Photoperiod manipulation is routinely used in the aquaculture industry with the aim to enhance growth by manipulating the timing of reproduction in several commercially important temperate fish species. However, there are clear gaps in our understanding of how photoperiod is perceived by the circadian axis and transmitted to the brain to alter reproduction. Furthermore, due to the wide range of environments inhabited by fish, it is unlikely that one single organization exists. It is therefore believed that comparative studies of temperate species “models” with tropical species such as the African catfish (Clarias gariepinus) that adapted to different environments characterized by weaker light signals can help in such an aim. A number of studies were therefore performed in this PhD project to expand our knowledge on circadian biology and environmental physiological effects in African catfish. The first aim was to characterize the circadian melatonin system in this species (chapter 3). Results clearly showed that the control of melatonin production by the pineal gland was very different in the African catfish as compared to temperate species such as salmon and trout. Indeed, melatonin production appeared to mainly depend on light stimuli perceived by the eyes as opposed to salmonids where light directly perceived by the pineal gland regulates its own melatonin production within photoreceptors. The main evidence was obtained in ophthalmectomised fish that were unable to synthesize and release melatonin into the blood circulation during the dark period. This was the first time that such a decentralized organisation, similar in a way to the mammalian system, was found in any teleost species. In vitro results also supported such findings as African catfish pineal glands in isolation were not able to normally produce melatonin at night as usually seen in all other fish species studied so far. This indirectly suggested that pineal gland photo-sensitivity might be different in this tropical species. Further studies were performed to better determine the amount of light that can be perceived by the African catfish pineal gland depending on light transmittance though the skull (where the pineal gland is located). Surprisingly, it appeared that catfish cranium act as a stronger light filter than in other species resulting in lower light irradiance of the pineal gland. This could explain, although it still needs to be further confirmed, why African catfish photic control of melatonin produced by the pineal would have evolved differently than in temperate species. The work then focused on better characterizing diel melatonin production and endogenous entrainment through exposure to continuous photic regimes (continuous light, LL or darkness, DD) (chapter 4). Daily melatonin profiles of fish exposed to 12L:12D photoperiod (routinely used in indoor systems) confirmed low melatonin production at day (<10 pg/ml) and increase at night (50 pg/ml) as reported in most vertebrate species studied to date. Interestingly, results also showed that melatonin production or suppression can anticipate the change from night to day with basal melatonin levels observed 45 mins prior to the switch on of the light. These observations clearly suggest the involvement of a clock-controlled system of melatonin secretion that is capable of anticipating the next photophase period. Furthermore, when constant light (LL) was applied, day/night melatonin rhythms were abolished as expected due to the constant photic inhibition of AANAT activity (e.g. one of the enzyme responsible for the conversion of serotonin into melatonin). However when fish were exposed to constant darkness (DD), a strong endogenous melatonin rhythm (maintained for at least 4 days and 18 days in catfish and Nile tilapia respectively) was found, demonstrating once again the presence of robust circadian oscillators in this species. The next aim of the doctoral project was then to investigate circadian behaviour of catfish through locomotor activity studies (Chapter 5). African catfish is again a very interesting “model” due to its reported nocturnal activity rhythmicity as compared to most other teleosts species. Locomotor activity is considered as a very useful tool to elucidate the mechanisms of circadian organization in both invertebrates and vertebrates circadian. Results first confirmed the nocturnal activity rhythms in the species. Furthermore, clear circadian endogenous rhythms were observed under constant light (LL) or darkness (DD) during several days before losing rhythmicity. Interestingly, the activity levels varied depending on the stocking density. Finally, the last aim of this project was to test the effects of a range of photoperiodic manipulations on growth performances, sexual development and reproductive performances in African catfish reared from eggs to puberty. Results did not show any differences at the early sages (up to 90 days post hatching) in growth performances nor mortality (high) between control 12L:12D and LL treatments. In contrast, during the juvenile-adult period (from 120 to 360 DPH), significant growth effects were observed, as previously reported in other catfish species, with fish under LL displaying lower growth rate, food consumption and feed conversion efficiency in comparison to most other treatments (12:12, LL, 6:6, 6:18, 12-LL and LL-12) especially 12l:12D. However, no major effects of the photoperiodic treatments were observed with all fish recruited into puberty and developing gonads although differences in the timing of gametogenesis could be observed, especially a delay (circa 2 months) in females exposed to short daylength (6L:18D and 6L:6D). As for egg quality, egg diameter was the only parameter to differ between treatments (slightly larger in egg batch from LL treated females). Overall, none of the photoperiodic regime suppressed maturation in African catfish as opposed to some temperate species. The work carried out during this PhD project clearly advanced our understanding of circadian rhythmicity, light perception and effects of photoperiod on physiology in a tropical species. Future studies are now required to further characterise the circadian system and link it to evolutionary trends within vertebrates.

Strawberry production in the United States is almost entirely done in open-fields. Recently, interest in off-season strawberry production using controlled environment (CE) systems such as greenhouses and soilless cultivation has increased in the US. However, strawberry production in greenhouses is relatively new in North America and available information about greenhouse strawberry production is limited. Plant physiological responses to the environment must be well understood to maximize production using CE systems. In the present research, photoperiodic and photosynthetic responses of strawberry plants in greenhouse were studied. To evaluate photoperiodic response eight cultivars of strawberry widely cultivated in North America were subjected to varied photoperiods under an average daily temperature of 17 °C. Short-day (SD) cultivars included 'Radiance', 'F-127', 'Shuksan' and 'Chandler', and day-neutral/ever-bearing (DN/EB) cultivars included 'Albion', 'Portola', 'Monterey' and 'San Andreas'. SD cultivars were subjected to treatments of 11-h, 12-h, 13-h and 14-h photoperiod for 8 weeks. DN/EB cultivars were subjected to separated treatments of 8-h, 11-h, 14-h and 17-h photoperiod for up to 10 weeks. After 8 and 10 weeks of photoperiodic treatments in SD and DN/EB cultivars respectively, shoot apical meristems (SAM) were observed under microscope and classified into one of twelve developmental stages (Indices: 0-11). All SD cultivars examined showed a critical photoperiod between 13 h and 14 h. DN/EB cultivars 'San Andreas', 'Albion' and 'Monterey' presented facultative long-day response with positive correlation between SAM developmental indices and photoperiod after 8 weeks of treatment. 'Portola' showed non-significant influences of photoperiodic treatments in flower primordial development, suggesting day neutral response. However, further experiments must be conducted to confirm cultivar responses and identify possible interactions between photoperiod and temperature. The effect of light intensity and plant sink/source balance on strawberry plant photosynthesis was also investigated. Measurements of leaf net photosynthetic rate (Pn), stomatal conductance, intercellular CO2 concentration (Ci) and transpiration rate under near-saturated photosynthetically photon flux of 1,000ï ­mol m-2 s-1 and ambient CO2 concentration in strawberry cultivars 'Albion' and 'Nyoho'; and tomato cultivar 'Speedella'(comparison purposes) were conducted monthly in greenhouse during May 2014, and monthly from September 2015 through May 2016 (winter/spring production season). Hourly measurements were recorded hourly from 9 AM to 4 PM. Potential source strength was determined by the number of leaves and the daily light integral (DLI, 400-700 nm) and sink load from flowers and fruits was represented as the number of flowers and fruits. Seasonal changes in daily maximum Pn were observed, as well as diurnal change in Pn in both strawberry cultivars. A significant positive correlation was determined between the estimated ratios of sink/source and the slopes representing diurnal linear decline of Pn. Also, Pn was negatively correlated with Ci but not significantly correlated with vapor pressure deficit (VPD) in greenhouse, suggesting diurnal decline in Pn was likely due to negative feedback of photosynthesis caused by unbalance of sink and source, and not to water stress from high VPD. The photosynthetic capacity as affected by seasonal changes in greenhouse environment and its diurnal change as affected by sink/source balance could help develop more effective practices in CE strawberry production to maximize production. Also, photoperiodic response revealed in this study for American strawberry cultivars will specify conditions to induce flowering in these economically important cultivars off-season.

Os relógios biológicos são fundamentais para a sincronização do comportamento dos organismos a mudanças no fotoperíodo. Todas as alterações rítmicas são determinantes para a sobrevivência da espécie uma vez que elas prevêem que os ajustes internos coincidam com a fase mais propícia do ciclo ambiental, permitindo aos organismos a capacidade de sincronizar esses eventos internos com os ciclos ambientais. Muitos desses ritmos biológicos são claramente associados ao ciclo claro-escuro, sendo este ciclo de grande importância para as espécies que possuem algum tipo de pigmento fotossensível. Os melanóforos de Xenopus laevis são fotossensíveis, respondendo à luz com dispersão dos grânulos de melanina, devido à presença de duas melanopsinas, Opn4x e Opn4m. As células pigmentares dos vertebrados heterotérmicos respondem com migração pigmentar a uma variedade de agentes, incluindo as endotelinas. Em peixes teleósteos, ETs induzem a agregação pigmentar em melanóforos, enquanto que em anfíbios, ET-3 induz a dispersão de grânulos de pigmentos em melanóforos de Xenopus laevis e de Rana catesbeiana, através da ativação de receptores ETc. Propusemos determinar o padrão temporal de expressão dos genes das melanopsinas e do receptor ETc em melanóforos dérmicos de X. laevis em cultura, bem como os efeitos temporais e dose- dependentes da endotelina sobre essa expressão. Demonstramos, através de ensaios de PCR quantitativo, que o tratamento de 12C:12E , somado a uma troca de meio, assim como o de endotelina-3 10-9 e 10-8M em escuro constante, foi capaz de sincronizar a expressão de Opn4x e Opn4m. Entretanto, o receptor ETc parece não ser sincronizado pelo ciclo claro-escuro, ou pelo tratamento hormonal. Dependendo da dose utilizada e do ZT analisado, ET-3 pode promover um aumento ou inibição da expressão gênica de Opn4x, Opn4m e ETc, indicando uma modulação de forma dose-dependente. Além disso, pode atuar como um agente sincronizador da expressão dos transcritos das melanopsinas. / The biological clocks are critical for synchronizing the behavior of organisms to changes in photoperiod. All rhythmic changes are crucial to the survival of the species since they provide for internal adjustments to coincide with the phase of the cycle most favorable. Many of these biological rhythms are clearly associated with the light-dark cycle, of major importance for species that have some type of photosensitive pigment. Melanophores of Xenopus laevis are photosensitive, responding to light with dispersion of melanin granules, due to the presence of two melanopsins, Opn4x and Opn4m. The pigment cells of ectothermic vertebrates respond with pigment migration to a variety of agents including the endothelins. In teleost fish, ETs induce pigment aggregation in melanophores, whereas in amphibians, ET-3 induces the dispersion of pigment granules in melanophores of Xenopus laevis and Rana catesbeiana, by activation of ETc. We proposed to determine the temporal pattern of gene expression of the ETc receptor and melanopsins in dermal melanophores of X. laevis in culture as well as the effects of endothelin-3 on the temporal expression of the 3 genes. Using quantitative PCR, we demonstrated that 12L: 12D regimen, combined with medium changes, as well as the treatment with 10-9 and 10-8M endothelin-3, was able to synchronize the expression of Opn4x and Opn4m. However, ETc receptor seems not to be synchronized by light-dark cycle, or hormone treatment. Depending on the dose and the ZT, ET-3 may promote an increase or inhibition of gene expression of Opn4x, Opn4m and ETc, indicating a dose-dependent modulatory effect. In addition, endothelin-3 may also act as a synchronizing agent of the melanopsins transcripts.

O sistema circadiano das aves é composto pela retina, a região homóloga aos núcleos supraquiasmáticos de mamíferos (NSQ) e a glândula pineal. A retina apresenta muitos eventos fisiológicos rítmicos, como por exemplo os movimentos das células fotorreceptoras em vertebrados não mamíferos, a expressão de opsinas, regeneração do cromóforo visual e produção e liberação de melatonina e dopamina. Todos estes eventos rítmicos são coordenados para prever alterações nas condições luminosas que ocorrem durante o dia, otimizando a função retiniana. Neste trabalho foi investigada a expressão de componentes chave de um sistema circadiano, incluindo os dois genes de melanopsina, Opn4x e Opn4m, os genes de relógio Clock e Per2, e os genes das enzimas chave da síntese de melatonina, N-Acetiltransferase, e de dopamina, Tirosina Hidroxilase, em células da retina de embriões de galinha. Culturas primárias de retina de embriões de galinha com 8 dias foram preparadas no ZT0 (quando as luz é acesa) e semeadas na densidade de 107 células por frasco de 25 cm2 . As células foram mantidas em ambiente úmido, com 5% CO2, a 40o C, em escuro constante, fotoperíodo 12C:12E, fotoperíodo 12C:12E seguido de escuro constante, ou em escuro constante na presença e na ausência de glutamato 100 &#956;M por 12 h. A extração de RNA total foi feita ao longo de 24 horas com intervalo de três horas tendo início no ZT0 do sexto dia. As amostras foram submetidas a RT-PCR seguido de PCR quantitativo para a quantificação de RNAm. Para confirmar a expressão da proteína OPN4x foi realizado ensaio imunohistoquímico com anticorpos anti-melanopsina de galinha desenvolvidos em coelho. Também foi feita a quantificação da concentração das proteínas OPN4x, CLOCK e TIROSINA HIDROXILASE através da técnica de Western Blot. A quantificação do RNAm em escuro constante não apresentou ritmos de transcrição para nenhum gene. Já as células mantidas em fotoperíodo 12C:12E apresentaram padrões rítmicos de transcrição para Clock, Per2, Opn4m, N-Acetiltransferase e Tirosina Hidroxilase. Glutamato 100 &#956;M foi eficaz em induzir ritmo em Clock, e inibiu drasticamente a expressão de Tirosina Hidroxilase e, apenas mais pontualmente, de Opn4x e Opn4m. Ensaios de viabilidade celular e fragmentação de DNA por citometria de fluxo demonstraram que essa inibição não foi resultante de ação tóxica ou apoptótica do glutamato. O neurotransmissor não teve qualquer efeito sobre a transcrição de Per2 e de N-Acetiltransferase. A quantificação protéica não indicou a presença de ritmo para CLOCK, OPN4x ou TIROSINA HIDROXILASE. A grande variabilidade inter-ensaios nos resultados de quantificação protéica sugere uma menor sensibilidade e precisão para esse método, quando comparado a PCR quantitativo. Nossos resultados indicam que as células de retina de embrião de 8 dias de galinha em cultura já contêm um relógio funcional, porém, este necessita do ciclo claro-escuro ou glutamato para sua sincronização. / The avian circadian system is composed by the retina, the mammalian homolog region of the supra-chiasmatic nucleus (SNC) and the pineal gland. The retina itself shows many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinaldehyde re-isomerization, melatonin and dopamine production and release. Altogether these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. In this work we investigated the expression of key components of a circadian system, including the two melanopsin genes, Opn4x, Opn4m, as well as the Clock, Per2, N-Acetyltransferase and Tyrosine Hidroxylase genes in chick embryo retinal cells. Primary cultures of chicken retina from 8-day-old embryos were prepared at ZT0 (lights on) and seeded at the density of 107 cells per 25 cm2 culture flask. The cells were kept in a humidified incubator in a 5% CO2 atmosphere at 40o C in constant dark, in 12L:12D, in 12L:12D followed by constant dark, or in constant dark in the absence or presence of 100 &#956;M glutamate for 12 h starting at ZT0 of the fifth day in vitro. Total RNA extraction was performed along 24 hours every three hours starting at ZT0 of the sixth day. The samples were submitted to RT-PCR followed by quantitative PCR for mRNA quantification. To analyze the Opn4x expression in these cells we performed an immunocytochemistry analysis with antibodies anti-chicken melanopsin developed in rabbit. We also quantified the protein levels of OPN4x, CLOCK AND TYROSINE HYDROXYLASE by Western Blot. The mRNA quantification showed no rhythm of transcription for any gene in cells kept in constant dark. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase and Tyrosine Hydroxylase presented rhythm patterns of transcription. 100 &#956;M glutamate was able to induce rhythmic expression of Clock, and strongly inhibited the expression of Tyrosine Hydroxylase and, just punctually, of Opn4x and Opn4m. Assays of cell viability and DNA fragmentation using flow cytometry demonstrated that the inhibition did not result of glutamate toxic or apoptotic actions. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. Protein quantification by Western Blot showed no rhythmic oscillation of CLOCK, OPN4x or TYROSINE HYDROXYLASE. The great variability inter-assays seen in the results of protein quantification suggests that this method is less precise and sensitive than quantitative PCR. The present data show evidences that chicken embryonic retinal cells contain a functional circadian Clock. However light-dark cycle or glutamate stimuli are needed to its synchronization.

Muitos vertebrados ectotérmicos ajustam suas cores corporais para serem confundidos com o ambiente, através da migração de pigmentos no interior de cromatóforos, regulada por sistemas neurais e/ou hormonais. Essas mudanças de coloração auxiliam no mimetismo, termorregulação, comunicação social e expressão de comportamentos como excitação sexual, agressividade e medo. Entretanto, cromatóforos de inúmeras espécies respondem diretamente à luz. Estudos sobre a resposta à luz nos melanóforos de Xenopus laevis levaram à descoberta do fotopigmento melanopsina, uma opsina que está presente na retina de todos os grupos de vertebrados, inclusive no homem. Vários hormônios podem regular o processo de mudança de cor nos vertebrados, dentre eles a melatonina, hormônio secretado pela glândula pineal. Este é o principal órgão responsável pela integração do sistema neuroendócrino dos vertebrados ao meio ambiente, traduzindo direta ou indiretamente a informação do fotoperíodo em sinal hormonal, coordenando assim os ritmos fisiológicos circadianos com o meio ambiente. Os objetivos deste trabalho foram: investigar se a expressão gênica das melanopsinas e do receptor de melatonina em melanóforos de Xenopus laevis apresenta variação temporal sob diferentes condições luminosas; verificar se a expressão gênica das melanopsinas e do receptor de melatonina em melanóforos de Xenopus laevis pode ser modulada por melatonina. Dados do trabalho demonstram que as melanopsinas em melanóforos de Xenopus laevis são sincronizadas aos ciclos de claro-escuro, expressando um robusto ritmo ultradiano com período de 16h para Opn4m e um ritmo circadiano com período de 25h para Opn4x. Curiosamente, essa ritmicidade só foi observada quando os melanóforos foram mantidos em ciclos 12C:12E e foram submetidos à troca de meio durante a fase clara do fotoperíodo. A constância na expressão gênica do receptor de melatonina Mel1, quer sob diferentes regimes de luz, quer sob tratamento por melatonina, sugere que esse gene é extremamente estável, não sofrendo alterações ao ser submetido a estímulos exógenos, podendo ser considerado um gene constitutivo. O tratamento com melatonina por 6h na fase clara do fotoperíodo, além de inibir drasticamente a expressão de Opn4x e Opn4m, aboliu a ritimicidade de ambas as melanopsinas. Nossos resultados indicam que os melanóforos de Xenopus laevis possuem um relógio funcional e podem ser caracterizados como relógios periféricos, porém necessitam do ciclo claro-escuro associado à troca de meio para exibirem sua sincronização. / Many ectothermic vertebrates adjust their body color to mimic the environment, through the pigment migration within chromatophores, regulated by neural and / or hormonal systems. These changes in color help in camouflage, thermoregulation, social communication and behaviors such as sexual arousal, agressiveness and fear. However, chromatophores of several species respond directly to light. Studies about light response in melanophores of Xenopus laevis have led to the discovery of the photopigment melanopsin, an opsin that is present in the retina of all vertebrate groups, including man. Various hormones may regulate the process of color change in vertebrates, among them melatonin, hormone secreted by the pineal gland. This is the main organ responsible for the integration of the neuroendocrine system of vertebrates to the environment, translating directly or indirectly the photoperiod information into hormonal signal, thus coordinating physiological circadian rhythms with the environment. The objectives of this work were: to investigate whether the gene expression of melanopsins and melatonin receptor in melanophores of Xenopus laevis exhibited temporal variation under different light conditions; to verify whether gene expression of melanopsins and melatonin receptor in melanophores of Xenopus laevis could be modulated by melatonin. Our data show that melanopsins in melanophores of Xenopus laevis are synchronized to light-dark cycles, expressing a robust ultradian rhythm with a period of 16h for Opn4m and circadian rhythm with a period of 25h for Opn4x. Interestingly, the rhythm was only observed when the melanophores were maintained in 12L: 12D regime and medium change was performed during the fotophase of photoperiod. The constancy in the expression of melatonin receptor Mel1c, either under different light regimes, or under treatment by melatonin, suggesting that this gene is extremely stable, not being altered by exogenous stimulus, and may be considered a constitutive gene. Treatment with melatonin for 6h during the fotophase of the photoperiod, drastically inhibit the expression of Opn4x and Opn4m, and abolished the rhythm of both melanopsins. Our results indicate that melanophores of Xenopus laevis possess a functional clock and can be characterized as peripheral clocks, but they need the light-dark cycle associated with change of medium to exhibit their synchronization.

Aggression is a feature of many clinical disorders including autism, Alzheimer’s disease, bipolar disorder, and schizophrenia. The available treatment options act to prevent impulsive aggression through modulation of GABAergic and dopaminergic pathways which come with metabolic and dyskinetic side effects. The mechanism underlying aggressive motivation, however, has not been elucidated. In addition, previous studies have been heavily biased towards males of various species. Mimicking changes in day length, or photoperiod, in the laboratory is a natural manipulation used to examine seasonal changes in aggressive behavior in many species. In response to the reduction in the duration of light exposure, animals undergo gonadal regression and become reproductively quiescent. During this non-breeding season in male photoperiod-responsive animals, aggressive behavior increases significantly. Although studies have shown offensive aggression remains elevated in female rodents, seasonal regulation of this behavior in females has not been thoroughly studied. The neuropeptide arginine-vasopressin (AVP) has been implicated in the facilitation of aggressive behavior in male rodents and fishes; therefore, it is useful to examine AVP as a modulator of seasonal aggression in females. Because the actions of AVP in female social behavior may be hormonally-dependent, we investigated the hormonal mechanisms that regulate the expression of AVP receptors and the behavioral actions of AVP on aggression. In addition to changes in gonadal steroid hormones during the non-breeding season, we identified photoperiod-dependent alterations in adrenal hormone secretion as AVP plays a role in regulation of hypothalamic-pituitary-adrenal axis (HPA) activity and anxiety-like behaviors in animal models.

Recent years have seen considerable advances in the study of biological rhythms and the underlying molecular mechanisms that drive the daily and seasonal physiology of vertebrates. Amongst teleosts the majority of work in this field has focused on the model species the zebrafish to characterise clock genes and the molecular feedback loop that underpins circadian rhythms and physiology. Daily profiles of clock gene expression in a wide variety of tissues and cell types are now relatively well described. However the zebrafish is a tropical species that does not display distinct seasonality and therefore may not be the species of choice to investigate the entrainment of circannual physiology. In contrast, Atlantic salmon is a highly seasonal teleost that displays considerable temporal organisation of most physiological processes. In salmonids photoperiod is widely known to synchronise physiology to the environmental conditions and as such photoperiod manipulation is routinely used by the salmon industry throughout the production cycle to control and manipulate spawning, smoltification and puberty. Previous studies in salmonid species have already identified a set of clock genes that are linked to these seasonal physiological processes. However, to date, the molecular mechanisms regulating daily and seasonal physiology are largely unknown despite the strong commercial relevance in the Atlantic salmon. In the Atlantic salmon, Davie et al (2009) was the first to report the photoperiod dependent circadian expression of clock genes (Clock, Bmal and Per2 and Cry2) in the brain of the Atlantic salmon. In the same investigation the expression of clock genes was reported in a wide variety of peripheral tissues, however 24h profiles of expression in peripheral tissues were not characterised. In order to examine further the role of seasonal photoperiod on the circadian expression of clock genes, the present work first aimed to characterise diel profiles of Clock, Per1 and Per 2 expression in the brain together with plasma melatonin levels in II Atlantic salmon acclimated to either long day (LD), short day (SD), 12L:12D (referred to as experiment 1 throughout) and SNP (referred to as experiment 2 throughout). Photoperiod dependent clocks were also investigated in peripheral tissues, namely in the fin and liver. Results showed circadian profiles of melatonin under all photoperiods. In experiment 1 both Clock and Per2 displayed significant circadian expression in fish exposed to LD. This is in contrast to previous results where rhythmic clock gene expression was observed under SD. In addition, clock gene expression differed in response to experimental photoperiod in the liver, and diel rhythm differed to that of the brain. No rhythmic expression was observed in the fin. Levels of plasma melatonin exhibited a circadian rhythm peaking during the nocturnal phase as expected. However the amplitude of nocturnal melatonin was significantly elevated under LD (experiment 1) and the SNP long day photoperiod and 2010 autumnal equinox samples (experiment 2). Overall results from these experiments suggested that the control of clock gene expression would be photoperiod dependent in the brain and the liver however photoperiod history is also likely to influence clock gene expression. Interestingly, the gradual seasonal changes in photoperiod under SNP did not elicit similar profiles of clock gene expression as compared to experimental seasonal photoperiods and clock gene expression differed between experimental photoperiod and SNP treatments. In experiment 2 significant seasonal differences were also observed in the amplitude of individual clock gene expression. The mechanisms underlying this and potential impact on seasonal physiology are unknown. Developmental changes such as the smoltification process or abiotic factors such as temperature or salinity should be further investigated. In mammals previous work has focused on the molecular switch for photoperiod response and regulation of thyroid hormone bioactivity via deiodinase mediated conversion of T4 to the biologically active form T3. In mammals and birds expression of key seasonal molecular markers i.e. Tsh, Eya3 and Dio2, are up-regulated hours after exposure to the first LD and III persist under chronic LD conditions. In order to confirm the involvement of these genes in the seasonal photoperiodic response in salmon, a microarray study was first carried out. Results displayed transcriptome level differences in the seasonal expression of a wide variety of target genes including Eya3 and Dio1-3 in relation to LD and SD photoperiod suggesting that these genes may have a conserved role in salmon. qPCR validations of selected genes of interest were then performed (Dio1, Dio2 and Dio3, Eya3 and Tshover diel cycles in fish exposed to LD and SD photoperiod (autumn acclimated fish). In addition an unrelated qPCR study was undertaken in salmon parr acclimated to LD, 12L12D and SD photoperiod (spring acclimated fish)(Dio2, Eya3 and Tsh. Consistent with findings obtained in other vertebrate species, circadian expression of Dio2 was observed under LD. However expression of Eya3 and Tsh appeared to be dependent on photoperiod history prior to acclimation to the experimental photoperiods as already suggested for clock gene expression in this thesis. This is potentially a consequence of direct regulation by clock genes. To our knowledge, this is the first report on the expression of key molecular components that drive vertebrate seasonal rhythms in a salmonid species. The thesis then focused on another key component of the photoneuroendocrine axis in fish, the pineal organ. In the Atlantic salmon, as in other teleosts the photoreceptive pineal organ is considered by many to be essential to the generation, synchronisation and maintenance of circadian and seasonal rhythms. This would be primarily achieved via the action of melatonin although direct evidence is still lacking in fish. In salmonids the production of pineal melatonin is regulated directly by light and levels are continually elevated under constant darkness. In non salmonid teleosts the rhythmic high at night/ low during day melatonin levels persists endogenously under constant conditions and is hypothesised to be governed by light and intra- pineal clocks. The aims of the present in vitro and in vivo trials were to determine if circadian clocks and Aanat2 expression, the rate limiting enzyme for melatonin IV production, are present in salmon, test the ability of the pineal to independently re-entrain itself to a different photoperiod and establish whether the candidate clock genes and Aanat2 expression can be sustained under un-entrained conditions. Expression of clock genes was first studied in vitro with pineal organs exposed to either 12L:12D photoperiod, reversed 12D:12L photoperiod and 24D. Clock gene expression was also determined in vivo, in fish exposed to 12L:12D. Results were then contrasted with an in vitro (12L:12D) investigation in the European seabass, a species displaying endogenous melatonin synthesis. Results revealed no rhythmic clock gene (Clock, per1 and per2) expression in isolated salmon pineals in culture under any of the culture conditions. In the seabass, Clock and Per1 did not also display circadian expression in vitro. However rhythmic expression of Cry2 and Per1 was observed in vivo in the salmon pineal. This suggested some degree of extra-pineal regulation of clocks in the Atlantic salmon. In terms of Aanat2 no rhythmic expression was observed in the Atlantic salmon under any experimental conditions while rhythmic expression of Aanat2 mRNA was observed in seabass pineals. This is consistent with the hypothesis that in salmonids AANAT2 is regulated directly at the protein level by light while in other teleosts, such as seabass, AANAT2 is also regulated by clocks at a transcriptional level.

Economic viability of Fraser River, Labrador Arctic charr (Salvelinus alpinus) aquaculture in Atlantic Canada may be greatly improved if grow-out could be completed in seawater (30 ppt), while having a low incidence of sexual maturation before harvesting. Growth and survival in seawater was investigated among individually PIT-tagged Arctic charr reared in tanks in the laboratory. Direct transfer from freshwater to brackish water (20 ppt), and then acclimation to 30 ppt was successful. The manipulation of photoperiod, temperature, and food ration can be used as practical applications in aquaculture to arrest maturation; this was investigated in two additional experiments. The most effective photoperiod was LD18:6 for 6 weeks starting December 21, which reduced maturation to 43% compared to 78% in controls. Restricted ration from December 21 through March 15 had no effect on maturation, however, rearing females in 5°C compared to 10°C reduced maturation to 15% compared to >80% in controls.

Canada is a global leader in flax production, but flax acreage in Canada remains limited since flax is not well adapted to the northern Prairies. Therefore, breeding early-flowering and early maturing flax cultivars that are adapted to the climate of the northern Prairies is one of the major strategies to expand flax acreage in Canada. The objective of this project is to understand flowering time in flax and generate early flowering genotypes that are adapted to the continental climate of the Canadian Prairies. This project examined photoperiod sensitivity in five Canadian flax cultivars (CDC Sorrel, CDC Bethune, Flanders, Prairie Thunder and Royal) and three M9 genotypes derived from 5-azacytidine (5-azaC) treatment (RE1, RE2 and RE3). To investigate how each cultivar or genotype responds to photoperiod changes, a reciprocal transfer experiment between long day and short day conditions was conducted. All cultivars and genotypes were photoperiod sensitive. However, the level of sensitivity and length of the sensitive phase varied among cultivars and genotypes. The five cultivars were more sensitive to photoperiod changes compared with the three mutant genotypes, while RE2, which was the earliest flowering genotype, was the least sensitive genotype. This project, in addition, examined the expression pattern of ELF4 (EARLY FLOWERING 4), a specific flowering-related gene. This experiment was conducted with three Canadian flax cultivars (CDC Sorrel, CDC Bethune and Royal) and one 5-azaC mutant genotype (RE2). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as a reference gene in RT-qPCR. Results of RT-qPCR demonstrated that CDC Sorrel and CDC Bethune had a similar expression pattern, while Royal and RE2 had a similar expression pattern. This project also sought to generate early-flowering genotypes by treating CDC Sorrel with 5-azaC as well as to introgress the early-flowering trait from RE genotypes into CDC Sorrel via hybridization. Mutant populations (M2, M3, bulk M3) and hybrid populations (F2, F3, and bulk F3) were grown and evaluated for time to flowering, maturity and height under latitude (53° N) field conditions in 2012 and 2013. 5-azaC treatment did not induce significant differences in flowering or maturity in the CDC Sorrel background. However, the early flowering trait was successfully introgressed into CDC Sorrel background since selected progeny lines flowered significantly earlier than the later flowering CDC Sorrel parental line.

Photoperiod is an important signal involved in the timing and co-ordination of many processes such as growth and reproduction in salmonids. Both growth and reproduction appear to be controlled by endogenous rhythms, which under natural conditions, is entrained by the seasonal changes in daylength, that is accurately reflected by the diel pattern of melatonin. This thesis investigates the influence of photoperiod on growth and reproduction in rainbow trout (Oncorhynchus mykiss), and examines the effect on plasma insulin-like growth factor-I (IGF-I). These studies aim to further our scientific understanding of the endocrine mechanisms controlling growth and reproduction and transfer this knowledge to commercial trout farming practices. Studies at fry (&lt;5g) and fingerling (&gt;25g) stages investigated the effect of exposure to constant long-days (LD18:6) and constant light (LL) on growth performance. In all experiments, there was a significant growth enhancing effect following exposure to extended light regimes relative to those under ambient or constant short-day photoperiods. Growth enhancement appeared to be caused through direct photo-stimulation or a phaseshift in an underlying endogenous rhythm dependent on the timing of photoperiod application. Measurement of plasma IGF-I accurately reflected growth rate during juvenile development. Furthermore, a clear autumnal increase in IGF-I was observed that was apparently up-regulated by long-day photoperiods. Additionally, it was shown that melatonin implants significantly reduced growth rate below that of unimplanted controls irrespective of photoperiod, but did not reduce circulating IGF-I levels suggesting that elevated melatonin levels masked the perception of daylength but did not act directly on the somatotropic axis to control growth. As size or one of its correlates (growth rate, energy balance or nutritional status) is regarded as an important determinant of the ability to undergo puberty, the interaction of natural (SNP) and advancing (ADV: long-short day) photoperiods with growth, IGF-I and reproduction was investigated in virgin female broodstock. Under SNP 63% of the population attained maturity while only 29% spawned in the ADV regime. Under SNP both size and growth rate in late spring/early summer appeared to determine whether an individual may initiate maturation. Conversely, under ADV, condition factor appeared to be a better predictor. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated for the first time in rainbow trout under natural conditions, and provides direct evidence for the relationship between maturation and IGF-I. IGF-I levels showed a negative correlation with testosterone as fish initiated maturation. Furthermore, IGF-I levels accurately reflected growth rate prior to the initiation of vitellogenesis, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that nutritional status, growth rate and/or size is sufficient to initiate maturation. In addition, maturing individuals under both photoperiods typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. In this sense, the observation of elevated levels for 3 months under SNP compared to only 1 month under ADV may reflect a reduction in the window of opportunity to initiate maturation under advancing photoperiods and hence explain the reduction in fish spawning. Finally, using a commercially available ELISA kit provided evidence of a possible detection method for measurement of a leptin-like peptide as absolute levels differed significantly between mature and immature fish. This provides preliminary evidence for a possible involvement of a leptin-like peptide in rainbow trout reproduction and energy homeostasis. Given the above evidence of a growth enhancing effect of photoperiod in covered systems, the transfer of constant light (LL) regimes to uncovered commercial trout farming practices was also examined. The application of LL regimes during either fry grow-out in tanks using floating lights or ongrowing to harvest size during winter in cages using submersible lights was investigated. In all trials conducted, the exposure to LL in autumn significantly enhanced winter growth rate and feeding efficiency by up to 30% and 25% respectively relative to those maintained under ambient light. Furthermore, the effect of light intensity was clearly shown to be an important requirement in successfully applying photoperiod regimes onto the ambient photoperiod in order to enhance commercial production in &quot;uncovered systems&quot;. These studies clearly indicate the importance of photoperiod in influencing growth and reproduction in rainbow trout. Furthermore, the use of plasma IGF-I as an indicator of growth provides a practical tool for studying growth-photoperiod-reproduction interactions in this species. However, further studies are necessary to further our understanding of the endocrine pathways governing physiological mechanisms, especially growth and reproduction. Overall, this work has provided important information to improve both scientific understanding and commercial development although it is clear that substantial research is still required.