggs, as it has been firstly defined by Geoffrey Parker. When ejaculates overlap in space and time, differences in characteristics that are key factors for the fertilization success may lead one ejaculate to overcome the rivals, generating differential males’ reproductive success. This mechanism, investigated in both externally and internally fertilizing species, is a powerful evolutionary force moulding an amazing variety of behavioural, morphological and physiological traits. Ejaculates are costly to produce and, thus, sperm expenditure drawns the attention for how males, to increase their reproductive success, may modulate their investment in response to different sperm competition levels. Sperm competition is expected to influence those traits driving sperm fertilization capabilities in a specific context. To date, theoretical and empirical studies have primarily and only focused on how sperm characteristics, i.e. number and quality, affect the fertilization success of competing males. Increasing evidence are suggesting that predictions on the outcome of sperm competition should not revolve only around the sperm component of the ejaculate. The seminal fluid often makes up a large part of an ejaculate and it may influence paternity success both directly and indirectly. Indeed, seminal fluid is already known to enhances sperm performance in several species as well as to indirectly influence paternity success, by decreasing female receptivity, increasing oviposition rate and forming mating plugs. Seminal fluids may also play a frontline role in sperm competition by directly affecting rivals’ sperm performance. For instance, in promiscuous ants and bees, seminal fluid incapacitates the sperm of rival males, while in other insects, it improves equally the survival of own and other sperm. This suggests that, unless a self/non-self-recognition mechanism evolves, the function of seminal fluid to enhance own sperm performance can be exploited by the sperm of rival males. In particular, when a male can his reproductive role while mating with a female, if advantaged or disadvantaged, he could strategically allocate his ejaculate to maximize the reproductive success. Theoretical analyses, still waiting for experimental tests, posit that selection should favour phenotypic plasticity in male expenditure on both sperm and seminal fluid components, specifically influencing that/those that affect more the ejaculate competitive weight. Clear evidence still lack, likely because, up to the present study, models and experimental works considered mostly internal fertilizers, where it is difficult to attribute sperm and seminal fluid to a specific individual. I overcame these problems by using as model species two fishes with external fertilization, the grass goby (Zosterisessor ophiocephalus) and the black goby (Gobius niger) as they show a similar mating system and comparable levels of sperm competition, but potentially differ in the likelihood for seminal fluid to influence competition contexts. In both species males display territorial-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted, with sneaker males experiencing the highest level producing a great number of sperm and less seminal fluid than territorial males. In the grass goby, sperm quality, in terms of velocity, viability and ATP content, does not vary between tactics, whereas, black goby sneakers produce sperm that are faster, more viable and richer in ATP than territorial males. In these two species, the dynamics of mating are potentially a crucial factor influencing the role of seminal fluid on the outcome of ejaculates competition. Indeed, grass goby sneakers enter inside the nest and may release their ejaculates in close proximity to those of the territorial male and to eggs. Thus, in this species I expected that the seminal fluid might have a competitive weigh, mediating sneaker and territorial ejaculates interplay. By contrast, in the black goby, sneakers are forced to release their ejaculate at the nest entrance and, thus, the opportunity for the mixing of territorial males’ and sneakers’ ejaculates does not occur or it is rare. On the basis of these preconditions, my PhD project pointed to i) verify in both species when and how the seminal fluid affects sperm performances, in terms of velocity, viability of own and rival sperm, making combinations of sperm and seminal fluid within and between males adopting different tactics; ii) deepen, in the grass goby, the proximate mechanisms driving sperm and seminal fluid interplay; iii) evaluate if the results from sperm performance give reliable insights on their fertilization ability and on the outcome of ejaculates competition in nature. Therefore, I performed in vitro fertilization tests, applying the same experimental design used in sperm performance trials. Secondly, considering that the paternity success of the grass goby has been already investigated from natural nests in a previous work, I concentrated on the black goby. I evaluated the fertilization success in the field through artificial nests located in natural breeding sites, by using molecular parentage analyses. In the grass goby, I found that sneaker’s sperm increase their performance, both in terms of velocity and fertilization rate, in presence of territorial male’s seminal fluid, while the performance of territorial male’s sperm is decreased in presence of sneaker’s seminal fluid. Appropriate control experiments demonstrate that this effect is not mediated by a self/non-self recognition mechanism. Investigating the proximate mechanisms driving sperm-seminal fluid cross interactions, we found that sneaker’ and territorial male’s ejaculates differ in seminal fluid protein content (quantitatively and qualitatively) and even in sperm quality, with sneaker sperm showing an higher oxygen consumption rate, a parameter rarely measured in sperm quality analyses. The deepening of sperm-seminal fluid proximate mechanisms is just at the beginning but I highlighted how the non-protein fraction of the seminal fluid (<3kDa) is crucial for sperm performance (velocity), despite even the protein fraction indicate a minor influence. In this species, sperm velocity results are perfectly mirrored by in vitro fertilization tests, hence sperm velocity is a reliable indicator of ejaculate fertilization ability. Considering the paternity success recorded in the field during a previous work, it seems that it is the territorial males nest guarding that finally determines their fertilization success, and thus, the distance at which sneakers are forced to release their ejaculates. Indeed, territorial males fertilization success positively correlate with their body size. In the black goby, where the ejaculates of competing males are released far from each other, seminal fluid does not affect the sperm performances of rival males, as expected. Despite the seminal fluid of territorial males significantly enhances their sperm speed, still sneaker sperm are significantly faster, regardless the seminal fluid present. Results from in vitro fertilization tests, apparently do not mirror sperm performance, since sneaker and territorial males fertilization rates do not significantly differ, probably because the low number of trials. However, I evidenced that sneaker and territorial males sperm differ in their swimming mode, with territorial male sperm moving in a significantly more linear trajectory. As a consequence, it could take the same time to sneaker and territorial male sperm to travel the same distance, even if those of sneaker have an higher speed. The analysis of the paternity distribution of territorial males in the field suggests that the distance at which sneakers are forced to release their ejaculates determines the number of eggs they fathered, as in the grass goby. Indeed, preliminary results from artificial nests in the field indicate that snakers stole more fertilizations close to the nest principal entrance, lowering the territorial male fertilization success in that area, but less in the rest of nest ceiling. However, territorial males parentage success is unexpectedly low, respect to that registered in the grass goby and across other fish species with a similar mating system. In addition, we found in two of four analysed nests few embryos sired by a neighbour territorial male. If the result would be confirmed by further analysis, it implies that territorial males may occasionally adopt sneaking behaviours, probably depending on the level of ejaculates competition determined by nests availability and male density. The territorial mating role would not appear as favoured as in other species with alternative mating tactics, especially considering that sneakers visits more than one nest. Further studies should be addressed to the investigation of territorial male paternity success along the breeding season. In both species, the spatial context in which the competition between ejaculates occurs proved to be important. The distance at which rival ejaculates are released determines the opportunity for the rival seminal fluid exploitation, and, consequently influences the strategy to maximize the fertilization success: through the number and/or the quality of the sperm, or taking advantage of the seminal fluid of a rival-tactic male. The seminal fluid proven itself to be one of the factor that may tip the balance in the ejaculates competition scenarios, that need to be investigated with a comprehensive multidisciplinary approach.
RIASSUNTO Negli ultimi quaranta anni, è emerso che i sistemi di accoppiamento nel mondo animale sono di tipo promiscuo o poliandrico molto più frequentemente di quanto non si credesse, rendendo necessarie una serie di considerazioni sulle implicazioni biologiche ed evolutive derivanti in particolare dal fatto che, in molte specie, le femmine si accoppiano con più di un maschio. La selezione sessuale è quel processo evolutivo che promuove la trasmissione dei geni che avvantaggiano dal punto di vista riproduttivo l’individuo che li esprime. Un sistema di accoppiamento poliandrico, o promiscuo, implica che la selezione sessuale possa proseguire anche dopo l’accoppiamento, fino al momento della fecondazione, e in alcuni casi anche successivamente. In questo contesto, il successo di fecondazione di un maschio non corrisponde necessariamente al suo successo di accoppiamento, ma può essere influenzato dai meccanismi di selettivi post-copulatori, quali la scelta criptica femminile e la competizione spermatica. Il primo meccanismo si realizza quando una femmina è in grado di favorire, “scegliere”, l’eiaculato di un maschio rispetto a quello degli altri maschi con cui si è accoppiata, e la scelta è stata definita criptica perché avviene all’interno delle vie riproduttive femminili. La competizione spermatica è stata definita come la competizione tra gli eiaculati di due o più maschi per fecondare uno stesso gruppo di uova. Questo secondo meccanismo è stato studiato sia in specie a fecondazione interna che esterna, e spinge l’evoluzione di tutti quei tratti morfologici, comportamentali e fisiologici che possono avvantaggiare l’eiaculato di un maschio rispetto agli eiaculati rivali in un determinato contesto di competizione. La produzione di spermi e liquido seminale comporta dei costi, e i maschi possono variarne l’investimento per massimizzare il proprio successo riproduttivo a seconda del livello di competizione spermatica percepito. Gli studi sperimentali e i modelli teorici si sono da sempre concentrati su come la competizione spermatica possa modellare il numero e la qualità degli spermi prodotti. Recentemente, tuttavia, la ricerca scientifica ha mostrato come la competizione tra gli eiaculati possa coinvolgere anche il liquido seminale. Questo, infatti, spesso costituisce una parte consistente dell’intero eiaculato ed è fondamentale per il mantenimento e le prestazioni degli spermi. Inoltre, nelle specie a fecondazione interna, è stato dimostrato che può indirettamente influenzare il successo di fecondazione di un maschio spingendo ad esempio le femmine a deporre un maggior numero di uova, oppure riducendo la loro ricettività e tendenza a riaccoppiarsi per limitare la competizione con gli spermi di altri maschi. Solo negli ultimi anni è stata presa in considerazione la possibilità che il liquido seminale di un maschio potesse agire direttamente sul successo di fecondazione degli eiaculati rivali. Per esempio, uno studio comparativo ha dimostrato che nelle specie di api e formiche dove il livello di competizione è più alto, il liquido seminale di un maschio favorisce le prestazioni dei propri spermi, e in maniera minore quelle degli spermi dei maschi rivali. In altre specie di insetti, invece, il liquido seminale di un maschio aumenta allo stesso modo la performance di tutti gli spermi contemporaneamente presenti. Questi risultati indicano che quando non si realizza un meccanismo di riconoscimento self/no-self tra gli spermi e il liquido seminale, le sue proprietà potrebbero essere sfruttate anche dagli spermi degli altri maschi in competizione. Di conseguenza, se, al momento dell’accoppiamento, un individuo riesce a stimare il grado di competizione che dovrà fronteggiare, potrebbe strategicamente regolare l’investimento nell’eiaculato a seconda che si trovi in una posizione favorita o sfavorita. I modelli teorici più recenti indicano che l’investimento nell’eiaculato può essere modellato su entrambe le sue componenti, a seconda di quanto spermi e liquido seminale influenzino il successo dell’intero eiaculato in un determinato contesto di competizione. Prima di questo progetto, le prove sperimentali a supporto delle predizioni derivate dai modelli sono state poche e i risultati contrastanti, probabilmente perché sia la ricerca teorica che quella sperimentale si sono prevalentemente concentrate su specie a fecondazione interna, dove è difficile separare il contributo di spermi e liquido seminale e distinguere tra gli eiaculati in competizione. Nel mio progetto ho usato quindi come modello due specie di pesci Teleostei che presentano fecondazione esterna, il go, Zosterisessor ophiocephalus e il ghiozzo nero, Gobius niger, che hanno un sistema di accoppiamento simile e livelli di competizione spermatica confrontabili. In entrambe le specie i maschi adottano tattiche riproduttive alternative, con i maschi dominanti, territoriali, che scavano un nido all’interno del quale la femmina depone le uova, e i maschi opportunisti, sneaker, che cercano di sfruttare gli accoppiamenti dei maschi territoriali. I maschi sneaker, che si confrontano con un più alto rischio di competizione dovendo sempre competere almeno con l’eiaculato del maschio territoriale, producono un maggior numero di spermi e meno liquido seminale rispetto ai maschi territoriali. Nel go, le prestazioni degli spermi non differiscono tra le due tattiche, mentre nel ghiozzo nero gli spermi dei maschi sneaker sono più veloci, longevi e presentano un maggior contenuto in ATP. Le due specie costituiscono un buon modello anche perché il liquido seminale ha una diversa possibilità di entrare in gioco nella competizione tra gli eiaculati a causa di alcune differenze legate alle modalità di accoppiamento. Infatti, nel go, i maschi sneaker riescono ad entrare nel nido e a rilasciare il proprio eiaculato vicino a quello del maschio territoriale e alle uova e, di conseguenza, in questa specie il liquido seminale potrebbe avere un ruolo nell’influenzare la competizione tra gli eiaculati. Nel ghiozzo nero, invece, i maschi sneaker sono spesso costretti a rilasciare il proprio eiaculato all’ingresso del nido, e di conseguenza il poco liquido seminale prodotto va verosimilmente incontro ad una rapida diluizione, e il grado di interazione tra spermi e liquido seminale di maschi aventi tattiche opposte è di conseguenza minore rispetto a quanto non accada nel go. Dato questo scenario, il mio progetto di dottorato ha voluto i) verificare, nelle due specie, quando e come il liquido seminale influenzi la competizione tra eiaculati, incrociando spermi e liquido seminale di maschi aventi la stessa o la tattica opposta e misurando le prestazioni degli spermi come velocità, vitalità e tramite test di fecondazione in vitro; ii) approfondire i meccanismi prossimi di interazione tra spermi e liquido seminale nel go, dove il liquido potrebbe effettivamente pesare sulle prestazioni degli eiaculati di maschi aventi tattiche opposte; e iii) controllare, nel ghiozzo nero, se i parametri considerati per misurare le prestazioni di un eiaculato sono indicativi del suo reale successo di competizione, andando quindi a misurare il successo di fecondazione dei maschi territoriali sul campo, tramite nidi artificiali ed analisi di paternità. Nel go, questa distribuzione della paternità nei nidi naturali è già stata valutata in un precedente studio. Nel go, è stato dimostrato un effetto tattica-dipendente del liquido seminale. Gli spermi dei maschi sneaker sono più veloci e mostrano un maggior successo di fecondazione quando è presente il liquido seminale di un maschio territoriale, mentre gli spermi dei maschi territoriali mostrano prestazioni inferiori, per gli stessi parametri, in presenza del liquido di un maschio sneaker, sempre rispetto ai valori misurati con il proprio liquido seminale. Un appropriato esperimento di controllo ha escluso che questi risultati siano dovuti ad un meccanismo di riconoscimento self/no-self tra spermi e liquido seminale. L’indagine dei meccanismi prossimi alla base dell’interazione tra spermi e liquido seminale ha evidenziato che l’eiaculato differisce tra maschi aventi tattiche opposte sia nella composizione del liquido seminale, per quanto riguarda il contenuto e il profilo proteico (differenze qualitative e quantitative), sia nelle prestazioni degli spermi misurate in termini di tasso di consumo dell’ossigeno, un parametro metabolico importante ma raramente considerato. Inoltre, nonostante le analisi sulla funzionalità del liquido seminale siano solo preliminari, hanno già dimostrato come sia determinante la presenza della frazione non proteica (<3kDa) per la velocità degli spermi, nonostante anche la frazione proteica (>3kDa) contribuisca, seppur in maniera minore. In questa specie, le prove di fecondazione in vitro rispecchiano i risultati emersi dai dati di velocità degli spermi, che di conseguenza risulta un buon indicatore della competitività dell’eiaculato. Considerando quanto emerso da uno studio precedente sulla distribuzione della paternità nei nidi naturali, sembra che il successo di fecondazione dei maschi territoriali sia determinato in ultimo dall’efficacia della difesa del nido, e cioè da quanto riescono a tenere a distanza i maschi sneaker durante l’accoppiamento. Infatti, il loro tasso di paternità correla positivamente con le loro dimensioni corporee. Nel ghiozzo nero, dove un maschio sneaker rilascia il proprio eiaculato a distanza rispetto a quello del maschio territoriale, non emerge nessun effetto tattica-dipendente del liquido seminale. Nonostante il proprio liquido seminale migliori le prestazioni degli spermi nei maschi territoriali, gli spermi dei maschi sneaker rimangono ancora significativamente più veloci e vitali. Le prove di fecondazione in vitro non rispecchiano i dati emersi, e non emerge alcuna differenza significativa nel successo di fecondazione delle due tattiche, anche se questo potrebbe esser dovuto alla scarsa numerosità dei dati. Tuttavia, in questa specie gli spermi dei maschi territoriali presentano un moto significativamente più lineare rispetto ai maschi sneaker e quindi è possibile che il loro nuoto sia in realtà più efficace, e possano percorrere la stessa distanza nello stesso tempo, nonostante gli spermi degli sneaker siano più veloci. L’analisi della distribuzione della paternità su campo suggerisce che anche nel ghiozzo nero sia la distanza a cui gli sneaker rilasciano il proprio eiaculato a determinare il risultato della competizione tra gli eiaculati. Infatti, i primi risultati mostrano che il successo di fecondazione del maschio territoriale è minore in corrispondenza dell’entrata principale del nido, dove è più probabile che i maschi sneaker riescano ad avvicinarsi e fecondare parte delle uova. Il successo di fecondazione del maschio territoriale all’interno di un nido è comunque basso (<50%) se confrontato con quello del go (>70%) o di altre specie di pesci aventi un sistema di accoppiamento simile. Inoltre in due nidi, alcune uova sono risultate fecondate da maschi territoriali che difendevano nidi vicini. Apparentemente quindi i territoriali parassiterebbero occasionalmente i nidi di altri maschi aventi la stessa tattica. Se il risultato venisse confermato, la posizione dei maschi territoriali nella competizione tra eiaculati potrebbe non apparire più come il ruolo favorito, considerando che i maschi sneaker visitano sistematicamente più di un nido. In entrambe le specie, il contesto spaziale in cui si realizza la competizione risulta decisivo. Il grado di interazione tra gli eiaculati di maschi aventi tattica opposta determina la possibilità che il liquido seminale possa esser sfruttato nell’ambito della competizione. Ne risulta che, nel go, dove il livello di interazione è alto, si crei l’opportunità per un maschio di sfruttare il liquido di un maschio avente tattica opposta, mentre nel ghiozzo nero, dove l’interazione è minima, i maschi siano costretti ad investire nel numero e qualità degli spermi prodotti o nella difesa del nido, a seconda della tattica. Il mio progetto di dottorato dimostra che il liquido seminale può effettivamente influenzare la competizione tra gli eiaculati a seconda del contesto, e che uno dei principali fattori da tenere in considerazione è il grado di interazione tra eiaculati rivali. L’approccio multidisciplinare adottato ha permesso di fare luce anche sui meccanismi prossimi di interazione tra spermi e liquido seminale.

This thesis explores the far-reaching impacts of sexual conflict over care on a suite of traits in the penduline tit family (Remizidae), further confirming the intricate relationships between parental care, mating systems and sexual selection. The results reveal the first genetic phylogeny of this family and suggest that uniparental care evolved once in this group. The transition to uniparental care is associated with rapid evolution of male plumage ornaments most likely driven by increased sexual selection. The results also suggest a relationship between male care and the likelihood of paternity on an evolutionary time-scale, as the biparental species exhibit much lower levels of promiscuity than the uniparental European penduline tit. Increased promiscuity was also found to impact sperm morphology in the penduline tits and allies with greater sperm length uniformity in more promiscuous species. This sperm trait was also discovered to co-vary with a sexually selected plumage trait in the European penduline tit suggesting potential interactions between female mate choice and male fertility. An investigation into genetic diversity within the Sylvioidea super-family finds large variation but does not suggest any link between promiscuity and genetic diversity as predicted if promiscuity maintains a higher effective population size in these passerines. The work highlights the interlinked relationships between parental care, mating systems and sexually selected traits, which are increasingly studied in concert. The consequences of sexual conflict over care appear to be far-reaching in the penduline tits, however the degree to which they feedback upon each other and the effect that it has on speciation remains to be seen. The penduline tits further prove their ability to provide valuable insight into the evolution of sexual conflict.

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.

The study of variation in arachnid genitalic structures has contributed to the fields of systematics and sexual selection. Simon (1892--1903) in his Histoire Naturelle des Araignees first divided the ecribellate higher spiders into two groups, the Haplogynae and Entelegynae, using reproductive morphology. Spider genitalia have been used as a taxonomic tool for distinguishing between taxa because of their species-specific morphological variation. Variation in spider genitalic morphology has inspired evolutionary biologists to test mechanisms of sexual selection by which the variation could evolve, ranging from Fisherian run-away selection, chase-away selection, and sperm competition. The Tetragnathidae are particularly interesting for a comparison between haplogyne and entelegyne reproductive morphologies. Within this entelegyne family, a reversal to haplogyny has occurred. Fifteen representative members of this family and four outgroup taxa were examined with scanning and transmission electron microscopy in order to describe the fine structure of spermathecae, including the distribution and density of spermathecal gland pores. While the function(s) of the glandular secretion are unknown, the distribution and density of spermathecal gland pores is discussed with regard to possible functions of the glandular secretion. The potential influence of spermathecal gland secretions on mating behavior and sperm competition is considered. Sperm release patterns have been examined in entelegynes, but previously were not available for haplogynes. The relationship between copulation duration and sperm release was determined in Tetragnatha versicolor, the first examination of sperm release patterns in any haplogyne spider. In this species, copulation duration is not proportional to sperm release. To examine the relative influence of spermathecal morphology and numerical sperm competition on paternity, sperm release and paternity was assessed in the entelegyne Nephila clavipes and the haplogyne Tetragnatha versicolor. The data clearly support differential sperm release between males as the cause of previously reported first-male advantage in Nephila clavipes and the mixed paternity found for N. clavipes and Tetragnatha versicolor in this study. The natural history, mating behavior, and sperm release were determined for a previously unstudied tetragnathid species, Glenognatha emertoni . This is the second examination of haplogyne sperm release behavior as well as the first description of an unusual escape behavior.

In the present research several aspects of the reproductive biology of the marine hermaphroditic bryozoan, Celleporell hyalin (Linnaeus, 1767) were investigated. First (see Chapter 2), aliquots, of different ages from a stock of allosperm suspension were used to fertilize a series of virgin ramets, so characterizing the decay in fertility of released sperm and any effects of sperm ageing on subsequent embryogenesis and larval metamorphosis. The effect of temperature on the above variables was also investigated. The fertile half-life of C. hvalina sperm was about 1-2 h, although significant decay in fertility occurred within a few minutes after release. Sperm ageing showed no deleterious effects on embryogenesis, larval viability, or metamorphosis. No clear effects of temperature on sperm ageing and fertilization success were found. Allosperm. storage was studied in colonies of C. hyalina. (L. ) under several experimental conditions (see Chapter 3). Recipient virgin colonies were exposed to sexually compatible allosperm suspension and the appearance of the last newly ovulated oocytes in the coelorn. was used to assess duration of sperm storage. The same experiments examined continuation of brooding cycle and brooding success throughout the period of allosperm storage. Similar obserVations were conducted on wild colonies of C. hyalina taken from the field and kept in reproductive isolation in the laboratory. Production of progeny in females zooids budded beyond the original colonial growing edge was taken as evidence of sperm movement. The results of the present study show that recipient colonies continue producing coelomic oocytes up to 5 weeks after exposure to allosperm suspension. Moreover, the progeny were produced not only by female zooids present at the moment of allosperm dosage but also by female zooids, budded later, beyond the limit of the original growing edge. Since oocytes were not present in control colonies exposed to selfsperm, the results of the present study indicate that recipient colonies store sexually compatible allosperm and transport them within the colony in order to produce viable progeny. The effect of water flow on both sperm release and fertilization success in colonies of C. hyalin (L. ) was studied (see Chapter 4). Maximum numbers of released sperm were found at low and zero water velocities. Moreover, protruded male lophophores were observed only under those conditions. Fertilization success was studied in virgin colonies of C. hyalina (L. ) exposed to compatible allosperm suspensions under different feeding activity and water flow conditions. Fertilization success was higher in colonies with more active feeding autozooids than in those with fewer feeding autozooids. High water flow conditions induced reduction in the proportion of protruded lophophores, and reduced the frequency of ovicells bearing progeny. Moreover, in all the experiments offspring were concentrated in areas of the colony bearing active feeding autozooids. The results of this study suggest that sperm release take place under similar conditions that enhance cross fertilization, with a possible role of feeding activity in bringing sperm to the proximity of receiver colonies. Sperm competition and female choice was investigated in virgin colonies of C. hyalin (L. ) exposed to sexually compatible allosperm cocktails (see Chapter 5). A microsatellite-based. genotyping system was used to determine paternity. Progeny were mainly the product of outcrossing. In a few cases, a small proportion of progeny was attributed to selffertilization. These results suggest that outcrossing is the main reproductive strategy in this species and that neither selective female choice nor sperm competition occur in C. hyalin . Cryptic incompatibility allowing a flexible mating strategy to produce out-crossed progeny Z) in the presence of allospenn and selfing when they were absent was not found. The effects of mating sequence and temporal interval between matings (2 or 48 h) on sperm precedence in double-mated individuals were studied (see Chapter 6). Paternity was determined by using a microsatellite-based genotyping system. Settled colonies produced after short mating showed evidence of sperm mixing and first-male precedence. However, last colonies produced after both short and long mating intervals showed evidence of first-male precedence. When analyses were conducted using all the sampled progeny, low incidence of paternity by the second sperm donor (P2) and absence of self fertilization were found. No effect of mating order on success of the second donor was found. Prevalence of outcrossing was also found. These results suggest that first-male precedence in C. hyalin may promote outcrossing under sperm limitation conditions, by acceptance of the first compatible allosperm to become available. The effects of exposure to sperm suspensions and stressors on sexual allocation in colonies were studied (see Chapter 7). In the first experiment the effect of a waterborne factor on receptor colonies was studied. Adult colonies were exposed to compatible allosperm suspensions that had been filtered through 0.45 Vrn pores potentially able to remove cellular sized particles. As a control, receptor colonies were exposed to non-filtered allosperin suspensions. Appearance and growth of oocytes occurred only in the coelorn. of the control colonies. The active factor is not a dissolved molecule, but a particle that can be removed from water by filtration through 0.45 [im pores. This result plus the absence of developing oocytes in the receptor colonies exposed to similar concentration of selfsperm, suggest the operation of self/honself recognition and an important and active role of allosperm in initiating colonial reproductive investment in C. hyalina. Prevention of colonial growth and others stressors were associated with production of basal male zooids. In other experiments, exposure to sperm suspensions of different degrees of genetic relatedness showed a virtual absence of production of progeny in those colonies exposed to closely related sperm (i. e. self and halfsibs). Finally, in experiments with sexually immature colonies, the onset of sexual reproduction was triggered by exposure to allosperm, resulting in the production of female zooids even before the appearance of male zooids. Contacts between colonies of different genetic relatedness were studied under laboratory conditions (see Chapter 8). Moreover, observations were made on colonies growing on their natural substrata. Five types of responses were observed, from total fusion to overgrowth. Maximum degree of fusion, or morphological fusion, was manifested as morphologic interconnection between the ad oining colonies (i. e. production of coalescent zooids). Fusion occurred in all contacts between colonies of the same genotype, between parental colonies and their progeny and, between full and half sib colonies. In most cases the production of coalescent zooids was found. Absence of fusion occurred in all contacts between unrelated colonies and between some of the half sibs. Observations on wild colonies growing in contact with each other failed to reveal any incidences of coalescence. Non-aggressive overgrowth was confined to dead areas of one colony overgrown by zooids of the other healthy colony, independent of the genetic relatedness of the pairs. Differences in the fusibility between isocontact and allocontact suggest that colony specificity exists in C. hyalin , as has been found in other sessile colonial marine organisms.

Bibliography: pages 163-179.
Spirostreptida millipedes comprise three families, the Harpagophoridae, Spirostreptidae and Odontopygidae. They are polygynandrous. Males transfer sperm via species-specific accessory genitalia called gonopods, that comprise three components, two of which, the emote and telopodite, are involved in processes of sperm transfer. The emotes function to translocate ejaculates from the penes to the vulvae, where they are stored. A delay between insemination and fertilisation provides an arena for syn- and postcopulatory sexual competition. These operate at the gametic level via sperm competition and cryptic female choice. By combining studies of genital form and function with single and double mating experiments, this study elucidates these processes in some southern African Spirostreptida millipedes.

Post-copulatory sexual selection is a strong evolutionary force, affecting morphological and behavioural traits in males and females in species with polyandrous mating systems. Many insects are subject to sperm competition; sperm from rival males compete to fertilise ova. Since sperm are finite, males should allocate them economically, tailoring ejaculate allocation to suit the reproductive potential of individual matings. Theory suggests when sperm competition risk is high, males should increase sperm numbers to achieve greater reproductive success than their rivals, but evidence of this expected fitness consequence of ejaculate allocation is largely lacking. In this thesis, I use Callosobruchus maculatus beetles to investigate the causes of ejaculate allocation patterns, and to examine whether ejaculate allocation does affect male reproductive success. In Chapter 3, I investigate the effect of rival male presence on ejaculate size and find that, while males grouped with rivals as adults produce bigger ejaculates, their increased effort unexpectedly does not lead to increased reproductive success. In Chapter 4, I examine whether larval conditions also affect ejaculate size, and find that, contrary to sperm competition theory, males reared under dense conditions produce smaller ejaculates than those reared solitarily, and that male reproductive success is consequently elevated in males reared at low larval densities compared to those reared at high densities. In Chapter 5, I then demonstrate that ejaculates produced by low density males contain more sperm than ejaculates produced by high density males, suggesting males do not respond to sperm competition level represented by larval density, but instead suffer resource limitation when reared at high density. In Chapter 6, I investigate the effects of water provision on ejaculate size, and find that males given water produce larger ejaculates, and females given water receive smaller ejaculates. Finally, I link my findings with those of other studies, and suggest my most important result is that plasticity of ejaculate allocation cannot be assumed to be an adaptive behaviour; studies directly measuring the fitness effects of male ejaculate allocation are needed, even when observed patterns conform to theory.

This thesis is an evaluation of the hypothesis that the spennathecae of spiders affects the sperm precedence patterns in a predictable way (Austad 1984). Spermathecae come in two varieties: cul-de-sac and conduit. Cul-de-sac spennathecae, according to the hypothesis, are supposed to lead to second male sperm priority and conduit to first male sperm priority . The hypothesis was evaluated both directly and indirectly. Direct measurements were made of paternity in two species, Pholcus phalangioides and Tetragnatha montana, both of which are cul-de-sac species. It was found that P. phalangioides complies with the predicted precedence pattern and thus does not disprove the hypothesis. This second male priority pattern was despite a much shorter mating time by second mating males. In T. montana no precedence pattern was found, with equal likelihood of first or second mating males of gaining paternity. There was in T. montana a possible influence of the duration of mating affecting the precedence pattern, with longer mating males gaining a higher paternity no matter what order they mated in. It is discussed whether or not this is due to sperm loading or genitalic stimulation (Eberhard 1985). Indirect evaluation of the hypothesis included an analysis of mating behaviour in Zygiella x-notata which is a conduit species and was chosen as a comparison to the two cul-de-sac species. In Z. x-notata it was found that there was no difference between mating duration in first and second mating males. Mating persistence is thus the same in first and second mating males, suggesting that the males cannot detect that the female is a denuded resource to second mating males. Hence first male priority may not be a factor in this species. Other indirect methods of evaluating the hypothesis involved charting the incidence of mate-guarding and mating-plugs. The expected pattern of mate-guarding was for conduit species to pre-mate guard and for cul-de-sac species to post-mate guard, because of the predicted sperm precedence patterns associated with the spermathecae. The predicted pattern was not found. In the case of mating-plugs it was predicted that these should be deployed by cul-de-sac species because it is in these species that second males are able to usurp paternity to a large extent. The opposite pattern was found with mating-plugs of various design being utilized by conduit species. It is postulated that mating-plugs are the mechanism by which first male priorities are established in conduit species, where this pattern is found. The absence of plugs in cul-de-sac species is possibly the reason that second males can cuckold. The additional data collected since 1984 reveal that patterns of paternity found in spiders seem to be more complex than was originally assumed by Austad (1984). Spermathecae are species-specific in character and this may reflect a species specificity in sperm precedence patterns. Thus the conduit I cul-de-sac dichotomy may not reflect a useful prediction of paternity patterns.

Over the last century, sexual selection has grown from a controversial theory into a vast field of theoretical and empirical research. Although Darwin outlined two major mechanisms within his theory, male-male competition and female mate choice, the latter has promoted a wealth of research by virtue of its complexity. Despite decades of research into how female preferences and sexually selected traits have evolved, there is still little consensus as to why females prefer the males they do. Preferences are thought to evolve from either direct selection on the preference, as females themselves benefit directly from mating with a preferred male, or through indirect selection on the preference via offspring fitness. In all cases however, female preferences should compensate for the costs of discriminating between potential mates, if they are to remain overall beneficial. The fitness benefits of mating with preferred males were investigated here using the fruitfly Drosophila simulans, employing a range of behavioural, phenotypic and quantitative genetic approaches. The findings presented here indicate that female Drosophila simulans do not gain directly from mating with a preferred male. Multiple mating can increase fecundity, although costs from male harassment can reduce the net benefit. They also indicate that females may benefit indirectly from mating with attractive males as attractiveness is heritable and sons of preferred males are themselves preferred. There is also evidence that attractive males are successful in both the pre- and post-copulatory sense, as preferred males are better sperm competitors than less-preferred males. However, although there appear to be benefits from preferred males via their sons, there appear to be no benefits from males via their daughters’ fitness. These findings collectively indicate that female preferences in Drosophila simulans are driven by indirectly selected benefits (via Fisherian sons), and that females benefit directly from mating multiply.

Background: Multidirectional interactions in social networks can have a profound effect on mate choice behavior; e.g., Poecilia mexicana males show weaker expression of mating preferences when being observed by a rival. This may be an adaptation to reduce sperm competition risk, which arises because commonly preferred female phenotypes will receive attention also from surrounding males, and/or because other males can copy the focal male's mate choice. Do P. mexicana males indeed respond to perceived sperm competition risk? We gave males a choice between two females and repeated the tests under one of the following conditions: (1) an empty transparent cylinder was presented (control); (2) another ("audience") male inside the cylinder observed the focal male throughout the 2nd part, or (3) the audience male was presented only before the tests, but could not eavesdrop during the actual choice tests (non-specific sperm competition risk treatments); (4) the focal male could see a rival male interact sexually with the previously preferred, or (5) with the non-preferred female before the 2nd part of the tests (specific sperm competition risk treatments). Results: The strength of individual male preferences declined slightly also during the control treatment (1). However, this decrease was more than two-fold stronger in audience treatment (2), i.e., with non-specific sperm competition risk including the possibility for visual eavesdropping by the audience male. No audience effect was found in treatments (3) and (5), but a weak effect was also observed when the focal male had seen the previously preferred female sexually interact with a rival male (treatment 4; specific sperm competition risk). Conclusion: When comparing the two 'non-specific sperm competition risk' treatments, a very strong effect was found only when the audience male could actually observe the focal male during mate choice [treatment (2)]. This suggests that focal males indeed attempt to conceal their mating preferences so as to prevent surrounding males from copying their mate choice. When there is no potential for eavesdropping [treatment (3)], non-specific specific sperm competition risk seems to play a minor or no role. Our results also show that P. mexicana males tend to share their mating effort more equally among females when the resource value of their previously preferred mate decreases after mating with a rival male (perceived specific sperm competition risk), but this effect is comparatively weak.

Marine and freshwater environments support numerous species of teleost fish with a wide and diverse range of reproductive strategies. Despite the considerable interest in fish reproduction, our knowledge regarding ejaculate traits and factors affecting them is limited. Using computer-assisted sperm analysis (CASA) I measured ejaculate traits (sperm swimming speed, motility, path trajectory, longevity and concentration) from sexually mature chinook salmon (Oncorhynchus tshawytscha) activated in freshwater and ovarian fluid. I also looked at these ejaculate traits in relation to measures of male quality (body condition) and investment into reproduction (relative testes mass). Furthermore, I determined the chemical composition of seminal and ovarian fluid and looked at the effect these fluids have on sperm behaviour. A considerable amount of intraspecific variation existed in all ejaculate traits measured, and investment into reproduction (relative testes mass) was dependent on male body condition, as males in better condition had relatively larger testes. However, these males did not have superior quality ejaculates or ejaculates with a higher density of spermatozoa; hence the potential reproductive advantage of having larger relative testes in this species remains unknown and requires further investigation. In addition, a positive relation between sperm longevity and sperm swimming speed was observed defying the expected trade-off between ejaculate traits according to theory. There was also a weak negative trend in our data between body condition and sperm swimming speed, linearity, and longevity. All sperm traits measured were greatly enhanced when activated in a solution containing ovarian fluid (a viscous fluid which is excreted with the egg batch during spawning) from female chinook salmon. Interestingly, sperm swimming speed activated in fresh water only accounted for < 12% of the observed variation in mean sperm swimming speed in ovarian fluid. This result suggests the sperm traits measured in fresh water are not relevant to those same traits measured in ovarian fluid, so caution should be applied when comparing the potential for individual males to fertilize ova when sperm traits are activated in water, especially in studies of sperm competition in an externally fertilising species. Sperm competition between males is known to strongly influence sperm and ejaculate traits, but less is known about female sperm choice after copulation via a process called cryptic female choice (CFC). In CFC, females may have the ability to favour the sperm of one male over another and bias fertilisation accordingly. To test whether ovarian fluid could act as a mechanism of CFC in an externally fertilising fish species, I measured sperm traits from each male activated in the ovarian fluids from different females. I found that mean sperm swimming speed, longevity, and path trajectory differed significantly among males, but most importantly, the pattern of within-male variation in these traits also varied significantly among males in response to different females’ ovarian fluids. This result suggests that ovarian fluid may be a potential mechanism of CFC whereby females differentially enhance the swimming speed of sperm from different males. In addition, I found that sperm longevity was negatively correlated with variation in [Ca²⁺] and [Mg²⁺] concentration in the ovarian fluid, while percent motility increased with increasing concentration of [Mg²⁺]. These observations provide a possible chemical basis for cryptic female choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males and thus their fertilisation success. This finding is particularly exciting, as we may have uncovered a potential mechanism of CFC in an externally fertilising species, which is poorly understood. In addition, results from this study suggest new directions for genetic studies to provide direct evidence for CFC. For example, does sperm selection via ovarian fluid promote favoured genetic combinations that enhance male reproductive success?

The aim of my study was to investigate the maintenance of variability for sperm production in the guppy, P. reticulata. Sperm number, which is the main determinant of SC outcome in the Trinidadian guppy population studied in my lab, shows high additive genetic variance despite strong directional selection and very high sire heritability and stronger condition dependence than other ejaculate traits; furthermore, multiply mated females produce higher quality offspring, suggesting that sperm c ompetition, and hence sperm number, may mediate genetic benefits to the female. To clarify the evolutionary processes associated with sperm allocation, I used bi-directional artificial selection for sperm production to 1) estimate the genetic correlations between sperm production and other sexually and naturally selected traits in both males and females; 2) investigate whether sperm production is associated with the rate of senescence and longevity; 3) estimate the contribution of partially recessive deleterious mutations to phenotypic variation in sperm production
Lo scopo di questa tesi è quello di valutare, in una specie nella quale sono noti in modo dettagliato i meccanismi di selezione sessuale pre- e postcopulatoria, le conseguenze evolutive dell’investimento maschile nella produzione di spermi, il principale carattere che predice il successo di competizione spermatica in questa specie. Considerazioni teoriche suggeriscono che l'allocazione differenziale in caratteri pre e post-copulatori costituisca un constraint evolutivo cruciale nell’ambito della selezione sessuale. A dispetto di questo mancano studi nei quali l’analisi di questi gruppi di caratteri sia valutata contemporaneamente in modo integrato. A questo scopo è stato effettuato un esperimento di selezione artificiale bidirezionale per la produzione spermatica al fine di: 1) Determinare se la selezione artificiale per la produzione di spermi ha un effetto correlato sull’espressione degli altri caratteri post-copulatori (velocità, vitalità e morfologia degli spermi). 2) Determinare se la selezione artificiale per la produzione di spermi ha un effetto correlato sull’espressione dei caratteri pre-copulatori, in particolare del pattern di colorazione del maschio, del comportamento sessuale e del grado di preferenza esercitato dalle femmine sul maschio. 3) Determinare se la selezione artificiale per la produzione di spermi ha un effetto correlato sulla fitness non riproduttiva del maschio, stimata sul tasso dl accrescimento, sulla taglia a maturità, e sulla sopravvivenza. 4) Determinare se la selezione artificiale per la produzione di spermi ha un effetto correlato sulla fitness delle femmine. A questo scopo sarà determinato il tasso di accrescimento, la fecondità e la condizione nelle femmine. 5) Determinare se un maggior investimento riproduttivo in età giovanile comporta dei costi in età avanzata a causa di una maggior senescenza e una ridotta longevità. 6) Determinare se una maggior produzione di spermi riflette una maggior qualità genetica del maschio attraverso un minor carico di mutazioni recessive deleterie espresse in importanti caratteri della fitness sia maschile (produzione di spermi, colorazione corporea, condizione) che femminile (fecondità), portate in omozigosi attraverso un esperimento di inbreeding sulle linee selezionate artificialmente

The honey possum Tarsipes rostratus, a marsupial endemic to South-Western Australia, feeds exclusively upon nectar and pollen. It is one of the smallest marsupials, with adult females (8-12g) significantly larger than adult males (6-9g). Honey possum males have the longest sperm (356µm) recorded for any mammaland the testes represent 4.2% body weight, amongst the largest recorded formammal species. These features suggest that sperm competition is an importantpart of the mating system. This study used a combination of field based studies,DNA analysis and histological examination of the female reproductive tract toinvestigate the life history, multiple paternity and reproduction of the honey possum innatural populations in the Fitzgerald River National Park (FRNP), on the south coastof Western Australia. This study drew upon earlier work on the honey possum in the FRNP in order to describe its life-history. The honey possum is short-lived (1-2 years), and attains sexual maturity whilst still growing. All four teats are occupied after birth, but the litter is reduced to 2 or 3 young during pouch life. The young have a relatively slow rate of growth. Breeding occurs continuously throughout the year, but is affected by the flowering phenologies of its foodplants. The greatest proportion of females with pouch-young occurs in winter; there are fewest pouch-young in autumn, a time of year when there is a dearth of flowers. Honey possums are essentially solitary animals, with no structured social unit, and male and female home ranges overlap. In captivity they are largely tolerant of one another, but larger females are behaviorally dominant to smaller females and to males. The densities and structure of the honey possum populations in the FRNP were analyzed from trapping data collected over 19 years. Population densities fluctuated significantly from season to season throughout the year, with changes in the flowering food resources available. There were also year-to-year differences in the intensity of those fluctuations, and these were significantly associated with rainfall in the previous year, and probably mediated through a lag effect in the flowering of the honey possum’s foodplants. The greatest densities of animals occurred over winter. In years following high rainfall, mean winter densities reached 88 individuals per hectare. The lowest densities occurred in spring, and in years following low rainfall mean spring densities fell to 8 individuals per hectare. Even at these lowest densities, there is still the potential for interaction between males and females. A succession from high to low, then back to high densities was seen during the three years of the present study (2000-2002) and this shadowed a similar succession of changes in rainfall. The proportion of females with pouch-young was significantly affected by the season, and by rainfall in the previous year. Years following low rainfall had a lower proportion of females in a condition to breed. The autumn dip in breeding that occurred in all years was exacerbated following dry years. Of those females that did breed in 2001, a time of low resources, there was no difference in the size of the litter compared to 2000 and 2002, times of higher resource availability. The sex-ratio of pouch young was at parity, but there was a slight bias towards males among both juveniles (56%) and adults (58%). This was probably due to the greater movements shown by males. Sex ratios were not affected by changes in rainfall and density. Male-biased dispersal was detected using genetic data and the movement patterns of males showed that they moved greater distances than females during their normal activity. Analysis of four microsatellite loci revealed extremely high levels of variation, with 28 to 50 alleles per locus and a mean expected heterozygosity of 0.95. These are amongst the highest seen in any microsatellite study of vertebrates. There was multiple paternity in 86% litters, using a minimum number of sires per litter method, and in 95% litters, using an estimated number of sires method based upon the relatedness of litter males. This indicates that multiple mating is frequent in female honey possums and is evidence for sperm competition. The estimated number of sires in a litter was often three or four. In 41% of cases, the number of sires was less than the number of young in the litter, indicating that some males were more successful at siring offspring than others. Nevertheless, no more than two offspring in a litter were known to have been sired by the same male. Despite marked fluctuations in density from high in 2000, to low in 2001, then high again in 2002, the level of multiple paternity remained equally high in all years. Embryonic diapause and female reproduction was investigated in the honey possum. All adult females examined, both with and without pouch-young, were either close to oestrus, had ovulated or were carrying conceptuses. The honey possum has a postpartum oestrus and it was evident that this occurs approximately 2-4 days after birth. Cleavage and formation of the unilaminar blastocyst appears to occur rapidly over approximately 5 days. Embryonic diapause proceeded in a two phase manner similar to other small possum species. The unilaminar blastocyst expanded rapidly at first; and then, from about 18-20 days after birth, the diameter of the blastocyst remained constant at approximately 1.2-1.8mm. No growth or development beyond the unilaminar stage was observed during pouch-life. The first signs of reactivation occurred during lactation, after pouch exit, and expansion of the blastocyst only occurred in one post-lactational female. The development of the corpus luteum appeared different to patterns described for other marsupials, but its formation coincided with the formation of the unilaminar blastocyst. The diameter of the corpus luteum remained constant throughout diapause. The histology of the reproductive tract was generally similar to other marsupials. There were no sperm storage crypts in the female reproductive tract. The length of pouch-life in the honey possum was 55-65 days, and the interval between litters of the same size varied between 65 and 100 days. Embryonic diapause may reduce the time between production of successive litters in the honey possum, but lifetime reproductive potential is reasonably low. Females had up to four litters over the period that they were captured. Thus, each litter represents a substantial proportion (25%) of a female’s lifetime reproductive output. Reproductive amortization occurred, with 61% loss overall, due to overproduction of ova, loss of conceptuses and reduction of the litter during lactation. The behavioural dominance of females suggests that multiple mating is an active strategy, and this presumably allows the genetic quality of their offspring to be maximized. Males that succeed in sperm competition may be of better intrinsic quality. Overproduction of conceptuses by females presents the opportunity for them to select those fertilized by intrinsically viable males or genetically compatible males. Sexually active males are present all year round. Females were not synchronous in their sexual receptivity, and this would lead to a skewed operational sex ratio, with more reproductive males than oestrous females. Since adult males are significantly smaller than adult females and possess no ornaments or armaments, it is unlikely that males overtly fight for access to females. Rather, males appear to monitor the reproductive status of females through smell, and probably compete in their ability to locate oestrous females. The risk and intensity of sperm competition is high, sexual selection for a large investment in spermatogenesis is evident and competition after copulation is probably an important factor in the mating system. It is likely that males, as well as females mate multiply, and the mating system is promiscuous.

The mating behaviour and reproductive ecology of the big-handed crab Heterozius rotundifrons was studied at Kaikoura between November 1997 and December 1998. H. rotundifrons was found at mean densities of 7.6 per m² (± 1.4) within the middle and low shore levels and varied little between seasons. The variance! mean ratio indicated that males and females aggregated within these shore levels. The sex ratio was significantly female biased during the majority of the year. Allometric growth rates indicated that males and females reached sexual maturity at 11 mm carapace width (CW). In males, spermatozoa production occurred between 9-9.99 mm CW. Ovigerous females were present every month except February. The first broods of the year were produced in March which coincides with a decrease in the female gonado-somatic index (GSI). These broods were incubated for approximately nine months whereas broods produced in August were incubated for only five months. Female brood production appeared to be cyclical, alternating between a winter incubation period and a summer incubation period. The completed cycle takes approximately three years with two broods produced during the cycle. Fecundity increased with female size but egg mortality was quite high (19%). Instantaneous mortality rate increased with increasing brood development. Females mate when recently moulted (soft-shelled). Although females moulted through out the year, in small numbers, there was a peak in female moulting during October and November. The operational sex ratio (OSR) was male biased during all months of the year. Females released an attractant prior to moulting which initiated pre-copulatory mate guarding by the male. Once the female moulted, copulation occurred approximately 6 h later. Copulation lasted for approximately 3 h and was followed by a period of post-copulatory mate guarding. Males increased the duration of post-copulatory mate guarding if another male was present. Large males out competed small males for receptive females. Males used their large cheliped to subdue competitors and to provide protection for the soft female. Mate guarding was shown to reduce cannibalism from other females. Spermatozoa are packaged in spermatophores within the vas deferens of males but are quickly dehisced within the spermathecae (ventral-type) of newly mated females. Last male to copulate probably achieves the highest level of paternity. Postcopulatory mate guarding by the male was found and ejaculates were found in discrete packets within the spermatheca. Sperm competition appeared to be important because large males displaced small males during copulation, males left when a female was still receptive and the females could retain sperm between moults. It is concluded that H. rotundifrons probably has a polygynous mating system in which males compete for soft females (female centered competition)

Reproductive success of males is strongly influenced by their investment in costly sexually selected traits. Fitness returns, however, are often context-dependent and vary with demographic parameters such as sex ratio and population density. Under conditions of environmental variability, the ability to modulate reproductive decisions on the social context is highly beneficial. As a result, phenotypic plasticity of sexually selected traits is widespread. The aim of my study was threefold. Firstly, I worked to expand our current knowledge on phenotypic plasticity in sexually selected traits both empirically, through a test of the effect of female availability on male mating effort in the nursery web spider, and theoretically, through a literature review on the subject of strategic adjustments of ejaculate quality. Secondly, I evaluated costs and benefits of the anticipatory upregulation of sperm production observed in male guppies as a response to perceived mating opportunities. Finally, I investigated how post-copulatory processes may shape male mate choice. The two species used in my study, the guppy, Poecilia reticulata, and the nursery web spider, Pisaura mirabilis, are particularly suited to investigate phenotypic plasticity of sexually selected traits because they both express costly reproductive traits and they experience environmental fluctuations in socio-sexual factors. When I explored the effect of female availability on male investment in pre- and post-copulatory traits, I found that P. mirabilis males do not respond to variation in this parameter. While males do not reduce their mating effort per partner as mating opportunities increase, they may respond instead by increasing their total reproductive budget. The literature review focusing on plasticity of ejaculate quality, besides showing the diversity of traits subject to adjustment and of stimuli triggering the response, highlighted the difficulty of estimating the fitness consequences of ejaculate plasticity because of the complexity of patterns of co-variation with other reproductive and non-reproductive traits. The experiments investigating costs and benefits of anticipatory ejaculate adjustments showed that the costs of plasticity are minor in guppies, as compared to the costs of phenotype. Furthermore, the trade-off between sperm production and pre-copulatory traits (courtship rate) appears to be stronger than the trade-off between sperm number and quality. Finally, my experiments on post-copulatory processes demonstrated a first male sperm precedence and an advantage of previous partners against novel ones in multiply mated female guppies. These findings brought an important contribution to the understanding of phenomena of male mate choice, such as mate choice copying, audience effect and Coolidge effect. In conclusion, the results of my study demonstrate how trade-offs between pre- and post-copulatory traits have crucial effects on costs and benefits of phenotypic plasticity in reproductive traits, highlighting the importance to adopt an integrative approach and to consider multiple traits and their interaction when studying sexual selection. My results also stress the need for a careful evaluation of episodes of post-copulatory selection when interpreting plasticity of both pre- and post-copulatory investment.
Il successo riproduttivo dei maschi è fortemente influenzato dal loro investimento in costosi tratti sottoposti a selezione sessuale. Tuttavia, i vantaggi in termini di fitness di tale investimento sono spesso condizione-dipendenti e variano al variare di parametri demografici quali la sex ratio e la densità di popolazione. In ambienti variabili, l’abilità di adattare le decisioni riproduttive al contesto sociale è altamente vantaggioso. Ne risulta che i tratti selezionati sessualmente sono frequentemente caratterizzati da plasticità fenotipica. L’obiettivo del mio studio era articolato in tre parti. In primo luogo, ho lavorato per espandere l’attuale conoscenza nel campo della plasticità fenotipica di tratti sottoposti a selezione sessuale, sia tramite un approccio sperimentale, testando l’effetto della disponibilità di femmine sullo sforzo riproduttivo di maschi di Pisaura mirabilis, sia tramite un approccio teorico, cioè raccogliendo la letteratura disponibile sul tema degli aggiustamenti strategici della qualità dell’eiaculato. In secondo luogo, ho valutato costi e benefici dell’aumento di produzione spermatica osservato nei maschi di guppy come risposta alla percezione di opportunità di accoppiamento. Infine, ho indagato l’effetto di processi post-copulatori su fenomeni legati alla scelta del partner. Le due specie utilizzate nel mio studio, il guppy, Poecilia reticulata, e il ragno P. mirabilis, sono particolarmente adatti per studiare la plasticità fenotipica di tratti sotto selezione sessuale in quanto entrambi esprimono costosi tratti riproduttivi e subiscono fluttuazioni del loro ambiente socio-sessuale. Lo studio degli effetti della disponibilità di femmine sull’investimento maschile in tratti pre- e post-copulatori ha rivelato che i maschi di P. mirabilis non rispondono a variazioni in questo parametro. Nonostante essi non riducano l’investimento riproduttivo allocato al partner all’aumentare delle opportunità di accoppiarsi, potrebbero invece rispondere aumentando il proprio budget riproduttivo totale. L’esame della letteratura sulla plasticità della qualità dell’eiaculato, oltre a mostrare la varietà dei tratti soggetti ad aggiustamenti e dei fattori di stimolo, ha evidenziato la difficoltà di stimare le conseguenze per la fitness di questo tipo di risposte, a causa della complessità dei pattern di interdipendenza con tratti riproduttivi e non. Gli esperimenti che hanno indagato costi e benefici degli aggiustamenti strategici dell’eiaculato hanno mostrato che in guppy i costi della plasticità sono ridotti, se confrontati con i costi del fenotipo. Inoltre, il trade-off tra produzione spermatica e tratti pre-copulatori (il tasso di corteggiamento) sembra essere più marcato di quello tra numero e qualità degli spermi. Infine, i miei esperimenti sui processi post-copulatori in femmine poliandriche hanno evidenziato una precedenza spermatica del primo maschio e un vantaggio dei partner precedenti rispetto a quelli nuovi. Queste scoperte portano un importante contributo alla comprensione di fenomeni osservati nel contesto della scelta maschile del partner, quali la copia della scelta, l’effetto audience e l’effetto Coolidge. In conclusione, i risultati del mio studio mostrano come i trade-off tra tratti pre- e post-copulatori abbiano importanti effetti sui costi e i benefici della plasticità fenotipica di tratti riproduttivi, evidenziando l’importanza di adottare un approccio integrativo e di considerare vari tratti e la loro interazione nello studio della selezione sessuale. I miei risultati sottolineano anche la necessità di un’attenta valutazione di episodi di selezione post-copulatoria durante l’interpretazione di casi di plasticità nell’investimento sia pre- e che post-copulatorio.

The mechanism of sperm transfer and sperm organization inside the spermatheca was investigated in two grasshoppers, (Scudder) and (DeGeer). The spermathecae were examined histologically from females whose copulations were interrupted at various prescribed intervals, either during their first or subsequent mating. Sperm organization inside the spermatheca from 24 to 120 hours after copulation had terminated was also investigated in . Scanning and transmission electron microscopy were used to investigate the distribution and morphology of small hair-like structures inside the spermathecae.

In both species sperm were first observed inside the spermatheca approximately 30 minutes after the initiation of copulation. The majority of sperm transferred into the spermatheca were in the form of sperm bundles, or spermatodesmes. In the rate at which sperm bundles were transferred appeared to decrease after 13 hours into copulation (average mating duration in the laboratory was 28 hours). In the rate of sperm transfer remained constant throughout copulation (average mating duration in the laboratory was 1.3 hours). The occurrence of both an abundance of individual sperm and sperm bundles was observed only in females of who had mated previously and had a second copulation of less than 9 hours in duration and in females of that had a second copulation less than 45 minutes in duration. As mating continued, fewer and fewer individual sperm were observed, and by 15 hours into copulation in only sperm bundles were observed inside the spermatheca. Therefore, it appears that the majority of the individual sperm in the above copulations are from prior matings, while most of the sperm bundles are from the last copulation. The interrupted matings of previously mated females indicate that at least some sperm bundles remain inside the spermatheca while individual sperm were removed by some mechanism, possibly sperm flushing by an excess of seminal fluid provided by the mating male.

In some sperm bundles remained intact for at least 7 days after the termination of copulation. The sperm bundles must disassociate into individual sperm prior to fertilizing the female's eggs, and therefore it may be the female that provides the mechanism or chemical stimulus to initiate sperm bundle disassociation prior to oviposition or prior to a second mating or both.

The results of this investigation suggest sperm competition, perhaps mediated by female choice, as a primary reason for lengthy copulations in . Male weight in has been documented to play a significant role in female choice and mating duration, whereas in , other factors such as nutrient transfer may play a significant role in female choice and mating duration. In , males may also act as mechanical plugs by remaining in copula for an extended time after a sufficient amount of sperm has been transferred, or males may be participating in the process of sperm removal by transferring an excess of seminal fluid to "flush-out" any sperm present from previous matings.

Evolution is not always straight-forward, as selection pressures may differ between different generations of the same species. This thesis focuses on the evolution of life history of the model species, the Green-veined White butterfly Pieris napi. In central Sweden P. napi has two generations per year. The directly developing summer generation is short-lived and time stressed, compared to the diapausing generation. In paper I polyandry, defined as female mating rate, was shown to differ between generations but was unaffected by environmental factors. In paper II both males and females of the direct developing generation were shown to eclose more immature than the diapausing generation, indicating larval time constraints. Consistent with this, diapausing males mated sooner than direct developers. Directly developing females, however, mated sooner after eclosion than diapausing females, even though they are more immature. This was shown to negatively affect fecundity, but can pay off when the season is short. Paper III shows that directly developing males have less sex pheromones at eclosion than diapausers, and the differences in sex pheromone production is consistent with developmental time constraints and the differences in mating system. In P. napi and other polyandrous butterflies, males transfer a large, nutritious ejaculate at mating. Large ejaculates confer advantages under sperm competition, but as they are costly, males should adjust ejaculate size to the risk of sperm competition. In paper IV we found that males transfer on average 20% larger spermatophores under high male competition than at low competition. The same effect could be observed if we added male sex pheromone to the air in a mating cage without male-male competition. Paper V shows that males of the two generations respond differently to an increase in male-male competition, with diapausing males transferring larger spermatophores than direct developers at high male competition risk.

At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper 5: Submitted manuscript.

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.