Correction: RNA profiling identifies novel, photoperiod-history dependent markers associated with enhanced saltwater performance in juvenile Atlantic salmon.

[This corrects the article DOI: 10.1371/journal.pone.0227496.].

RNA profiling identifies novel, photoperiod-history dependent markers associated with enhanced saltwater performance in juvenile Atlantic salmon.

Atlantic salmon migrate to sea following completion of a developmental process known as smolting, which establishes a seawater (SW) tolerant phenotype. Smolting is stimulated by exposure to long photoperiod or continuous light (LL) following a period of exposure to short photoperiod (SP), and this leads to major changes in gill ion exchange and osmoregulatory function. Here, we performed an RNAseq experiment to discover novel genes involved in photoperiod-dependent remodeling of the gill. This revealed a novel cohort of genes whose expression rises dramatically in fish transferred to LL following SP exposure, but not in control fish maintained continuously on LL or on SP. A follow-up experiment revealed that the SP-history dependence of LL induction of gene expression varies considerably between genes. Some genes were inducible by LL exposure after only 2 weeks exposure to SP, while others required 8 weeks prior SP exposure for maximum responsiveness to LL. Since subsequent SW growth performance is also markedly improved following 8 weeks SP exposure, these photoperiodic history-dependent genes may be useful predictive markers for full smolt development.

Sequencing and characterization of a multi-organ Arctic charr transcriptome: A toolbox for investigating polymorphism and seasonal life in a high Arctic fish.

The Arctic charr (Salvelinus alpinus L.) inhabits fresh water ecosystems of the high North. The species has developed a strong phenotypic plasticity and variability in life history characteristics which has made this species an attractive model for investigations on phenotype plasticity, morph formation and ecological speciation. Further, the extreme seasonal variations in environmental conditions (e.g. food availability) in the high North induce seasonal changes in phenotype, which require precise timing mechanisms and physiological preparations. Individual gating of life-history strategies (e.g. formation of resident and sea-migrating morphs) and transitions (e.g. maturation) depends on conditional traits (size/energy status) at specific assessment time windows, and complex neuroendocrine regulation, which so far is poorly understood. In the absence of a reference genome, and in order to facilitate the investigation of the complex biological mechanisms of this unique fish model, the present study reveals a reference transcriptome for the Arctic charr. Using Roche 454 GS FLX+, we targeted various organs being either at the crossroads of many key pathways (neuroendocrine, metabolic, behavioral), of different ontological origins or displaying complementary physiological functions. The assemblage yielded 34,690 contigs greater than 1000bp with an average length (1690bp) and annotation rate (52%) within the range, or even higher, than what has been previously obtained with other teleost de novo transcriptomes. We dramatically improve the publically available transcript data on this species that may indeed be useful for various disciplines, from basic research to applied aspects related to conservation issues and aquaculture.

Seasonal Differences in Relative Gene Expression of Putative Central Appetite Regulators in Arctic Charr (Salvelinus alpinus) Do Not Reflect Its Annual Feeding Cycle.

The highly seasonal anadromous Arctic charr (Salvelinus alpinus) was used to investigate the possible involvement of altered gene expression of brain neuropeptides in seasonal appetite regulation. Pro-opiomelanocortin (POMCA1, POMCA2), Cocaine and amphetamine regulated transcript (CART), Agouti related Peptide (AgRP), Neuropeptide Y (NPY) and Melanocortin Receptor 4 (MC4-R) genes were examined. The function of centrally expressed Leptin (Lep) in fish remains unclear, so Lep (LepA1, LepA2) and Leptin Receptor (LepR) genes were included in the investigation. In a ten months study gene expression was analysed in hypothalamus, mesencephalon and telencephalon of immature charr held under natural photoperiod (69°38'N) and ambient temperature and given excess feed. From April to the beginning of June the charr did not feed and lost weight, during July and August they were feeding and had a marked increase in weight and condition factor, and from November until the end of the study the charr lost appetite and decreased in weight and condition factor. Brain compartments were sampled from non-feeding charr (May), feeding charr (July), and non-feeding charr (January). Reverse transcription real-time quantitative PCR revealed temporal patterns of gene expression that differed across brain compartments. The non-feeding charr (May, January) had a lower expression of the anorexigenic LepA1, MC4-R and LepR in hypothalamus and a higher expression of the orexigenic NPY and AgRP in mesencephalon, than the feeding charr (July). In the telencephalon, LepR was more highly expressed in January and May than in July. These results do not indicate that changes in central gene expression of the neuropeptides investigated here directly induce seasonal changes in feeding in Arctic charr.

Rhythmic life of the Arctic charr: adaptations to life at the edge.

High latitudes are characterized by strong seasonal changes in environmental conditions, including temperature and food availability. To cope with these changes, many high latitude species have developed circannual oscillators that enable them to anticipate and prepare for forthcoming environmental changes and synchronize seasonal events (e.g. reproduction) to environmental fluctuations. The Arctic charr (Salvelinus alpinus) is the world's northernmost freshwater fish species with a distribution largely confined within the Arctic. In the northernmost part of its distribution they have developed an anadromous life-history strategy implying annual, seaward migrations in the summer to utilize the rich feeding opportunity in the sea. Overwintering in freshwater is characterized by anorexia and energy conservation. The seaward migration in early summer is preceded by physiological and behavioral changes (smolting), by which they develop seawater tolerance (hypoosmoregulatory ability) and migratory behavior. When migrating to the sea, Arctic charr have regained a strong appetite and within 4-6weeks in the sea they may have doubled their body weight and increased their body fat stores several-fold, in anticipation of the resources needed for reproduction in the autumn and overwintering. All these processes are regulated independently of environmental changes; captive offspring of anadromous charr kept in freshwater displays seasonal changes in seawater tolerance and strong seasonal changes in food intake and growth even when they are continuously fed in excess and held at a constant water temperature in freshwater. A correct timing of these events is crucial for their survival in the Arctic and the Arctic charr seems to possess timing mechanisms that include endogenous, circannual oscillator(s) entrainable by photoperiod. The entrainment mechanism may be linked to diel melatonin rhythms, which in this species exactly mirror overground photoperiod, even during the winter residence in lakes with thick ice and snow. Little is known, however, about how photoperiod, melatonin and putative endogenous clock(s) interact in the generation of seasonal rhythms in fish, and downstream neuroendocrine mechanisms leading to physiological changes. The anadromous Arctic charr seems ideal as a model for studying such mechanisms.

Long-term fasting in the anadromous Arctic charr is associated with downregulation of metabolic enzyme activity and upregulation of leptin A1 and SOCS expression in the liver.

The life strategy of the anadromous Arctic charr (Salvelinus alpinus) includes several months of voluntary fasting during overwintering in freshwater, leading to emaciation prior to seawater migration in spring. In this study we compared changes in condition, substrate utilization and liver metabolism between captive anadromous charr subjected to food deprivation during late winter and spring, and conspecifics fed in excess. In March, nine out of the 10 sampled fed fish had not eaten, indicating that they were in a voluntary anorexic state. In June, the fed fish were eating and all had higher body mass, condition factor and adiposity than in March. In fasted fish there were only small decreases in body mass, condition factor and adiposity between March and May, but all these parameters decreased markedly from May to June. The fasted fish were depleted in fat and glycogen in June, had suppressed activity of hepatic enzymes involved in lipid metabolism (G6PDH and HOAD) and seemed to rely on protein-derived glucose as a major energy source. This was associated with upregulated liver gene expression of leptin A1, leptin A2, SOCS1, SOCS2 and SOCS3, and reduced IGF-I expression. In an in vitro study with liver slices it was shown that recombinant rainbow trout leptin stimulated SOCS1 and SOCS3 expression, but not SOCS2, IGF-I or genes of enzymes involved in lipid (G6PDH) and amino acid (AspAT) metabolism. It is concluded that liver leptin interacts with SOCS in a paracrine fashion to suppress lipolytic pathways and depress metabolism when fat stores are depleted.

Seasonal appetite regulation in the anadromous Arctic charr: evidence for a role of adiposity in the regulation of appetite but not for leptin in signalling adiposity.

The aim of this study was to investigate whether the seasonal feeding cycle of the anadromous Arctic charr (Salvelinus alpinus) is regulated by a lipostatic mechanism and if leptin (Lep) might act as an endocrine signal of adiposity. Offspring of anadromous Arctic charr with a body mass of 121 g were divided into two treatment groups; one was given feed in excess from March to November, and the other was fasted between April and early June and fed in excess thereafter. In the continuously fed group there was an 8-fold increase in body mass, and a doubling of percentage body fat, from March to August, after which there was no further increase. Fish in the other group lost weight and body fat during fasting, but grew rapidly on being fed, and had partially compensated for their deficit in body mass by August. Differences in percentage body fat between treatment groups were eliminated by August, providing evidence for a lipostatic regulation of feeding and energy homeostasis in Arctic charr. Neither liver total LepA gene expression nor plasma Lep concentrations correlated positively with fish adiposity, so there was no evidence that Lep acts as a signal of adiposity in this species. On the other hand, there was a strong increase in liver LepA1 gene expression at the end of the fasting period, concomitant with fat mobilization and increased plasma glucose, indicating that LepA1 may play a role in regulating metabolic processes associated with fasting.

Leptin and ghrelin in anadromous Arctic charr: cloning and change in expressions during a seasonal feeding cycle.

Anadromous (sea-migrating) Arctic charr (Salvelinus alpinus) display pronounced seasonal variations in food intake and growth and is an interesting model for studying mechanisms of appetite regulation. In this study cDNAs encoding for ghrelin (GHRL) and leptin (LEP) in Arctic charr were cloned, after which stomach GHRL and liver LEP mRNA expressions were examined by qPCR during a seasonal feeding cycle of semi-wild anadromous Arctic charr. The fish were captured as they returned from summer feeding in seawater and transferred to an indoor tank where they were fed in excess until October the year after. Growth rate was low in late winter, increased in late spring and reached a peak during summer, and then declined during autumn, when the fish became sexually mature. The changes in growth rate were associated with corresponding changes in the proportion of fish that had been eating at each sampling date, and whole body lipid status. Stomach GHRL mRNA expression was high in late winter, decreased to a nadir in mid-summer and increased again to a high level in early autumn. Liver LEP mRNA remained low during winter, spring and early summer, after which there was a gradual, 7-fold increase until October. The seasonal changes in ghrelin and leptin support a role of these hormones in the long-term regulation of energy homeostasis in the anadromous Arctic charr. It cannot be excluded, however, that the increase in liver leptin expression during autumn is related to sexual maturation.

Keeping track of time under ice and snow in a sub-arctic lake: plasma melatonin rhythms in Arctic charr overwintering under natural conditions.

Although photoperiod is considered as a major environmental cue for timing of seasonal events in fish, little is known about the photic information perceived by fish in different aquatic environments. The strongly seasonal Arctic charr, Salvelinus alpinus, reside in lakes covered by thick ice and snow throughout the dark winter in the north. In the present study, we have measured diel changes in their plasma melatonin concentrations from September to June in Lake Storvatnet (70 degrees N), northern Norway. In addition, we have measured the in vitro melatonin production of Arctic charr pineal glands held at experimental light conditions. From September to April a diel profile in plasma melatonin was seen in the charr in Lake Storvatn, with highest concentrations at night. This profile reflected the prevailing above-surface photoperiod, even in February when there were minimal changes in sub-surface irradiance between day and night. In June, plasma melatonin was low throughout the 24-hr cycle, despite there being a marked sub-surface difference in irradiance between night and day. At this time the irradiance in night probably remained above the threshold for suppression of melatonin production. The in vitro experiments revealed no endogenous rhythm in the pineal melatonin secretion, supporting the conclusion that the diel profile seen in the Arctic charr in their natural habitat was driven by ambient photoperiod. In conclusion, the Arctic charr appear to keep track of time even under the extreme conditions of high latitudes during winter, when lakes have thick ice and snow cover.