Photoperiod regulation of molecular clocks and seasonal physiology in the Atlantic salmon (Salmo salar)

Abstract

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. Post hoc in silico analysis of the Aanat 2 5’ regulatory region revealed the absence of a functional E-box element in the salmon in comparison to other teleosts, including the European seabass, confirming the absence of clock regulation of Aanat2 mRNA in salmon. Although it is crucial to first characterise the molecular mechanisms regulating daily and seasonal rhythms, understanding how these mechanisms impact on the animal’s physiology is critical. One such aspect is the circadian regulation of fatty acid metabolism and cholesterol homeostasis ultimately impacting fat deposition in commercially produced Atlantic salmon. This is an area of considerable research interest both in terms of human health and improving the sustainability of commercial salmon feed. In mammals a number of genes involved in V liver lipid and cholesterol homeostasis are rhythmically expressed under the control of clock genes via Rev-erb . The aim of the present work was to determine diel mRNA expression patterns of selected genes involved in cholesterol homeostasis (Srebp 1, Srebp 2, Fas, Lxr, Elovl5, Hmgcr and D6 Fad) together with circadian clocks (Bmal1, Clock, Per 1, Per 2 and Rev-erb ) in the liver of the Atlantic salmon. Results demonstrated significant circadian expression of Srebp 1 and Bmal 1, similar to previous results in mice, Lxr also exhibited significant circadian expression. Additionally the gene coding for the rate limiting enzyme in cholesterol synthesis, Hmgcr, was significantly elevated during the day. This is in contrast to mammals where mRNA expression and protein activity was elevated during the night. Also in contrast to results obtained in mammals, Per1, Per2, Fas, and Reverb  did not display significant circadian rhythmicity in salmon. This investigation represents the first attempt to correlate 24h profiles of clock gene expression to a functionally important process in peripheral tissues, lipid metabolism, which is an area of considerable interest for future research in this commercially important species. This thesis has significantly advanced knowledge on the expression of clock and seasonal genes in response to photoperiod information in the Atlantic salmon. Moreover it has given an important insight into the expression of clock genes in multiple tissue types and how clocks can regulate important physiological processes. However research is still in the early days and much work is needed to understand such a complex network in this highly seasonal and commercially important species.