Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Impact of temperature on sea bass, Dicentrarchus labrax, retina: Fatty acid composition, expression of rhodopsin and enzymes of lipid and melatonin metabolism.

Teleost fish are ectothermic vertebrates. Their metabolism, physiology and behavior rely on the external temperature. This study, on the retina of the sea bass Dicentrarchus labrax, reports on the impact of temperature on the fatty acid composition and mRNA abundance of key enzymes of lipid metabolism: fatty acid desaturase-2 (FADS2), fatty acid elongase-5 (ELOVL5), sterol regulatory element-binding protein-1 (SREBP-1), triglyceride lipase and phospholipase A2 (PLA2). We also report on the effects on the photopigment molecule rhodopsin and on enzymes of the melatonin synthesis pathway, namely arylalkylamine N-acetyltransferases 1a and 1b and acetylserotonin methyltransferase. Juvenile fish were placed for 30 days at 18, 23 or 28 °C. At 23 °C, the fatty acid composition of D. labrax retina showed, as generally reported for the retina of other fish species, particularly high amounts of docosahexaenoic (DHA), palmitic and oleic acids. The fatty acids composition was not significantly (P > 0.05) altered between 23 and 28 °C, but did increase at 18 °C compared to 23 and 28 °C. At 18 °C there were noticeable increases in total DHA, ecosapentaenoic, arachidonic, oleic, linoleic, palmitoleic and stearic acids. A negative correlation was found in the abundance of neutral (NL) vs. polar (PL) lipids: 18 °C induced an increase in NL and a decrease in PL, while 28 °C induced higher PL with decreased NL. In NL the changes affected mainly triglycerides. FADS2 and ELOVL5 mRNA abundance decreased from 18° to 28 °C while SREBP-1 and triglyceride lipase mRNA remained stable. Conversely PLA2 mRNA was more abundant at 23 than at 18 and 28 °C. Temperature increased and decreased rhodopsin mRNA abundance, at 28 °C and 18 °C respectively, while there was no effect on mRNA from the melatonin synthesis enzymes. In conclusion the data indicate a temperature induced redistribution of fatty acids among the lipid classes that might affect the physical properties of the plasma membranes as well as functions associated with photoreception or generation of intracellular second messengers. In addition, the results suggest that temperature targets only the proteins and activities of retinal melatonin production. This study opens new lines of investigation related to the role temperature and fatty acids play in fish visual perception. They are relevant in the context of the global warming of seas affecting both the wild and the aquaculture species.

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya.

Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.

Subfunctionalization of arylalkylamine N-acetyltransferases in the sea bass Dicentrarchus labrax: two-ones for one two.

Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N-acetyltransferase (Aanat: serotonin→N-acetylserotonin) and acetylserotonin-O-methyltransferase (Asmt: N-acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost-specific whole-genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co-expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N-acetylserotonin and N-acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.

Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

Somatotropic axis genes are expressed before pituitary onset during zebrafish and sea bass development.

The somatotropic axis, or growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis, of fish is involved in numerous physiological process including regulation of ionic and osmotic balance, lipid, carbohydrate and protein metabolism, growth, reproduction, immune function and behavior. It is thought that GH plays a role in fish development but conflicting results have been obtained concerning the ontogeny of the somatotropic axis. Here we investigated the developmental expression of GH, GH-receptor (GHR) and IGF-1 genes and of a GH-like protein from fertilization until early stages of larval development in two Teleosts species, Danio rerio and Dicentrarchus labrax, by PCR, in situ hybridization and Western blotting. GH, GHR and IGF-1 mRNA were present in unfertilized eggs and at all stages of embryonic development, all three displaying a similar distribution in the two species. First located in the whole embryo (until 12 hpf in zebrafish and 76 hpf in sea bass), the mRNAs appeared then distributed in the head and tail, from where they disappeared progressively to concentrate in the forming pituitary gland. Proteins immunoreactive with a specific sea bass anti-GH antibody were also detected at all stages in this species. Differences in intensity and number of bands suggest that protein processing varies from early to later stages of development. The data show that all actors of the somatotropic axis are present from fertilization in these two species, suggesting they plays a role in early development, perhaps in an autocrine/paracrine mode as all three elements displayed a similar distribution at each stage investigated.

Identification of two arylalkylamine N-acetyltranferase 1 genes with different developmental expression profiles in the flatfish Solea senegalensis.

The existence of two arylalkylamine N-acetyltransferase 1 (Aanat1) genes in the genome of some teleosts has been reported recently by in silico analysis. However, there are no data concerning the similarities and/or differences between them and many questions remain to be answered, such as their expression sites, development, or kinetics. Here, we report the cloning of Aanat1a and Aanat1b cDNAs from the sole retina and show for the first time that at least three Aanat genes are expressed in a vertebrate species. Because melatonin is involved in fish ontogeny, we analyzed the developmental transcript levels of Aanat1a and Aanat1b by quantitative real-time PCR, showing their inverse and stage-specific expression patterns. Aanat1a was more abundant during early than late larval stages. Before metamorphosis, nocturnal expression was higher. At metamorphosis, Aanat1a expression decreased and lost these day-night variations. In contrast, the abundance of Aanat1b transcripts, low during early developing stages, rose significantly throughout metamorphosis. This situation seemed to apply to the adult because Aanat1a expression was lower than Aanat1b expression in the retina of adults, where the former did not exhibit day-night variations, while the latter did so with much higher nocturnal transcript levels. In situ hybridization analysis detected Aanat1a and Aanat1b messengers in the outer and inner nuclear layers of retina. The differences in abundance and distinct day-night expression patterns between Aanat1a and Aanat1b during sole development suggest different functions for these two enzymes as well as the existence of interactions between the melatoninergic and thyroid hormone systems during flatfish metamorphosis.

Melatonin and osmoregulation in fish: A focus on Atlantic salmon Salmo salar smoltification.

Part of the life cycle of several fish species includes important salinity changes, as is the case for the sea bass (Dicentrarchus labrax) or the Atlantic salmon (Salmo salar). Salmo salar juveniles migrate downstream from their spawning sites to reach seawater, where they grow and become sexually mature. The process of preparation enabling juveniles to migrate downstream and physiologically adapt to seawater is called smoltification. Daily and seasonal variations of photoperiod and temperature play a role in defining the timing of smoltification, which may take weeks to months, depending on the river length and latitude. Smoltification is characterised by a series of biochemical, physiological and behavioural changes within the neuroendocrine axis. This review discusses the current knowledge and gaps related to the neuroendocrine mechanisms that mediate the effects of light and temperature on smoltification. Studies performed in S. salar and other salmonids, as well as in other species undergoing important salinity changes, are reviewed, and a particular emphasis is given to the pineal hormone melatonin and its possible role in osmoregulation. The daily and annual variations of plasma melatonin levels reflect corresponding changes in external photoperiod and temperature, which suggests that the hormonal time-keeper melatonin might contribute to controlling smoltification. Here, we review studies on (i) the impact of pinealectomy and/or melatonin administration on smoltification; (ii) melatonin interactions with hormones involved in osmoregulation (e.g., prolactin, growth hormone and cortisol); (iii) the presence of melatonin receptors in tissues involved in osmoregulation; and (iv) the impacts of salinity changes on melatonin receptors and circulating melatonin levels. Altogether, these studies show evidence indicating that melatonin interacts with the neuroendocrine pathways controlling smoltification, although more information is needed to clearly decipher its mechanisms of action.

Transient Receptor Potential-Vanilloid (TRPV1-TRPV4) Channels in the Atlantic Salmon, Salmo salar. A Focus on the Pineal Gland and Melatonin Production.

Fish are ectotherm, which rely on the external temperature to regulate their internal body temperature, although some may perform partial endothermy. Together with photoperiod, temperature oscillations, contribute to synchronizing the daily and seasonal variations of fish metabolism, physiology and behavior. Recent studies are shedding light on the mechanisms of temperature sensing and behavioral thermoregulation in fish. In particular, the role of some members of the transient receptor potential channels (TRP) is being gradually unraveled. The present study in the migratory Atlantic salmon, Salmo salar, aims at identifying the tissue distribution and abundance in mRNA corresponding to the TRP of the vanilloid subfamilies, TRPV1 and TRPV4, and at characterizing their putative role in the control of the temperature-dependent modulation of melatonin production-the time-keeping hormone-by the pineal gland. In Salmo salar, TRPV1 and TRPV4 mRNA tissue distribution appeared ubiquitous; mRNA abundance varied as a function of the month investigated. In situ hybridization and immunohistochemistry indicated specific labeling located in the photoreceptor cells of the pineal gland and the retina. Additionally, TRPV analogs modulated the production of melatonin by isolated pineal glands in culture. The TRPV1 agonist induced an inhibitory response at high concentrations, while evoking a bell-shaped response (stimulatory at low, and inhibitory at high, concentrations) when added with an antagonist. The TRPV4 agonist was stimulatory at the highest concentration used. Altogether, the present results agree with the known widespread distribution and role of TRPV1 and TRPV4 channels, and with published data on trout (Oncorhynchus mykiss), leading to suggest these channels mediate the effects of temperature on S. salar pineal melatonin production. We discuss their involvement in controlling the timing of daily and seasonal events in this migratory species, in the context of an increasing warming of water temperatures.

Pituitary Hormones mRNA Abundance in the Mediterranean Sea Bass Dicentrarchus labrax: Seasonal Rhythms, Effects of Melatonin and Water Salinity.

In fish, most hormonal productions of the pituitary gland display daily and/or seasonal rhythmic patterns under control by upstream regulators, including internal biological clocks. The pineal hormone melatonin, one main output of the clocks, acts at different levels of the neuroendocrine axis. Melatonin rhythmic production is synchronized mainly by photoperiod and temperature. Here we aimed at better understanding the role melatonin plays in regulating the pituitary hormonal productions in a species of scientific and economical interest, the euryhaline European sea bass Dicentrarchus labrax. We investigated the seasonal variations in mRNA abundance of pituitary hormones in two groups of fish raised one in sea water (SW fish), and one in brackish water (BW fish). The mRNA abundance of three melatonin receptors was also studied in the SW fish. Finally, we investigated the in vitro effects of melatonin or analogs on the mRNA abundance of pituitary hormones at two times of the year and after adaptation to different salinities. We found that (1) the reproductive hormones displayed similar mRNA seasonal profiles regardless of the fish origin, while (2) the other hormones exhibited different patterns in the SW vs. the BW fish. (3) The melatonin receptors mRNA abundance displayed seasonal variations in the SW fish. (4) Melatonin affected mRNA abundance of most of the pituitary hormones in vitro; (5) the responses to melatonin depended on its concentration, the month investigated and the salinity at which the fish were previously adapted. Our results suggest that the productions of the pituitary are a response to multiple factors from internal and external origin including melatonin. The variety of the responses described might reflect a high plasticity of the pituitary in a fish that faces multiple external conditions along its life characterized by marked daily and seasonal changes in photoperiod, temperature and salinity.

Evolution of AANAT: expansion of the gene family in the cephalochordate amphioxus.

BACKGROUND: The arylalkylamine N-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue. RESULTS: We have found that the amphioxus genome contains seven AANATs, all having non-vertebrate type features. This and the absence of AANATs from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the AANATs may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent AANATs in Branchiostoma lanceolatum (bl) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANAT alpha and bfAANAT delta' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs. CONCLUSIONS: The substrate selectivity profiles of bfAANAT alpha and delta' are relatively broad, including alkylamines, arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.

Continuous light and melatonin: daily and seasonal variations of brain binding sites and plasma concentration during the first reproductive cycle of sea bass.

The present study reports on the daily and seasonal variations in plasma melatonin concentration, and also in optic tectum and hypothalamus melatonin binding sites, in male European sea bass maintained under natural photoperiod (NP) or continuous light (LL) from early stages of development. Samples were collected on a 24-h cycle, at four physiological phases of their first annual reproductive cycle, i.e., pre-spermatogenesis, spermatogenesis, spermiation and post-spermiation. Under NP, (1) plasma melatonin levels were higher at night than during the day regardless of the year period, and the duration of the signal matched the duration of the dark phase; (2) daily variations in Kd and Bmax were found in the optic tectum, but only during spermiation, with the acrophase being 180° out of phase with the plasma melatonin variations; and (3) significant seasonal Kd and Bmax changes were seen in the hypothalamus. Under LL, (1) plasma melatonin showed no elevation during the subjective night; and (2) Kd and Bmax exhibited seasonal variations in the hypothalamus. These results led to the conclusion that long-term exposure to LL affected both plasma melatonin and receptor oscillations; particularly, LL disrupted the receptor density circadian oscillation found in the optic tectum during spermiation under NP. This oscillation appears to be important for sea bass to pursue gametogenesis until full spermiation. The persistence of both daily and seasonal variation of receptor affinity and density in the hypothalamus under LL indicates that these variations are controlled by internal circadian and circannual clocks that do not involve melatonin.

Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations.

Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the synchronization of circadian and circannual rhythms. In this study we have cloned three melatonin receptor subtypes (MT1, MT2 and Mel1c) in the Senegalese sole and analyzed their central and peripheral tissue distribution. The full-length MT1 (1452 nt), MT2 (1728 nt) and Mel1c (1980 nt) cDNAs encode different proteins of 345, 373, 355 amino acids, respectively. They were mainly expressed in retina, brain and pituitary, but MT1 was also expressed in gill, liver, intestine, kidney, spleen, heart and skin. At peripheral level, MT2 expression was only evident in gill, kidney and skin whereas Mel1c expression was restricted to the muscle and skin. This pattern of expression was not markedly different between sexes or among the times of day analyzed. The real-time quantitative PCR analyses showed that MT1 displayed higher expression at night than during the day in the retina and optic tectum. Seasonal MT1 expression was characterized by higher mRNA levels in spring and autumn equinoxes for the retina, and in winter and summer solstices for the optic tectum. An almost similar expression profile was found for MT2, but differences were less conspicuous. No day-night differences in MT1 and MT2 expression were observed in the pituitary but a seasonal variation was detected, being mRNA levels higher in summer for both receptors. Mel1c expression did not exhibit significant day-night variation in retina and optic tectum but showed seasonal variations, with higher transcript levels in summer (optic tectum) and autumn (retina). Our results suggest that day-night and seasonal variations in melatonin receptor expression could also be mediating circadian and circannual rhythms in sole.

Effects of Melatonin on Anterior Pituitary Plasticity: A Comparison Between Mammals and Teleosts.

Melatonin is a key hormone involved in the photoperiodic signaling pathway. In both teleosts and mammals, melatonin produced in the pineal gland at night is released into the blood and cerebrospinal fluid, providing rhythmic information to the whole organism. Melatonin acts via specific receptors, allowing the synchronization of daily and annual physiological rhythms to environmental conditions. The pituitary gland, which produces several hormones involved in a variety of physiological processes such as growth, metabolism, stress and reproduction, is an important target of melatonin. Melatonin modulates pituitary cellular activities, adjusting the synthesis and release of the different pituitary hormones to the functional demands, which changes during the day, seasons and life stages. It is, however, not always clear whether melatonin acts directly or indirectly on the pituitary. Indeed, melatonin also acts both upstream, on brain centers that control the pituitary hormone production and release, as well as downstream, on the tissues targeted by the pituitary hormones, which provide positive and negative feedback to the pituitary gland. In this review, we describe the known pathways through which melatonin modulates anterior pituitary hormonal production, distinguishing indirect effects mediated by brain centers from direct effects on the anterior pituitary. We also highlight similarities and differences between teleosts and mammals, drawing attention to knowledge gaps, and suggesting aims for future research.

Exposure to Artificial Light at Night and the Consequences for Flora, Fauna, and Ecosystems.

The present review draws together wide-ranging studies performed over the last decades that catalogue the effects of artificial-light-at-night (ALAN) upon living species and their environment. We provide an overview of the tremendous variety of light-detection strategies which have evolved in living organisms - unicellular, plants and animals, covering chloroplasts (plants), and the plethora of ocular and extra-ocular organs (animals). We describe the visual pigments which permit photo-detection, paying attention to their spectral characteristics, which extend from the ultraviolet into infrared. We discuss how organisms use light information in a way crucial for their development, growth and survival: phototropism, phototaxis, photoperiodism, and synchronization of circadian clocks. These aspects are treated in depth, as their perturbation underlies much of the disruptive effects of ALAN. The review goes into detail on circadian networks in living organisms, since these fundamental features are of critical importance in regulating the interface between environment and body. Especially, hormonal synthesis and secretion are often under circadian and circannual control, hence perturbation of the clock will lead to hormonal imbalance. The review addresses how the ubiquitous introduction of light-emitting diode technology may exacerbate, or in some cases reduce, the generalized ever-increasing light pollution. Numerous examples are given of how widespread exposure to ALAN is perturbing many aspects of plant and animal behaviour and survival: foraging, orientation, migration, seasonal reproduction, colonization and more. We examine the potential problems at the level of individual species and populations and extend the debate to the consequences for ecosystems. We stress, through a few examples, the synergistic harmful effects resulting from the impacts of ALAN combined with other anthropogenic pressures, which often impact the neuroendocrine loops in vertebrates. The article concludes by debating how these anthropogenic changes could be mitigated by more reasonable use of available technology - for example by restricting illumination to more essential areas and hours, directing lighting to avoid wasteful radiation and selecting spectral emissions, to reduce impact on circadian clocks. We end by discussing how society should take into account the potentially major consequences that ALAN has on the natural world and the repercussions for ongoing human health and welfare.

Effects of a temperature rise on melatonin and thyroid hormones during smoltification of Atlantic salmon, Salmo salar.

Smoltification prepares juvenile Atlantic salmon (Salmo salar) for downstream migration. Dramatic changes characterize this crucial event in the salmon's life cycle, including increased gill Na+/K+-ATPase activity (NKA) and plasma hormone levels. The triggering of smoltification relies on photoperiod and is modulated by temperature. Both provide reliable information, to which fish have adapted for thousands of years, that allows deciphering daily and calendar time. Here we studied the impact of different photoperiod (natural, sustained winter solstice) and temperature (natural, ~ + 4° C) combinations, on gill NKA, plasma free triiodothyronine (T3) and thyroxine (T4), and melatonin (MEL; the time-keeping hormone), throughout smoltification. We also studied the impact of temperature history on pineal gland MEL production in vitro. The spring increase in gill NKA was less pronounced in smolts kept under sustained winter photoperiod and/or elevated temperature. Plasma thyroid hormone levels displayed day-night variations, which were affected by elevated temperature, either independently from photoperiod (decrease in T3 levels) or under natural photoperiod exclusively (increase in T4 nocturnal levels). Nocturnal MEL secretion was potentiated by the elevated temperature, which also altered the MEL profile under sustained winter photoperiod. Temperature also affected pineal MEL production in vitro, a response that depended on previous environmental acclimation of the organ. The results support the view that the salmon pineal is a photoperiod and temperature sensor, highlight the complexity of the interaction of these environmental factors on the endocrine system of S. salar, and indicate that climate change might compromise salmon's time "deciphering" during smoltification, downstream migration and seawater residence.

Iodothyronine deiodinases and thyroid hormone receptors regulation during flatfish (Solea senegalensis) metamorphosis.

Thyroid hormone-induced metamorphosis seems to represent an ancestral feature of chrordates (urochordates, cephalochordates and vertebrates), but also of nonchordate animals. Although thyroid hormones and thyroid hormone receptor profiles during metamorphosis have been analyzed in different vertebrate taxa, including fish, developmental expression and activity of type 2 (dio2, D2) and type 3 (dio3, D3) iodothyronine deiodinases, two key enzymes in anuran metamorphosis, remain unknown in any fish species. The aim of this work was to investigate the development of thyroid hormone system during the metamorphosis of a flatfish species, the Senegalese sole, focusing on the deiodinases developmental profile. We have cloned sole D2 and D3 and analyzed several parameters of thyroid hormones system in pre-, early-, middle-, and late-metamorphic larvae. Both deiodinases contain in their catalytic centers an UGA triplet encoding for a selenocystein (Sec) residue as expected. Left eye migration and rotation in body position were associated with a significant increase in both thyroid hormones and thyroid hormone receptors at the middle-late metamorphic stages. Although dio2 expression slightly increased during metamorphosis, D2 activity augmentation was much more significant. Sole dio3 expression declined only slightly, whereas the D3 activity clearly decreased at mid-late metamorphic period. This developmental profile of deiodinases sustained the rise of thyroid hormones levels observed during sole metamorphosis. No clear cut daily rhythms were observed in the parameters analyzed although it seemed that thyroid hormone system was more active during daytime, in particular at late metamorphic stages. These developmental changes point out the importance not only of thyroid hormones and their receptors but also of dio2 and dio3 in mediating flatfish metamorphosis, as it has been described in amphibians.

Cloning and expression of arylalkylamine N-acetyltranferase-2 during early development and metamorphosis in the sole Solea senegalensis.

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in teleost fish, one retinal specific, Aanat1, and the other one pineal specific, Aanat2, being the latter the main enzyme responsible of the plasma nocturnal melatonin increase in fish. In anurans melatonin has been involved in metamorphosis through antagonizing thyroid hormone function; however, no available data reports a relationship between melatonin system and metamorphosis in fish. In this study, we have cloned the AANAT2 (SsAanat2) in a flatfish, Solea senegalensis, and studied its sites of expression and developmental expression pattern by in situ hybridization and Real Time PCR. These studies allowed us to demonstrate a specific signal in the pineal gland of sole larvae from 2 days post-fertilization (dpf), which was evident until post-metamorphosis. Immunohistochemical analysis on the hybridized slides showed that the sole pineal Aanat2 expressing cells corresponded to pineal photoreceptor cells. Real Time PCR was performed in animals kept under natural photoperiod and sampled at different stages from 0 to 21 dpf (including pre-, early-, middle- and late-metamorphic stages) and at midlight (ML) and middark (MD) daytimes. Sole Aanat2 expression was higher at MD than at ML from 2 dpf and at most developmental stages analyzed. The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf. A significant 60-fold reduction in Aanat2 expression was seen just before metamorphosis demonstrating, for the first time in a vertebrate species, that the expression of pineal AANAT and thyroid hormones levels exhibit an inverse pattern during metamorphosis.

Melatonin effects on the hypothalamo-pituitary axis in fish.

Melatonin, a hormonal output signal of vertebrate circadian clocks, contributes to synchronizing behaviors and neuroendocrine regulations with the daily and annual variations of the photoperiod. Conservation and diversity characterize the melatonin system: conservation because its pattern of production and synchronizing properties are a constant among vertebrates; and diversity because regulation of both its synthesis and modes of action have been profoundly modified during vertebrate evolution. Studies of the targets and modes of action of melatonin in fish, and their parallels in mammals, are of interest to our understanding of time-related neuroendocrine regulation and its evolution from fish to mammals, as well as for aquacultural purposes.