The nonapeptide family of hormones is deeply conserved in evolution. In teleost fishes, as in all vertebrates, two nonapeptide families exist. These are vasotocin (avp) and oxytocin (oxt). While vasotocin has been shown to regulate individual aspects of reproductive physiology in several teleost species, an integrative assessment of its role on male and female reproduction is currently lacking even in widely used fish models, such as the zebrafish (Danio rerio). Taking advantage of the genetic tractability of the zebrafish, and its emerging status as model to study reproductive physiology, I generated avp -/- mutants using a CRISPR/Cas9 based approach to determine reproductive consequences in female and male zebrafish. Following the identification of a female-specific reproductive phenotype which manifests as a reduction in oocyte release and decreased quivering behaviour, I investigated the potential mechanistic basis at the level of the gonad. In avp -/- ovaries, significantly fewer eggs were present compared to WT fishes. When comparing the distribution of oocyte maturation stages, a significantly lower percentage of stage I and higher percentage of stage V oocytes was present in avp-/- ovaries. The altered distribution in oocyte maturation stages coincided with significant decreases in ovarian transcript abundance of nanos2, a germ-cell specific marker suggesting a possible role for Avp in germ-cell maintenance. Additionally, I observed a decrease in the ovarian concentration of the prostaglandin PGF2, which coincided with a reduction in ovarian transcript abundance of pla2g4ab, a paralogue of the phospholipase A2 involved in mobilizing arachidonic acid, a precursor of PGF2,. Together, these finding suggests a role for Avp in PGF2 -mediated ovulation. Because Avp has pleiotropic effects and may thus affect female reproductive physiology indirectly, we assessed somatic growth, a key regulator of sexual maturation in zebrafish, as well as aspects of the endocrine stress axis known to affect oocyte growth in avp -/- mutants. While avp -/- mutants did not exhibit differences in somatic growth up to sexual maturation or GSI, mutants exhibited hypercortisolism. While other zebrafish knock-out mutants exhibiting persistent hypercortisolism do not share the observed reproductive phenotype, future studies investigating potential contributions of pleiotropic Avp effects are nevertheless warranted. Overall, I demonstrate that avp, while not essential, affects female reproductive success, at least iii in part by regulating oocyte maturation. This finding is in line with the recent findings from other vertebrate and invertebrate species, suggesting an evolutionarily ancient role in these processes. It is anticipated that such novel insights into the regulation of female oocyte maturation have in addition to increasing our understanding of female reproduction, translational potential for captive breeding (aquaculture, species conservation) and ecotoxicology (insight into mode of action of specific EDCs).

Two distinct nonapeptide systems, consisting of the vasotocin- and oxytocin-related peptides have evolved in vertebrates, and their role in male reproduction is well-described in mammals. In contrast, their comparative role in reproduction in basal vertebrate species, and teleost fishes in particular, has not been investigated in great detail. Using two cyprinid species, the zebrafish (D. rerio) and the goldfish (C. auratus), I address the hypothesis that the teleost nonapeptides vasotocin and isotocin stimulate male cyprinid reproductive physiology by affecting central neuronal and/or peripheral endocrine pathways. To test this hypothesis in zebrafish, an indeterminate breeder, I conducted pharmacological inhibition experiments employing vasotocin and isotocin-specific antagonists in males, a treatment predicted to inhibit reproductive success in mating trials. Because nonapeptides can act both as central peptide neuromodulators and as secreted hormone, I further quantified indices of male courtship behavior (nudging, circling and chasing) and major androgens (testosterone and 11-keto-testosterone) as key endocrine indices of the male reproductive axis. Together, these experiments revealed a dose-dependent, differential inhibition of spawning success, with significant reductions (-65%) in egg fertilization rate observed in pairs in which males had been i.p. injected with 5 ng/g vasotocin and significant reductions (-79%) observed at 500 ng/g i.p injected isotocin. In either case, these partial inhibitions of reproductive success were correlated with significant decreases in specific indices of male courtship behavior, but not endocrine indices, suggesting that individual nonapeptides mediate their effects via central modulation of behavioural neurocircuits. Interestingly, a co-administration of vasotocin and isotocin antagonists completely abolished reproductive success, however this effect was neither correlated with decreases in male courtship behavior, nor endocrine indices, suggesting a separate mode of action, possibly at the level of male pheromone release. To further probe the role of nonapeptides in male zebrafish reproduction, I subsequently tested the hypothesis that nonapeptide systems are acutely activated by key reproductive cues, specifically the releaser pheromone PGF2α, which serves as a chemoattractant and acutely stimulates male reproductive behavior in male cyprinids. Using a chemoattractant choice assay in conjunction with immunohistochemistry and gene expression approaches, I determined whether male zebrafish are attracted to pheromonal cues and acutely activate isotocinergic neurons in the short term and/or regulate nonapeptide gene expression in the longer term. My results show that individual male zebrafish are attracted to PGF2α in an acute choice test. Furthermore, an increase in p-ERK immunoreactivity, a marker of neuronal activation, was observed in the olfactory bulb 10 min following exposure, suggesting a specific response to the pheromone compared to EtOH vehicle. However, no co-localization of p-ERK and IT-positive perikarya was observed in the preoptic area (POA), refuting the hypothesis that PGF2α exposure acutely activates isotocinergic neurons in zebrafish. Analysis of whole brain relative mRNA transcript abundance revealed that PGF2α exposure time-dependently regulates whole brain isotocin, but not vasotocin transcript abundance, suggesting secondary longer-term effects of PGF2α exposure on the isotocinergic system. Using an analogous experimental approach, I further tested the hypothesis that nonapeptides stimulate male reproductive physiology in goldfish, a determinate breeder. Sexually mature male goldfish pretreated with saline or vasotocin or isotocin antagonists were exposed to saline or PGF2α-injected stimulus females and male courtship behavior (chasing, circling), endocrine indices (circulating testosterone) and milt release were quantified. Both nonapeptide antagonists reduced strippable male milt quantity in response to PGF2α-injected females, suggesting a neuronal or hormonal action of both nonapeptides on goldfish milt release. Together, I show that nonapeptides contribute to male reproductive physiology in two species of cyprinids with different reproductive tactics. However, the mode of action may differ from one species to another, with evidence suggesting that nonapeptides play a role in the regulation of reproductive behavior and, possibly, male pheromone, release in zebrafish, while effects on male goldfish seem to be exclusively related to the release of milt. Future studies should compare other teleost species with specific reproductive biology and focus on the gonadal roles of nonapeptides in sperm maturation and/or release.

In my master's thesis, I deal with reproduction of central European wolf spiders of the genus Alopecosa. Here I present patterns and duration of courtship behaviour and copulation of 15 members of the genus Alopecosa. Such information were never published before. I recognized 17 courtship elements, six of them are described here for the first time. Duration of copulation, number of palpal insertions and number of hematodochal expansions of 15 species were examined. The obtained data were analyzed, courtship and copulations among species and species groups are discussed. Courtship behaviour of two sibling species, A. striatipes and A. mariae, is compared, as well as differences in reproduction of Czech and Italian populations of A. accentuata. Based on courtship behaviour, I propose placing Alopecosa psammophila in a striatipes group. Stridulatory apparatus on male pedipalps was not detected, however, some pegs on vetral side of the abdomen in males of Alopecosa pinteroum were found. These pegs could serve as a source of vibratory signals. The thesis provides some new remarks on ecology of members of the genus Alopecosa and contains faunistical information, including those on the recent occurrence of some of the rarest Czech spiders.

Four experiments are presented that explore social interactivity in a visually communicating species: the pigeon, Columba livia. A closed-loop teleprompter system was used to isolate, control and manipulate social contingency in a natural courtship interaction. Experiment 1 tested different ways to measure pigeon behaviour and developed an automatic method for measuring the pigeon’s circle walking display using motion energy analysis. In Experiment, 2 the subject’s courtship behaviour towards the video image of an opposite sex partner streamed live (Live), was compared with their behaviour towards a pre-recorded video image of the same partner (Playback). The only difference between the Live and Playback condition was the presence or absence of social contingency. The results showed that pigeons behaved interactively: their behaviour was determined, in part, by the social contingencies between visual signals. To investigate what types of social contingencies are behaviourally relevant, the effects of the partner’s facing direction and the timing of social contingencies on behaviour were investigated in Experiment 3 and 4, respectively. To manipulate partner facing direction, the camera was rotated so that the partner appears to be courting 90° away from the subject. To manipulate social timing, three time delays, 1, 3 and 9s, were implemented within the closed-circuit communication. In Experiment 4, the context-specificity of interactive behaviour was also investigated by testing behavioural sensitivity to social contingency and timing in both opposite sex and same sex social interactions. The results showed that the partner’s facing direction did not significantly influence behaviour, whereas the timing of social contingencies had a significant impact on behaviour: in courtship only. These findings suggest that temporal relations between display bouts in courtship are behaviourally relevant. A post hoc analysis was then used to evaluate the behavioural relevance of two social contingencies in particular: partner responsiveness and simultaneous display. The results showed that females may be sensitive to the male responsiveness in courtship. Also, simultaneous display appeared to create a perceptual interaction which acted to potentiate the courtship dynamic. These studies provide a basis for further developing the pigeon and its courtship behaviour as a model for visual communication. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2011-04-25 14:37:19.888

Dans un contexte de sélection sexuelle, les systèmes de communication permettant l’attraction et la stimulation du partenaire sexuel et la compétition entre individus du même sexe sont indispensables. Ceci est particulièrement vrai chez les espèces à système d’appariement polygyne de type lek, où les mâles rassemblés dans l’espace sont en compétition pour l’accès aux femelles. Chez notre sujet d’étude, l’outarde houbara nord-africaine les males réalisent des parades incluant des démonstrations visuelles et des vocalisations appelées booms, sur des sites espacés les uns des autres par des distances importantes dans un système qualifié de lek explosé. Notre objectif était d’étudier les systèmes de codage-décodage des informations exprimées durant la parade des mâles chez cette espèce. Nous avons démontré l’existence d’un codage de l’identité individuelle porté par des paramètres visuels et des paramètres acoustiques des vocalisations. Des associations significatives entre des paramètres des booms et des caractéristiques physiques et comportementales des mâles susceptibles de refléter leur qualité, ont également été mises en évidence. Ainsi, les mâles qui produisent les booms les plus graves avaient les masses les plus importantes et paradaient le plus intensément. Lorsque des interactions agonistiques étaient simulées expérimentalement en diffusant des leurres acoustiques, les mâles avaient des réponses comportementales différentes en fonction de la fréquence des booms diffusés. Ainsi le niveau fréquentiel des booms des autres mâles est effectivement perçu et décodé par les individus en compétition. Par ailleurs, plusieurs paramètres des booms sont génétiquement déterminés et héritables, et pourraient porter une information sur l’apparentement entre individus.Certains paramètres sont également liés à la consanguinité des individus. Nous avons montré également que les booms sont particulièrement bien adaptés à la communication à grande distance. En effet, les booms sont de très basse fréquence, se propagent à des distances supérieures à 640 m, et les paramètres supportant le codage-décodage de l’information sont résistants à la propagation et produits de manière redondante. Enfin, les signaux acoustiques et des signaux visuels et semblent agir en synergie (multimodalité) ce qui pourraient améliorer l’efficacité de la communication à grande distance. Ainsi notre étude a démontré que des informations complexes sont transmises durant la parade des mâles au travers de signaux acoustiques et visuels au sein d’un réseau de communication à grande distance. / Résumé en anglais ;In a context of sexual selection, the communication between individuals is essential to optimize both intra-sexual (competition between male) and inter-sexual (mate choice) processes. This is particularly true in species with lek mating systems, where both processes reach their full expression. The North African Houbara bustard, Chlamydotis undulata undulata, is a lekking bird where courting males aggregate in a so called ‘exploded-lek’, i.e. where aggregation is detectable only when mapped over a large area. During the breeding season, males perform a conspicuous and spectacular courtship including visual and acoustic (booms) signals. Our aim was to decipher the coding-decoding systems of the courtship in this species. We found that males can be individually discriminated on the basis of visual and acoustic parameters. The booms produced during the courtship are also related to males’ characteristics susceptible to reflect their quality. Individuals who produced the lowest frequency booms were those with the greatest weights and performed the highest intensity courtships. Playback experiment shows that the frequency content of the booms was perceived and decoded by males during agonistic interaction, leading to significantly different behavioural responses according to the frequency of the signals played back. Furthermore, several acoustic parameters of the booms are significantly heritable through genetic transmission and these parameters are also related to the level of inbreeding. Finally, we showed that the booms and the acoustic parameters supporting information are particularly well adapted to the transmission of information at long range as required in a long distance communication network constituted by an exploded lek. First, booms are of very low-frequency and propagate up to 640 m. Second, coding-decoding of information relies on sequentially redundant and propagation-resistant features. Third, acoustic signals are combined with visual signalling (multimodality) which may improve the long distance transmission of the information. This study brought evidence that complex information is conveyed by visual and acoustic signals during the courtship of males within a long distance network.