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

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 Tshover 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

Impact of dietary phosphorous in diploid and triploid Atlantic salmon (Salmo salar L.) with reference to early skeletal development in freshwater

In order to assess the effect of dietary phosphorus (P) in reducing vertebral malformations and improving freshwater (FW) performance in triploid Atlantic salmon (Salmo salar), both triploid and diploid Atlantic salmon were fed three different dietary P inclusion levels (low: 4.9, medium: 7.7, and high: 9.7 g available P kg−1) from first feeding until smolt. Somatic and skeletal response was assessed at fry (~0.5 g), parr (~5 g) and smolt (~45 g) stages. Triploid parr initially grew faster on the high P diet, while groups fed low P resulted in a significantly higher weight at smolt. Image analysis of double stained Alcian blue and Alizarin red S fry revealed that low P fed triploid fish presented less well mineralised vertebrae, and significantly more malformed vertebrae in both parr and smolt stages following x-ray radiographic assessment. Triploid parr fed high and medium P had similar numbers of malformed vertebrae relative to their diploid counterparts but greater numbers than at smolt. Low P fed triploids had the highest prevalence of jaw and vertebral malformations as well as the highest number of deformed vertebrae in the central caudal vertebral region, which was more pronounced at parr than at smolt. Shorter vertebrae dorso-ventral lengths were observed throughout the spinal column (R1–R4) in parr fed low P and only in the caudal region (R3) at smolt. In parr, both ploidies showed reduced phosphate homeostasis protein fgf23 gene expression in vertebrae when fed low P diets, while triploids showed greater down-regulation of osteogenic factors (alp, opn and igf1r) between diets relative to diploids, suggesting possible greater active suppression of mineralisation and reduced osteogenic potential in triploids. No effects of diet or ploidy on gene expression were evident at smolt. Comparisons between development stages suggest early P supplementation in triploids is crucial for skeletal development. Ultimately, reducing vertebral deformities observed at smolt with higher P supplementation in triploids could contribute towards improving skeletal performance and welfare of the stocks in the marine phase

Response of triploid Atlantic salmon (Salmo salar) to commercial vaccines

While triploid Atlantic salmon represent a practical and affordable solution to the issues associated with sexual maturation in the salmonid aquaculture industry, empirical evidence suggests triploids are more susceptible to disease and vaccine side-effects than diploids. With vaccination now part of routine husbandry, it is essential their response be studied to confirm their suitability for commercial production. This study tested the response of triploid and diploid Atlantic salmon to vaccination with commercially available vaccines. Triploid and diploid Atlantic salmon siblings were injected with one of three commercial vaccines (or sham-vaccinated) and monitored for performance throughout a commercial production cycle. Sampling at smolt and harvest was undertaken along with individual weight and length assessments through the cycle. Antibody response to Aeromonas salmonicida vaccination was similar in both ploidy, with a positive response in vaccine-injected fish. For both adhesions and melanin, analysis found that higher scores were more likely to occur as the anticipated severity of the vaccine increased. In addition, for adhesion scores at smolt and melanin scores at smolt and harvest, triploids were statistically more likely to exhibit high scores than diploids. Triploids maintained a significantly higher body weight during freshwater and until 11 months post-seawater transfer, with diploids weighing significantly more at harvest. Growth, represented by thermal growth coefficient (TGC), decreased in both ploidy as the severity of adhesions increased, and regression patterns did not differ significantly between ploidy. Vertebral deformity prevalence was consistently higher in triploids (smolt 12.3 ± 4.5%; harvest 34.9 ± 5.9%) than diploids (smolt 0.8 ± 0.5%; harvest 15.9 ± 1.9%), with no significant difference between vaccine groups in each ploidy. This study demonstrates that triploids respond as well to vaccination as diploids and provides further supporting evidence of triploid robustness for commercial aquaculture

A temperature shift during embryogenesis impacts prevalence of deformity in diploid and triploid Atlantic salmon (Salmo salar L.)

The study investigated the effects of a temperature shift during embryogenesis on diploid and triploid Atlantic salmon (Salmo salar L.) embryo development and juvenile skeletal deformities. From fertilization, sibling populations were incubated under one of three temperatures (6, 8 or 11°C) until 400 °days when all fish were then reared under a common temperature until smolt. Survival was negatively impacted by increasing temperatures irrespective of ploidy. There was no effect of incubation temperatures on growth in diploids, but triploids incubated at 6°C had improved growth rates (thermal growth coefficient, TGC: 6°C: 1.05, 8°C: 0.94, 11°C: 0.48). Fish from 11°C in both ploidies showed increased jaw and vertebral deformity prevalence. In response to the temperature change at 400 °days post fertilization, upregulation of bmp2, bmp4, col2a1, mmp13, opn and sparc, and downregulation of ocn further suggest that bone and cartilage formation is compromised after experiencing a thermal shift. The data show that temperature profile during embryogenesis strongly influences future growth and deformity prevalence. Triploids appear to require a lower incubation temperature than the current industry standard of 8°C to promote better overall performance; however, a thermal shift during embryogenesis was shown to impact expression of important developmental genes

Investigating the kisspeptin system in the hermaphrodite teleost gilthead seabream (Sparus aurata)

The kisspeptin system, a known regulator of reproduction in fish, was investigated during two key phases within the gilthead seabream (Sparus aurata) life cycle: protandrous sex change and larval ontogeny. Seabream specific partial cDNA sequences were identified for two key targets, kissr4 and kiss2, which were subsequently cloned and qPCR assays developed. Thereafter, to examine association in expression with sex change, a group of adult seabream (2+ years old) undergoing sex change were sampled for gene expression at two different periods of the annual cycle. To study the kisspeptin system ontogeny during early life stages, transcript levels were monitored in larvae (till 30 days-post-hatch, DPH) and post-larvae (from 30 till 140 DPH). During sex change, higher expression of kissr4 and kiss2 was observed in males when compared to females or individual undergoing sex change, this is suggestive of differential actions of the kisspeptin system during protandrous sex change. Equally, variable expression of the kisspeptin system during early ontogenic development was observed. The higher expression of kissr4 and kiss2 observed from 5 DPH, with elevations at 5–20 and 90 DPH for kissr4 and at 5, 10, 20, and 60 DPH for kiss2, is coincident with the early ontogeny of gnrh genes previously reported for seabream, and possibly related with early development of the reproductive axis in this species

Comparative study of pineal clock gene and AANAT2 expression in relation to melatonin synthesis in Atlantic salmon (Salmo salar) and European seabass (Dicentrarchus labrax)

The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 &2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway

IL CANTO DI EZZO

In mammals, several genes involved in liver lipid and cholesterol homeostasis are rhythmically expressed with expression shown to be regulated by clock genes via Rev-erb 1α. In order to elucidate clock gene regulation of genes involved in lipid metabolism in Atlantic salmon (Salmo salar L.), the orphan nuclear receptor Rev-erb 1α was cloned and 24 h expression of clock genes, transcription factors and genes involved in cholesterol and lipid metabolism determined in liver of parr acclimated to a long-day photoperiod, which was previously shown to elicit rhythmic clock gene expression in the brain. Of the 31 genes analysed, significant daily expression was demonstrated in the clock gene Bmal1, transcription factor genes Srebp1, Lxr, Pparα and Pparγ, and several lipid metabolism genes Hmgcr, Ipi, ApoCII and El. The possible regulatory mechanisms and pathways, and the functional significance of these patterns of expression were discussed. Importantly and in contrast to mammals, Per1, Per2, Fas, Srebp2, Cyp71α and Rev-erb 1α did not display significant daily rhythmicity in salmon. The present study is the first report characterising 24 h profiles of gene expression in liver of Atlantic salmon. However, more importantly, the predominant role of lipids in the nutrition and metabolism of fish, and of feed efficiency in determining farming economics, means that daily rhythmicity in the regulation of lipid metabolism will be an area of considerable interest for future research in commercially important species

Twenty-four hour expression of fatty acid metabolism genes in the liver of salmon parr acclimated to LD photoperiod.

<p>Results are displayed in relation to Zeitgeiber time (ZT), where ZT 0 is the onset of light. Gene expression data is displayed as the percentage of the mean ± SEM and includes the spread of the data. The presence of a cosine wave denotes a significant circadian rhythm by acro analysis. The grey bar at the bottom of the graph represents the dark period. The presence of different letters represents statistically significant differences between samples by ANOVA and Tukey’s test (P<0.05).</p

Twenty-four hour expression profiles of transcription factors involved in the regulation of lipid metabolism in the liver of salmon parr acclimated to LD photoperiod.

<p>Results are displayed in relation to Zeitgeiber time (ZT), where ZT 0 is the onset of light. Gene expression data is displayed as the percentage of the mean ± SEM and includes the spread of the data. The presence of a cosine wave denotes a significant circadian rhythm by acro analysis. The grey bar at the bottom of the graph represents the dark period. The presence of different letters represents statistically significant differences between samples by ANOVA and Tukey’s test (P<0.05).</p

Twenty-four hour expression of lipoprotein metabolism genes in the liver of salmon parr acclimated to LD photoperiod.

<p>Results are displayed in relation to Zeitgeiber time (ZT), where ZT 0 is the onset of light. Gene expression data is displayed as the percentage of the mean ± SEM and includes the spread of the data. The presence of a cosine wave denotes a significant circadian rhythm by acro analysis. The grey bar at the bottom of the graph represents the dark period. The presence of different letters represents statistically significant differences between samples by ANOVA and Tukey’s test (P<0.05).</p