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Pheromones are chemosensory cues released by an individual to cause a behavioral or physiological change in a conspecific. These changes can range from increasing a female's receptivity to altering hormone secretions in the body. The red-legged salamander (Plethodon shermani) is an emerging non-mammalian model for understanding the evolution of chemical communication due to their well-characterized pheromones. Plethodontid salamanders secrete pheromones from their submandibular gland, called mental gland pheromones. Previous studies showed that mental gland pheromones increased receptivity in females during courtship and also increased corticosterone (CORT) concentrations in males. CORT is a metabolic hormone that mediates vertebrate stress responses. To further understand the neuroendocrine mechanisms involved in behavioral and physiological responses to pheromones, I conducted several experiments. First, I investigated whether pheromones altered levels of two neuromodulators in the brain, gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT) in P. shermani. GnRH is both a neuromodulator and hormone, and has strong effects on reproductive behavior. Additionally, AVT has broad behavioral effects in vertebrates, particularly in newts. I found that application of mental gland pheromones altered the number of GnRH-immunoreactive neurons, but had no effect on the number of AVT-immunoreactive neurons. Second, I examined the effects of AVT and AVP antagonist on courtship and mating in dusky salamanders (Desmognathus spp.). There was no effect of AVT or antagonist on reproductive behaviors in dusky salamanders. Finally, I investigated the role of increased CORT concentrations in male P. shermani. I first validated a non-invasive method to transdermally deliver CORT through a dermal patch. I then used dermal patches to exogenously elevate plasma CORT and examine the effects of CORT on behavior (activity and chemoinvestigation) and metabolism. CORT had no effect on behavior, but increased metabolic rates in male red-legged salamanders. Together these studies provide insight into the mechanisms by which pheromones work to alter behaviors and physiological functions in vertebrates. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

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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.

The ability of an animal to respond and adapt to stimuli is necessary for its survival and involves plasticity and coordination of multiple levels of biological organization, including behavior, tissue organization, hormones, and gene expression. Each of these levels of response is complex, and none of them responds to stimuli in isolation. Thus, to understand how each system responds, it is necessary to consider its role in the context of the entire organism. Here, I have used the African cichlid fish Astatotilapia burtoni and its extraordinary phenotypic plasticity to investigate how animals respond to a change in social status from subordinate to dominant and attempted to integrate these multiple levels of biological response, as well as the roles of several candidate neuromodulators,. First, I have described how male A. burtoni become more aggressive and reproductive during their transition to dominance as well as increasing circulating levels of testosterone and estradiol and the histological organization of their testes. I then mapped the distribution of expression of two behaviorally relevant neuropeptides, arginine vasotocin and isotocin, and their respective receptors, throughout the A. burtoni brain, and found that they were highly expressed in several brain areas important for social behavior and decision-making. I then investigated the role of arginine vasotocin in social status and behavior via pharmacological manipulation and qPCR, showing the importance of arginine vasotocin in controlling the transition to dominance. Lastly, I investigated the role of aromatase, testosterone, and estradiol in male A. burtoni, both in stable dominant males and in males as they transition to dominance, using pharmacological manipulation and quantitative radioactive in situ hybridization, illustrating that estradiol synthesis during dominance is dependent on aromatase activity and necessary for aggressive behavior. / text

The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing hormone (CRH) are involved in the hypothalamic-pituitary-interrenal (HPI) axis. During stress, the HPI axis is activated and cortisol is released into the blood. In addition to their role in the HPI axis, AVT and CRH also have behavioural effects. The roles of AVT and CRH in stress responses and agonistic behaviour were studied in this thesis, using two different models. In the first model, two strains of rainbow trout (Onchorhynchus mykiss) divergent in stress-induced release of cortisol were investigated. This was done by observing behaviour and stress responses under different conditions. These strains were found to have divergent stress coping strategies based on the observed behaviour and levels of plasma cortisol. This divergence in behaviour could be associated with the CRH system, since the mRNA levels of CRH differed between the strains during stress. However, no differences between strains were observed in AVT or its receptor expressions. In the second model, non-selected rainbow trout were paired and the effect of intracerebroventricular (icv) injections of an active substance (AVT, CRH or the CRH related peptide Urotensin-I (UI)) on fights for dominance was investigated. One fish of the pair received the active substance icv and the other received saline icv. Fish receiving AVT became subordinate in accordance with the suggestion that AVT attenuates aggression in territorial vertebrates. Fish receiving CRH became subordinate whereas UI showed no effect on fights for dominance. Further, both CRH and UI induced an anxiety-related behaviour similar to non-ambulatory motor activity in rats. In addition, CRH appeared to affect the dopaminergic and serotonergic systems. In this thesis, it is suggested that CRH is involved in the behavioural modulation of the stress coping strategies in teleost fish. Further, AVT and CRH seem to act inhibitory on aggressive behaviour.

The aim of this study was to compare the effects of arginine vasotocin (AVT) administration in the endangered boreal toad (Bufo boreas boreas) and common Fowler’s toad (Bufo fowleri). The objectives of this study were to determine if AVT could elicit: 1) calling, and 2) amplexus behaviors. Toads were paired into single male:female groups and administered AVT at varying concentrations: 0.1, 1.0, 5.0, 10.0 and 25.0 μg/g and in different combinations: 1) only male was treated; 2) only female was treated, and 3) both male and female treated. AVT failed to stimulate any breeding behavior in the boreal toad, but the administration of AVT to both B. Fowleri genders significantly affected the duration of amplexus (p<0.0347). Also, the concentration of AVT significantly affected the length of amplexus (p<0.0429) and call frequency (p<0.0294). These results will be valuable for breeding programs where animals are failing to show natural reproductive behavior.

Hermaphroditism, associated with territoriality and dominance behavior, is common in the marine environment. Male sex-specific coloration patterns and behavior are particularly evident in species where males are territorial and guard harems of females such as wrasses and groupers. Protogynous hermaphrodites that change sex from female to male are good models to study sexual behavior and related changes in the brain due to their abilities to reorganize their sexual phenotype as adults. Two hormones produced in the brain and implicated in the process of sex-specific behavior and reproductive development are arginine vasotocin (AVT) and gonadotropin releasing hormone (GnRH). While a wealth of data exists regarding these hormone systems separately, little is known about linkage between these two systems. Especially there is no data tracking these two systems together in any protogynous fish. This study was conducted to test the hypothesis that coordinated interactions between AVT and GnRH facilitate the process of behavioral and gonadal sex change in the rock hind Epinephelus adscensionis. Four topics were addressed to investigate the relationship between behavior and reproduction: i) rock hind sex change, sexual characteristics and conditions causing sex change to occur in captivity were detailed as a basis for examining the AVT system and GnRH during this process, ii) the distribution of a vasotocin V1a type receptor identified in rock hind brain was examined for the first time in a fish species using a custom designed antibody then the receptor protein was co-localized with GnRH producing cells within the brain to confirm that a pathway exists for AVT action on GnRH, iii) levels of AVT, AVT receptors, and GnRH messenger RNA (mRNA) were compared between male and female rock hind phenotypes, and iv) female rock hind at early stages of sex change were compared for brain mRNA expression of AVT, AVT receptors, and GnRH to determine the order of hormonal change during the process of sexual inversion in this species. This study provides a better understanding of the relationship between sex-specific behavior and reproductive development via AVT and GnRH systems that are conserved in all vertebrates. / text

Les amphibiens sont des vertébrés liés aux alternances entre la vie aquatique et la vie terrestre et aux alternances saisonnières. Le développement embryonnaire commence par une phase aquatique et se termine par une métamorphose qui donnera une forme juvénile terrestre pouvant rester dans le milieu aquatique chez certaines espèces retournées secondairement à la vie aquatique. Les cycles de reproduction sont régulés, entre autres, par les hormones hypophysaires: LH et FSH (gonadotropes) et PRL (prolactine). La régulation hydrominérale des vertébrés est également assurée par des hormones neurohypophysaires telles que l'arginine vasopressine (AVP), l'ocytocine (OT), l'arginine vasotocine (AVT), la mésotocine (MST) et l'isotocine (IST). Le but du travail effectué dans le cadre de cette thèse a été d'apporter quelques éléments de compréhension des liens pouvant exister entre la reproduction et la régulation hydrominérale. Les cycles de reproduction de Typhlonectes compressicauda sont soumis à des alternances saisonnières caractérisées par une saison des pluies et une saison sèche.Dans le présent travail nous avons mis en évidence les principales hormones impliquées dans la régulation de la balance hydrique (AVT, MST). Parallèlement, une étude précise de la structure des reins avec la mise en évidence des récepteurs de différentes hormones concernées a été menée. La mise en évidence par biochimie (western blot et dosage) des différentes hormones et de leurs récepteurs (immunohistochimie) a également été effectuée chez Xenopus laevis, espèce aquatique représentant un modèle d'étude conventionnel, de manière à apprécier l'implication de ces hormones lorsque l'animal termine sa métamorphose / The amphibians are vertebrates the cycle of life being related to both the alternations between aquatic and terrestrial phases and seasonal alternations. Embryonic development begins with an aquatic phase and completed with a metamorphosis, at the end of which terrestrial juveniles animals can persist in the aquatic environment in secondary aquatic species. The breeding cycles are regulated by the pituitary hormones: LH, FSH (gonadotropins) and PRL (prolactin). The hydromineral regulation of vertebrates is also ensured by neurohypophysial neurohormones such as arginine vasopressin (AVP), oxytocin (OT), vasotocin (AVT), mesotocin (MST), and isotocin (IST).The work in the context of this thesis was to bring some elements for understanding the relationships that may exist between the reproduction and hydromineral regulations.The reproductive cycles in Typhlonectes compressicauda are submitted to seasonal alternations characterized by a rainy season and a dry season. In the present work we highlighted the main hormones involved in the regulation of hydric balance in amphibians (AVT, MST). In parallel, a precise study of the structure of the kidneys with the identification of receptors of various hormones concerned, was performed. The identification of both various hormones and their receptors by biochemistry (Western blot, assay) and immunohistochemistry was also performed on Xenopus laevis, an aquatic species representing also a conventional model for the study, in way to appreciate the implication of these hormones when the animal completes its metamorphosis

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