Search

In the wild, the environment does not remain constant, but periodically oscillates so that temperature rises in the daytime and drops at night, which generates a daily thermocycle. The effects of thermocycles on thermal tolerance have been previously described in fish. However, the impact of thermocycles on daytime-dependent thermal responses and daily rhythms of temperature tolerance and sensing expression mechanisms remain poorly understood. This study investigates the effects of two rearing conditions: constant (26 °C, C) versus a daily thermocycle (28 °C in the daytime; 24 °C at night, T) on the thermal tolerance response in zebrafish. Thermal tolerance (mortality) was assessed in 4dpf (days post fertilization) zebrafish larvae after acute heat shock (39 °C for 1 h) at two time points: middle of the light phase (ML) or middle of the dark phase (MD). Thermal stress responses were evaluated in adult zebrafish after a 37 °C challenge for 1 h at ML or MD to examine the expression of the heat-shock protein (HSP) (hsp70, hsp90ab1, grp94, hsp90aa1, hspb1, hsp47, cirbp) and transient receptor potential (TRP) channels (trpv4, trpm4a, trpm2, trpa1b) in the brain. Finally, the daily rhythms of gene expression of HSPs and TRPs were measured every 4 h for 24 h. The results revealed the larval mortality rates and the expression induction of most HSPs in adult zebrafish brain reached the highest values in fish reared under constant temperature and subjected to thermal shock at MD. The expression of most HSPs and TRPs was mainly synchronized to the light/dark (LD) cycle, regardless of the temperature regime. Most HSPs involved in hyperthermic challenges displayed diurnal rhythms with their acrophases in phase with warm-sensing thermoTRPs acrophases. The cold-sensing trpa1b peaked in the second half of the light period and slightly shifted toward the dark phase anticipating the acrophase of cirpb, which is involved in hypothermic challenges. These findings indicated that: a) thermal shocks are best tolerated in the daytime; b) the implementation of daily thermocycles during larval development reduces mortality and stress-cellular expression of HSPs to an acute thermal stress at MD; c) daily rhythms need to be considered when discussing physiological responses of thermal sensing and thermotolerance in zebrafish.

The seasonally changing photoperiod controls the timing of reproduction in most fish species, however, the transduction of this photoperiodic information to the reproductive axis is still unclear. This study explored the potential role of two candidate neuropeptide systems, gonadotropin-inhibitory hormone (Gnih) and kisspeptin, as mediators between the pineal organ (a principle transducer of photoperiodic information) and reproductive axis in male European sea bass, Dicentrarchus labrax. Two seven-day experiments of pinealectomy (Px) were performed, in March (end of reproductive season) and August (resting season). Effects of Px and season on the brain expression of gnih (sbgnih) and its receptor (sbgnihr), kisspeptins (kiss1, kiss2) and their receptors (kissr2, kissr3) and gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) and the main brain receptor (gnrhr-II-2b) genes, plasma melatonin levels and locomotor activity rhythms were examined. Results showed that Px reduced night-time plasma melatonin levels. Gene expression analyses demonstrated a sensitivity of the Gnih system to Px in March, with a reduction in sbgnih in the mid-hindbrain, a region with bilateral connections to the pineal organ. In August, kiss2 levels increased in Px animals but not in controls. Significant differences in expression were observed for diencephalic sbgnih, sbgnihr, kissr3 and tegmental gnrh2 between seasons. Recordings of locomotor activity following surgery revealed a change from light-synchronised to free-running rhythmic behavior. Altogether, the Gnih and Kiss2 sensitivity to Px and seasonal differences observed for Gnih and its receptor, Gnrh2, and the receptor for Kiss2 (Kissr3), suggested they could be mediators involved in the relay between environment and seasonal reproduction.

Fish have evolved a biological clock to cope with environmental cycles, so they display circadian rhythms in most physiological functions including stress response. Photoperiodic information is transduced by the pineal organ into a rhythmic secretion of melatonin, which is released into the blood circulation with high concentrations at night and low during the day. The melatonin rhythmic profile is under the control of circadian clocks in most fish (except salmonids), and it is considered as an important output of the circadian system, thus modulating most daily behavioral and physiological rhythms. Lighting conditions (intensity and spectrum) change in the underwater environment and affect fish embryo and larvae development: constant light/darkness or red lights can lead to increased malformations and mortality, whereas blue light usually results in best hatching rates and growth performance in marine fish. Many factors display daily rhythms along the hypothalamus-pituitary-interrenal (HPI) axis that controls stress response in fish, including corticotropin-releasing hormone (Crh) and its binding protein (Crhbp), proopiomelanocortin A and B (Pomca and Pomcb), and plasma cortisol, glucose, and lactate. Many of these circadian rhythms are under the control of endogenous molecular clocks, which consist of self-sustained transcriptional-translational feedback loops involving the cyclic expression of circadian clock genes (clock, bmal, per, and cry) which persists under constant light or darkness. Exposing fish to a stressor can result in altered rhythms of most stress indicators, such as cortisol, glucose, and lactate among others, as well as daily rhythms of most behavioral and physiological functions. In addition, crh and pomca expression profiles can be affected by other factors such as light spectrum, which strongly influence the expression profile of growth-related (igf1a, igf2a) genes. Additionally, the daily cycle of water temperature (warmer at day and cooler at night) is another factor that has to be considered. The response to any acute stressor is not only species dependent, but also depends on the time of the day when the stress occurs: nocturnal species show higher responses when stressed during day time, whereas diurnal fish respond stronger at night. Melatonin administration in fish has sedative effects with a reduction in locomotor activity and cortisol levels, as well as reduced liver glycogen and dopaminergic and serotonergic activities within the hypothalamus. In this paper, we are reviewing the role of environmental cycles and biological clocks on the entrainment of daily rhythms in the HPI axis and stress responses in fish.

The brain-pituitary-gonadal (BPG) axis regulates the activation of the endocrine machinery that triggers reproduction, which is a typical rhythmic process. In this research we focused on investigating the daily expression rhythms of the key reproductive genes involved in the BPG axis and the liver of zebrafish. To this end, male and female zebrafish were subjected to a stimulating photoperiod with a 14 h light:10 h dark cycle. Brain, pituitary and gonads, as well as female liver samples, were taken every 4 h during a 24 h cycle. The results revealed that most genes exhibited statistically significant daily rhythms. Most of the brain reproductive genes (gnrh2, gnrh3, kiss1, kiss2 and gnrhr3) displayed a daily rhythm of expression with a nocturnal acrophase (between Zeitgeber Time [ZT] 14:34 h and ZT18:34 h, lights off at ZT = 14 h). The male kiss2 gene presented neither significant rhythms nor daily variations, while the male gnrh3 and female kiss2 genes exhibited diurnal peaks of expression at ZT06:34 h and ZT04:34 h, respectively. In contrast, the pituitary genes (fshβ, lhβ, gnrhr2) showed daily rhythms of expression with an acrophase during the light phase (between ZT02:10 h and ZT10:35 h). The female gnrhr3 gene exhibited neither significant rhythms nor daily variations. The male gnrhr3 gene presented a nocturnal acrophase (ZT14:32 h). The gonad genes (star, cyp17a1, 20βhsd, lhr, fshr, cyp19a1a, foxl2, amh, dmrt1 and 11βhsd) revealed statistically significant daily rhythms with nocturnal acrophases, except for female cyp17a1a (ZT06:21 h) and 20βhsd (ZT05:19 h). Lastly, the female liver genes presented daily rhythms with a maximum peak of expression around the transition phase from darkness to light (ZT01:00 h for erα and at ZT23:09 h for vtg2). These findings are consistent with the daily reproduction rhythms displayed by zebrafish, which are timed by the reproductive axis. Considering that reproductive success is critical for survival of the species, the knowledge of the rhythms of the endocrine BPG machinery provides useful information to understand the reproduction process and to establish optimal protocols and conditions for reproductive treatments.

Despite numerous studies about fish nutrition and lipid metabolism, very little is known about the daily rhythm expression of lipogenesis and lipolysis genes. This research aimed to investigate the existence of daily rhythm expressions of the genes involved in lipid metabolism and their synchronization to different light/dark (LD) and feeding cycles in zebra fish liver. For this purpose, three groups of zebra fish were submitted to a 12:12 h LD cycle. A single daily meal was provided to each group at various times: in the middle of the light phase (ML); in the middle of the dark phase (MD); at random times. After 20 days of acclimation to these experimental conditions, liver samples were collected every 4 h in one 24-h cycle. The results revealed that most genes displayed a significant daily rhythm with an acrophase of expression in the dark phase. The acrophase of lipolytic genes (lipoprotein lipase - lpl, peroxisome proliferator-activated receptor - pparα and hydroxyacil CoA dehydrogenase - hadh) was displayed between ZT 02:17 h and ZT 18:31 h. That of lipogenic genes (leptin-a - lepa, peroxisome proliferator-activated receptor - pparγ, liver X receptor - lxr, insulin-like growth factor - igf1, sterol regulatory element-binding protein - srebp and fatty acid synthase - fas) was displayed between ZT 15:25 h and 20:06 h (dark phase). Feeding time barely influenced daily expression rhythms, except for lxr in the MD group, whose acrophase shifted by about 14 h compared with the ML group (ZT 04:31 h versus ZT 18:29 h, respectively). These results evidence a strong synchronization to the LD cycle, but not to feeding time, and most genes showed a nocturnal acrophase. These findings highlight the importance of considering light and feeding time to optimize lipid metabolism and feeding protocols in fish farming.

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects RIDIGEST (AGL2011-23722) and AQUAGENOMICS (Consolider-Ingenio Program CDS2007-0002) with FEDER/ERDF contribution.

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.

The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p < .05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT] = 14.5 and 14.4 h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT = 1.2, 2.4, and 3.4 h, respectively), and crhbp and lactate at the end of the dark phase (ZT = 22.3 and 23.0 h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2 ± 10.7 ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6 ± 11.1 ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture.

The light–dark cycle and feeding can be the most important factors acting as synchronizers of biological rhythms. In this research we aimed to evaluate synchronization to feeding schedule of daily rhythms of locomotor activity and digestive enzymes of tilapia. For that purpose, 120 tilapias (65.0 ± 0.6 g) were distributed in 12 tanks (10 fish per tank) and divided into two groups. One group was fed once a day at 11:00 h (zeitgeber time, ZT6) (ML group) and the other group was fed at 23:00 h (ZT18) (MD group). The fish were anesthetized to collect samples of blood, stomach and midgut at 4-hour intervals over a period of 24 h. Fish fed at ML showed a diurnal locomotor activity (74% of the total daily activity occurring during the light phase) and synchronization to the feeding schedule, as this group showed anticipation to the feeding time. Fish fed at MD showed a disruption in the pattern of locomotor activity and became less diurnal (59%). Alkaline protease activity in the midgut showed daily rhythm with the achrophase at the beginning of the dark phase in both ML and MD groups. Acid protease and amylase did not show significant daily rhythms. Plasma glucose showed a daily rhythm with the achrophase shifted by 12 h in the ML and MD groups. These results revealed that the feeding time and light cycle synchronize differently the daily rhythms of behavior, digestive physiology and plasma metabolites in the Nile tilapia, which indicate the plasticity of the circadian system and its synchronizers.

Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase–activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28–0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture., This research was funded by projects AGL2010-22139-C03-01 and AGL2013-49027-C3-1-R granted by the Spanish Ministry of Economy and Competitiveness (MINECO) to F.J. Sánchez-Vázquez, and by project AGL2013-48835-C2-1-R granted by MINECO to J.M. Mancera. J.F. López-Olmeda was funded through a research fellowship granted by MINECO (Juan de la Cierva Program).

14 pages, 5 figures, 2 tables, Fish present daily and seasonal rhythms in spawning and plasmatic levels of steroids that control reproduction. However, the existence of the rhythms of expression of the genes that underlie the endocrine mechanisms responsible for processes such as steroidogenesis and reproduction in fish have still been poorly explored to date. Here we investigated the daily pattern of the expression of key genes involved in sex steroid production that ultimately set the sex ratio in fish. Adult zebrafish were maintained under a 12:12 h light-dark cycle at a constant temperature of 27°C and were sampled every 4 h during a 24-hour cycle. The expression of key genes in the gonads and brains of female and male individuals were analyzed. In gonads, the expression of aromatase (cyp19a1a, ovarian aromatase) and the antimüllerian hormone (amh, testis) was rhythmic, with almost opposite acrophases: ZT 5:13 h (in the light phase) and ZT 15:39 h (at night), respectively. The expression of foxl2 (forkhead box L2) was also rhythmic in the ovary (acrophase located at ZT 5:02 h) and the expression of dmrt1 (doublesex and mab-3-related transcription factor 1) was rhythmic in testes (acrophase at ZT 18:36 h). In the brain, cyp19a1b (brain aromatase) and cyp11b (11beta-hydroxylase) presented daily differences, especially in males, where the expression peaked at night. These results provide the first evidence for marked time-of-the-day-dependent differences in the expression of the genes involved in sex ratio control, which should be considered when investigating processes such as reproduction, sex differentiation and steroidogenesis in fish, This research was funded by Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project “Cronosolea” AGL2010-22139-C03-01 and “Solembryo” AGL2013-49027-C3-1-R, cofunded with FEDER fund; and by Project 18963/JLI/13 granted by the Agencia de Ciencia y Tecnología de la Región de Murcia (Fundacion Seneca) and a research fellowship granted by MINECO (Juan de la Cierva Program) to JFLO

Biological rhythms are driven by circadian oscillators, which are ultimately controlled by the cyclic expression of clock genes. Cryptochromes (CRY), blue light photoreceptors, belong to the negative elements of the transcriptional feedback loop into the molecular clock. This paper describes the cloning and characterization of two cryptochromes (cry1 and 2) in European seabass, which is considered an interesting chronobiology model due to its dual (diurnal/nocturnal) behavior. The cloned cDNA fragments encoded for two proteins of 567 and 668 amino acids, which included the FAD-binding and the DNA-photolyase domains. Moreover, both proteins had a high homology with cryptochrome proteins (Cry) of other teleost fish. These cry1 and 2 genes were expressed in several tissues of seabass (brain, liver, heart, retina, muscle, spleen, gill and intestine). In addition, the daily expression of cry1 was rhythmic in brain, heart and liver with the acrophase around ZT 03:15 h (after the onset of lights). Similarly, the cry2 daily expression was rhythmic in liver, peaking at ZT 03:28 h, whereas in brain the acrophase was at ZT 11:08 h (shortly prior to the offset of lights). These findings provide new elements to help understanding the functioning of the molecular clock of seabass.

Seabass is a fish species with dual (diurnal/nocturnal) feeding behavior, although little is known about changes in its molecular clock, physiology and metabolism linked to this dual behavior. In the research described here possible differences in clock gene expression in central (brain) and peripheral (liver) oscillators, and in physiology (blood glucose and amylase activity in mid-intestine) were studied in seabass with diurnal or nocturnal self-feeding patterns under LD 12:12h (light:dark) (lights on=Zeitgeber Time (ZT) 00:00h). The results revealed that per1 expression in brain shows daily rhythmicity with the acrophase (Φ) around the lights offset (ZT 12:00h, Cosinor, p0.01) in both diurnal and nocturnal seabass. In liver, per 1 daily levels of expression were higher in diurnal fish (univariate GML, p0.02). Daily blood glucose variations were observed in both groups (ANOVA I, p0.01), with higher glucose levels occurring at night in nocturnal as well as in diurnal fish, although only diurnal seabass displayed a significant daily rhythm (Φ=ZT 16:52h, Cosinor, p0.02). The highest values of amylase activity coincided with the feeding-phase of fish; that is, in nocturnal seabass the maximum was reached at ZT 18:00h (ANOVA I, p0.01), whereas in diurnal seabass the Φ was ZT 03:39h (Cosinor, p0.02). In short, our findings indicated that the feeding rhythm (diurnal vs. nocturnal) strongly influenced the daily patterns of digestive function and clock gene expression in the liver (feeding-entrained clock), but not in the brain (light-entrained clock).

Circadian rhythms are common in a wide variety of physiological processes. Our knowledge of biological rhythms in fish can be exploited to improve some key aspects of aquaculture such as feed optimization and fish welfare. The aim of this paper was to investigate daily rhythms in behavioural and physiological variables of the European eel. To this end, we first analysed the daily rhythms in locomotor activity measured by means of infrared photocells and feeding activity by means of self-feeders. The eels were then submitted to scheduled feeding (single meals once a day at a fixed time), and the levels of cortisol, glucose and digestive enzymes were analysed. Locomotor activity was predominantly nocturnal (63 % of daily activity occurring at night) with peaks located around light transitions, whereas feeding activity was less clearly defined. Cortisol and glucose showed daily rhythms, with acrophases located in the middle and at the beginning of the light phase, respectively, which should be carefully considered when using these parameters as welfare indicators. Significant variations were found during the day in acid protease levels, which increased in anticipation of mealtime. Taken together, these results describe for the first time a picture of the circadian physiology of the European eel, providing hints to improve feeding methods and welfare assessment.

An extensive network of endogenous oscilla- tors governs vertebrate circadian rhythmicity. At the molec- ular level, they are composed of a set of clock genes that participate in transcriptional-translational feedback loops to control their own expression and that of downstream out- put genes. These clocks are synchronized with the environ- ment, although entrainment by external periodic cues remains little explored in Wsh. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal XatWsh Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high iden- tities with their respective teleost clock genes, and Per- Arnt-Sim or basic helix-loop-helix binding domains were recognized in their primary structure. They showed a wide- spread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression proWles were inverted in retina and optic tectum compared to liver. Such diVerences suggest the existence of tissue-dependent zeit- gebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their diVerent phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity.

The pineal organ of fish is a photosensory and neuroendocrine epithalamic structure that plays a key role in the temporal organisation of physiological and behavioural processes. In this study performed in the European sea bass, Dicentrarchus labrax, we provided an in-depth description of the macroscopic and microscopic anatomy of the pineal organ and identified the presence of photoreceptor and presumed melatonin-producing cells using histological and immunohistochemical techniques. In addition, we analysed in the pineal the day–night expression (using quantitative real-time PCR) of two key enzymes in the melatonin-synthesising pathway; arylalkylamine-N-acetyltransferase 2 (AANAT2) and hydroxyindole-O-methyltransferase (HIOMT). The pineal complex of sea bass consisted of a narrow and short pineal stalk that adopts a vertical disposition, a small-sized pineal end vesicle firmly attached to the skull by connective tissue, a parapineal organ and a convoluted dorsal sac. Immunohistochemical study showed the presence of abundant serotonin-positive cells. Cone opsin-like and rod opsin-like photoreceptor cells were also evidenced in the pineal stalk and vesicle. Both Aanat2 and Hiomt were expressed in sea bass pineal organ. Aanat2 exhibited higher nocturnal transcript levels, while no significant day–night differences were found for Hiomt. These results, together with ongoing studies analysing neural and neurohormonal outputs from the pineal organ of sea bass, provide the basic framework to understand the transduction integration of light stimulus in this relevant species for marine aquaculture.

To investigate daily feeding rhythms in zebrafish, the authors have developed a new self-feeding system with an infrared photocell acting as a food-demand sensor, which lets small-size fish such as zebrafish trigger a self-feeder. In this paper, the authors used eight groups of 20 fish. Locomotor activity rhythms were also investigated by means of infrared sensors. Under a 12 h:12 h light (L)-dark (D) cycle, zebrafish showed a clear nocturnal feeding pattern (88.0% of the total daily food-demands occurring in the dark phase), concentrated during the last 4 h of the dark phase. In contrast, locomotor activity was mostly diurnal (88.2% of total daily activity occurring in the light phase). Moreover, both feeding and locomotor rhythms were endogenously driven, as they persisted under free-running conditions. The average period length (τ) of the locomotor and feeding rhythms was shorter (τ = 22.9 h) and longer (τ = 24.6 h) than 24 h, respectively. During the time that food availability was restricted, fish could only feed during ZT0-ZT12 or ZT12-ZT16. This resulted in feeding activity being significantly modified according to feeding time, whereas the locomotor activity pattern remained synchronized to the LD cycle and did not change during this trial. These findings revealed an independent phasing between locomotor and feeding activities (which were mostly nocturnal or diurnal, respectively), thus supporting the concept of multioscillatory control of circadian rhythmicity in zebrafish.

The regulation of feed intake is very complex and involves interaction among the circadian and homeostatic control systems within the central nervous system, the gastrointestinal tract and the environment. The hypothalamus, which receives, integrates and transmits relevant internal and external signals, is recognized as the primary centre of regulation of feed intake. The neuroendocrine factors that originate from the hypothalamus either stimulate or inhibit feed intake so that nutritional demands of the organism can be ful¢lled and energy balance can be achieved. Appetite regulation is a physiological mechanism in which a variety of neurohormones interact and ¢sh show diierent feeding behaviour (e.g. diurnal, nocturnal). This complicated system is very sensitive to any disturbance. Fish in farms and ¢sh in a natural environment are equipped with the same combination of neurohormones to regulate feed intake, but they meet diierent challenges, particularly with regard to the type of feed and feeding schedule. In this review, the neurohormonal regulation of feed intake is analysed in ¢sh in terms of entrainment of their circadian feeding rhythms and while exposed to diierent stressors in captivity.

Feeding cycles entrain biological rhythms, which enable animals to anticipate feeding times and so maximize food utilization and welfare. In this article, the effect of mealtime was investigated in two groups of sea bream (Sparus aurata): one group received a single daily meal at random times during the light period (random daytime feeding, RDF), whereas the other group received the meal during the light period but at the same time (scheduled daytime feeding, SDF). All the fish showed diurnal behavior, although the SDF group showed a lower percentage of diurnalism (84.4% vs. 79.5% in RDF and SDF respectively) and developed food anticipatory activity some hours before the mealtime. In addition, the mean daily locomotor activity of the RDF group was significantly higher than that of the SDF group (3132 vs. 2654 counts/day, respectively). Although the mean weight differed between both groups on day 30 (115.7 g and 125.6 g in RDF and SDF respectively), these differences had disappeared by day 60. Plasma cortisol and glucose significantly differed in both groups (cortisol: 71.8 vs. 8.7 ng/ml, glucose: 53.7 vs. 43.8 mg/dl in RDF and SDF, respectively), whereas lactate did not differ significantly. The results obtained suggest that altering the feeding time (scheduled vs. random) affects the behavior and physiology of sea bream, indicating that a single daily feeding cycle (compared to random) is beneficial for fish welfare because they can prepare themselves for the forthcoming feed.

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B(max)) at mid-light (ZT06) and mid-dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2-[(125)I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid-light (189.5+/-46 pg/ml) than mid-dark (455.5+/-163 pg/ml). Values of B(max) were statistically significantly higher in the optic tectum (5.6+/-0.6 and 12.3+/-1 fmol/mg prot, at mid-light and mid-dark, respectively) and in the cerebellum (7.7+/-1.1 and 10.6+/-1.3 fmol/mg prot, at mid-light and mid-dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down-regulation.

The present research aimed to investigate the existence of clock gene expression rhythms in tilapia, their endogenous origin, and how light and feeding cycles synchronize these rhythms. In the first experiment, two groups of fish were kept under an LD cycle and fed at two different time points: in the middle of the light (ML) or in the middle of the dark (MD) phase. In the second experiment, fish fed at ML was fasted and kept under constant lighting (LL) conditions for 1 day. In both experiments, the samples from central (optic tectum and hypothalamus) and peripheral (liver) tissues were collected every 3 h throughout a 24 h cycle. The expression levels of clock genes bmal1a, clock1, per1b, cry2a, and cry5 were analyzed by quantitative PCR. All the clock genes analyzed in brain regions showed daily rhythms: clock1, bmal1a, and cry2a showed the acrophase approximately at the end of the light phase (ZT 8:43-11:22 h), whereas per1b and cry5 did so between the end of the dark phase and the beginning of the light phase, respectively (ZT 21:16-4:00 h). These rhythms persisted under constant conditions. No effect of the feeding time was observed in the brain. In the liver, however, the rhythms of clock1 and cry5 were influenced by feeding, and a shift was observed in the MD fish group (ZT 3:58 h for clock1 and 11:20 h for cry5). This study provides the first insights into the molecular clock of tilapia, a very important fish species for aquaculture. It also reveals the endogenous origin of clock gene rhythms and the ability of feeding time to shift the phase in some clock genes in the peripheral, but not the central, oscillator.

The melatonin daily rhythm provides the organism with photoperiod-related information and represents a mechanism to transduce information concerning time of day. In addition, the duration and amplitude of the nocturnal elevation gives information about duration and thus the time of year. In this study, we investigate the existence of an annual rhythm of plasma melatonin in the Senegal sole. Differences in plasma melatonin levels between fish kept at a controlled temperature (17-20 degrees C) and those exposed to the environmental temperature cycle (11.5-25 degrees C) were also examined throughout the year. Spawning was registered in both groups to determine the time of year in which reproductive rhythms occurred. Our results pointed to the existence of an annual rhythm of plasma melatonin at mid-darkness (MD), with the highest levels (203 +/- 44 pg/mL) observed when water temperature reached 25 degrees C. Water temperature influenced nocturnal, but not diurnal melatonin. Daily melatonin rhythms showed seasonal differences, with higher mean nocturnal levels during the summer solstice (138 +/- 19 pg/mL) and autumn equinox (149 +/- 49 pg/mL). When animals were kept at a constant temperature throughout the year, plasma melatonin levels differed from those observed in fish exposed to the environmental temperature cycle. Regarding the reproductive rhythms, spawning was observed at the end of spring in sole kept under natural temperature conditions, whereas no spawning at all was registered in sole reared at a constant temperature. In short, both photoperiod and temperature affected melatonin production in the Senegal sole, transducing seasonal information and controlling annual reproductive rhythms.

Melatonin has several known physiological functions, the main one being synchronization of daily and seasonal rhythms. In addition, melatonin has been reported to influence food intake and behavioral rhythms with varying results depending on the species. The aim of this research was to evaluate the effects of intraperitoneal melatonin injection on food intake and locomotor activity in two different fish species: goldfish (diurnal) and tench (nocturnal), under different light regimes: constant light (LL) conditions or LD 12:12, with melatonin administration at mid-light (ML), mid-dark (MD), and after a 1-h light pulse at MD. In addition to these acute tests, in the case of goldfish we also investigated the effects of daily melatonin administration for 1 week. Our results indicated that acute melatonin administration significantly decreased goldfish food intake (16-52% inhibition, depending on the light regime) and locomotor activity (55-100%), with the chronic treatment inducing a similar total food intake inhibition that persisted for 7 days. In tench, a nocturnal fish species, acute melatonin administration at MD and ML reduced food intake (37% and 29%, respectively), while locomotor activity was not affected at MD and slightly increased at ML. Taken together, these results indicated that melatonin reduced food intake in both species, while its effects on locomotor activity depended on the time of administration (light or dark phase) and the activity patterns of the species.

Melatonin has a number of physiological functions in addition to light-dark transduction. In recent years, many in vivo and in vitro studies in rodents have revealed an important antioxidant activity of melatonin, both directly and indirectly. Nevertheless, the potential effects of melatonin as an antioxidant in fish remain unknown. The aim of this research was to evaluate the capacity of melatonin injections (3 mg/kg) to attenuate oxidative damage after submitting goldfish to oxidative stress caused directly by hydrogen peroxide (H2O2) baths and indirectly by hypoxia and subsequent reoxygenation, as well as the locomotor activity. The results revealed that melatonin decreased lipid damage in muscle after hypoxia/reoxygenation (1.22 vs. 2.27 nmoles lipid peroxides/g tissue), but not in liver. Mortality caused by oxidative stress was not attenuated by melatonin. Surprisingly, melatonin caused an increase of mortality (50 vs. 95%) when administered before hypoxia. Locomotor activity was also affected by melatonin but not by the administration of the vehicle, suggesting a sedative effect of melatonin in goldfish. In conclusion, melatonin administration provoked slight effects on lipid peroxidation and mortality resulting from oxidative stress, with reduction of locomotor activity in relation to the vehicle.

Seasonal and circadian changes have been observed in dietary selection and feeding behavior of fish. It is known that the light-dark cycle is the principal mediator of the production of pineal-derived melatonin in fish, and also that the digestive tract synthesizes and secretes melatonin and indeed is the principal extrapineal source of this secretor product, suggesting that melatonin could be involved in the processes of feeding and/or digestion. Fish are capable of regulating their energy intake from separate sources of macronutrients, but the mechanisms of this selection process are unknown. In the present paper, we explored the effect of melatonin administered orally to European sea bass on their selection of encapsulated macronutrients. Melatonin doses of 0.1, 0.5, and 2.5 mg/kg body weight were administered in gelatin capsules. The voluntarily ingested melatonin was absorbed into the fish's plasma, 45 min after the administration reaching a level depending on the dose that was up to 26 times greater than the controls with the highest dose of melatonin. The indole produced a dose-dependent inhibition of total food intake of 9, 26, and 34%, respectively, and also modified the pattern of macronutrient selection. Carbohydrate intake was significantly reduced (by 17, 33, and 42% for the three doses, respectively), but the observed reductions in fat and protein intake were not statistically significant. On the contrary, fat percentage significantly increased for the highest dose, but no changes were observed in the protein or carbohydrate percentages for any of the melatonin doses. In conclusion, orally administered melatonin affected both the amount of food consumed and the pattern of macronutrients selected. This is the first evidence for the existence of neurohumoral mediators involved in the selection of macronutrients in fish.

Reproduction in fish is cyclical and timed to guarantee the survival of the offspring. Seasonal variations in reproductive hormones of fish have been deeply investigated in fish over the last years. However, there are few studies regarding the daily changes in reproductive hormone profiles in teleosts. The aim of the present research was to investigate the effects of photoperiod manipulation on melatonin and reproductive hormones (pituitary sbGnRH, pituitary LH and plasma LH, testosterone [T], and 11-ketotestosterone [11KT]) daily rhythms in male sea bass, kept in net cages under farming conditions in winter (9L:15D). Fish were distributed in two groups, one under constant long photoperiod (18L:6D) and the other under natural photoperiod. The photoperiod strongly influenced the daily melatonin profile, so that the duration of the nocturnal melatonin rise was longer in the control group than in the group exposed to the artificial photoperiod (18L:6D). A daily rhythm was observed in the pituitary sbGnRH profile in both groups, showing the lowest levels during the dark period. A daily rhythm of pituitary LH was detected in the control group, which was suppressed in the group under long photoperiod. Daily variations in plasma LH were observed, the highest levels being found in the dark phase in both groups, although this profile was significantly altered by artificial light, maintaining a fixed relationship between the first nocturnal rise of melatonin and the nocturnal peaks of plasma LH in both groups. Plasma T levels showed significant fluctuations in their daily cycle following a sinusoidal pattern with an acrophase around sunrise in both groups, without any influence of light regime. No significant daily variations in plasma levels of 11-KT were observed in none of the groups. Our results provide the first evidence of the presence of daily variations in pituitary sbGnRH content, pituitary and plasma LH, and plasma T in sea bass. Artificial lights suppressed the circulating melatonin and significantly affected the daily rhythm of LH storage and release., This research was funded by the MCYT Projects No. 1FD97-1699 and AGL2001-0593-(03-01) to Dr. F.J. Sánchez-Vázquez and Dr. S. Zanuy. Authors thank CULMAREX, S.A. for offering us their facilities and its R&D assistant (M.C. Marı́n), for her help.

The transduction of seasonal information from the environment (i.e., photoperiod and water temperature) into melatonin rhythms was studied in sea bass. Plasma and ocular melatonin (N-acetyl-5-methoxytryptamine) was determined in autumn, winter, spring and summer (experiment 1) under natural culture conditions, and in the summer and winter solstices under both natural and "6-month out-of-phase" photoperiods (experiment 2). At each sampling, 48 sea bass were sacrificed at a rate of 6 fish every 3 hr and the level of melatonin was determined in plasma and eye cup samples by ELISA. In experiment 1, significant diel changes were observed in plasma melatonin, with nocturnal melatonin varying from 144 pg/mL (summer) to 23 pg/mL (autumn), while diurnal melatonin remained low, around 8 pg/mL throughout the year. In experiment 2, the photoperiod length was shown to control the duration of the nocturnal melatonin rise, while the water temperature determined the amplitude of the melatonin rhythm. Ocular melatonin peaked during daytime in autumn and winter, but no significant changes were detected in summer and spring. In conclusion, plasma melatonin rhythms in sea bass reflect the pineal capacity to integrate seasonal information and supply precise calendar information, which may synchronize different physiological processes such as annual reproduction and feeding rhythms.

The influence of two different fasting periods on macronutrient self-selection during refeeding was studied in sea bass. Twelve groups of three sea bass were kept under laboratory conditions with a 12:12 light–dark cycle and at 22.5°C. Each aquarium was provided with three self-feeders containing three different diets made up of two macronutrients: protein–fat, protein–carbohydrate, and carbohydrate–fat. The fish were allowed to acclimatise to the diets and laboratory conditions in 4 weeks, during which they selected a baseline diet of 51% protein, 16.5% carbohydrate, and 32.5% fat, in terms of digestible energy. The fish were then deprived of food for 6 days, after which they were permitted to refeed. When food demands stabilised, they were subjected to total food deprivation for another 2 weeks. The total energy demanded from all the macronutrients increased significantly during the first day after both fasting periods. The proportion of macronutrient self-selection after fasting periods remained unchanged in respect to the baseline, except for protein energy which remained high in the two first days of refeeding after the second fasting period (the 51% of total demanded energy from protein during the baseline period rising to 60%). In short, after two short fasting periods of different length, sea bass showed a compensatory ingestion of energy, with a slightly increased demand from protein after the longest fasting period.

Most animals when presented with food on a periodic basis develop feeding anticipatory activity (FAA). The objective of the present study was to investigate the synchronization of activity rhythms to light and feeding in single goldfish given a single daily meal and exposed to different light regimes. In the 1st experiment, the group of fish fed at the end of the day showed a longer FAA (228 ± 27 min) than the group fed 1 h after lights-on (97 ± 31 min). In the 2nd experiment, goldfish under conflicting zeitgebers, LD (T = 26) and feeding (T = 24) cycles, initially synchronized to the light cycle although feeding time gradually assumed greater importance as the experiment progressed. In the 3rd experiment, after altering the feeding cycles by advancing or delaying feeding time by 4 h in LD 0.25:23.75, most goldfish resynchronized to the feeding stimulus and developed FAA. In conclusion, the authors' results revealed that FAAin goldfish is driven by an endogenous timing system that is entrainable by periodic feeding and influenced by the light regime.

Under free-running conditions, most rainbow trout displayed circadian feeding rhythms, although the expression of circadian rhythmicity depended on the experimental condition: 16·7% of fish under constant dim light (LL dim), 66·1% under a 45 :45 min light-dark cycle (LD pulses), and 83·8% under constant light (LL). Under LD pulses, the period length of the free-running rhythms for feeding was significantly shorter (21·9 ± 0·7 h, n=8) than under LL (26·2 ± 0·3 h, n=10). Period length for locomotor activity under LL was 25·8 ± 0·6 h (n=4). Under LD conditions, the daily demand-feeding profile was always confined to the light phase and chiefly composed of two main episodes, directly after lights on (light elicited) and in anticipation to lights off (endogenous). Contrasting to feeding, the diel locomotor activity profile varied remarkably: a diurnal activity pattern at the bottom, while a clearly nocturnal pattern at the surface. These results contribute to a better understanding of feeding and locomotor rhythms of rainbow trout, providing evidence for the existence of a biological clock involved in their circadian control. This finding contrasts with the previously recorded lack of an endogenous oscillator in the pineal organ driving the rhythmic secretion of melatonin, which suggests different locations from the pineal for the circadian pacemakers in this species.

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream (Sparus aurata L.) submitted to air exposure for 30s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm of corticotropin-releasing hormone (crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression of crh-binding protein (crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i) cytochrome c oxidase 4 showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii) peroxiredoxin (prdx) 3 and 5 (prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii) uncoupling protein 1 showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses. © 2014 Informa Healthcare USA, Inc. All rights reserved., This research was supported by the project AQUAGENOMICS (Consolider-Ingenio 2010 Programme) and CRONOSOLEA from the Spanish Ministry of Economy and Competitiveness (MINECO).

Melatonin production by the fish pineal is under the control of photoperiod. In addition, in some fish species pineal melatonin production is self-sustained by an intrapineal circadian pacemaker, not in salmonids, though. The aim of this research was to test melatonin release in vitro by the pineal of sea bass held under LD 12:12 and DD conditions to determine the endogenous nature of melatonin rhythm. Circadian rhythmicity (τ = 28.3 h) was found since rhythmic melatonin release persisted on DD.

The effect of intracoelomic tagging of an acoustic telemetry transmitter (1.65% ratio of tag mass in the air to fish mass in the air) on behavioral (food intake and locomotor activity) and physiological (blood glucose and plasma cortisol) parameters of gilthead seabream was investigated. To this end, fish (289 ± 53 g, mean ± SD) were divided into 3 experimental groups: control (C), transmitter (T, inserted surgically) and sham group (S, subjected to surgery but without transmitter insertion). Blood was extracted during surgery and 9 days later. Throughout the trial, fish were fed by means of self-feeders and locomotor activity was measured by means of an infrared photocell. Two days after the first manipulation, a significant decrease in food intake could be observed in all the experimental groups. The fact that food intake was not affected after the second manipulation seems to indicate that a learning process took place for handling. The rhythmicity of feeding and locomotor activity was not affected by handling in any experimental group. However, group T showed increased plasma cortisol levels 9 days after surgery. In conclusion, while most behavioral parameters were not affected by handling, the plasma cortisol levels of seabream 9 days after insertion of the transmitter indicated a physiological impact that should be taken into account in long-term radiotracking studies, since such an operation could have negative effects on wild individuals after the end of the tracking experiments.

18 p., 5 figures, 1 table and references, Photoperiod and temperature are known as the main synchronizers of seasonal reproduction in fish. This paper studied the role of photoperiod on the synchronization of F1 Senegal sole reproduction rhythms. Fish were maintained under constant short-photoperiod (9L:15D) from the winter solstice onwards (experimental group) or under naturally-changing photoperiod (control group), and water temperature naturally oscillated in both groups. Blood samples were collected during the reproduction season at pre-spawning (March), spawning (April) and post-spawning (May) to determine the endocrine status. Spawning events and egg quality parameters were also monitored. The results revealed a significant increase in nocturnal melatonin concentration from March to May in the control group, while in the experimental group such seasonal change did not occur. As to plasma levels of vitellogenin, testosterone, estradiol and 11keto-testosterone, differences between groups were found mostly in March, while in April and May levels were often similar. Spawning was observed in both groups, although the experimental group started slightly earlier and also finished earlier than the control group, perhaps as a result of the increase in sex steroids and VTG observed at pre-spawning. Briefly, reproduction rhythms persisted in the absence of the natural lengthening of photoperiod, although photoperiod manipulation altered the seasonal modulation of melatonin, increased sex steroids and vitellogenin at pre-spawning, and slightly advanced the timing of spawning., This work was financed by the MEC (project Reprosolea AGL2004-07984-CO2-01/ACU granted to FJSV and project AGL2006-13777-C03-01 to EM) and by SENECA (Project 05690/PI/07 to FJSV). CO received a FPI Fellowship from the MEC.

28 p., 6 figures and references, The effect of water temperature during the development of fish larvae on sex differentiation is well known, but not so well known is the impact of the daily thermocycles. Our aim was to investigate the effect of early exposure of Senegal sole larvae to different temperature cycles on gonad development, sex ratio and sex steroid (11-ketotestosterone, 11-KT; estradiol, E2; and testosterone, T) content in muscle extracts of juveniles. From 1 to 97 DPH (days post-hatching) fish larvae and post larvae were subjected to three temperature regimes: TC (Thermophase-Cryophase); CT (Cryophase-Thermophase); and constant temperature. In fish exposed to TC, sex determination occurred earlier, since 90% of soles were males/females at 110 DPH, whereas 45% of fish under CT were undifferentiated at that time. Fish under TC showed the highest growth rates, followed by fish under constant temperature and by fish under CT, the differences being statistically significant between the TC and CT groups. Regarding sex ratio, juveniles exposed to TC showed a higher proportion of females than fish under CT or constant temperature. Under TC, fish showed the highest concentration of E2, while 11-KT concentration was highest in fish under CT and constant temperature. Fish under constant temperature and CT showed higher T levels than those under TC. These results provide the first insights into the effect of daily thermocycles on sex differentiation in fish, and underline the key role of natural environmental cycles on the control of sex ratios during larval development, which may be applied to the manipulation of sex ratio in aquaculture., This research was funded by the Spanish Ministry of Science and Innovation (MICINN) by projects “Circasole” AGL2007-66507-C02-02 and “Reprosole” AGL2006-13777-C03. AQUAGENOMICS 28502 (Consolider-Ingenio Program), and FUNDACIÓN SENECA by project 05690/PI/07 awarded to Francisco Javier Sánchez Vázquez and Generalitat Valenciana project ACOMP/2006/202 to Evaristo Mañanós. Thanks are also due to COST ACTION 867 (Fish Welfare) and FA0801 (Larvanet), where part of these results were presented and discussed

Food provided on a periodic basis can act as a potent synchronizer, being a stronger zeitgeber than light for peripheral oscillators in mammals. In fish, however, little is known about the influence of feeding time on the circadian pacemaker and the relationship between central and peripheral oscillators. The aim of this research was to investigate the influence of mealtime on the activity rhythms, and on central (brain) and peripheral (liver) oscillators in zebrafish. The authors tested different feeding times under a light-dark (LD) cycle and the endogenous origin of food-anticipatory activity (FAA) by feeding zebrafish at a fixed time under constant bright-light conditions (LL). The authors then measured locomotor activity and the expression of the clock gene per1 in animals under a LD cycle and fed at random times during the light phase, with restricted feeding at the mid-light phase (ML) or with restricted feeding during the mid-dark phase (MD). Finally, the authors measured locomotor activity and per1 expression in fish maintained under LL under either random feeding or scheduled feeding. Zebrafish displayed FAA in all the groups fed at a fixed time but not when feeding was randomly scheduled. Under LL, fish entrainment persisted, and when released under fasting conditions FAA free-ran with a circa-24-h period. The expression of per1 in the brain of fish under LD showed a daily rhythm with the acrophase (peak time) at the end of the dark phase regardless of feeding schedule. This brain rhythm disappeared in LL fish under both random feeding and scheduled feeding. Feeding at MD advanced the phase of per1 in the liver by 7 h compared with the ML-fed group phase (23:54 versus 07:23 h, respectively). In addition, under LL scheduled feeding entrained the rhythms of per1 expression in the liver. This study reveals for the first time that scheduled feeding entrains peripheral oscillators in a fish species, zebrafish, which is a powerful model widely used for molecular genetics and for the study of basic clock mechanisms of the vertebrate circadian system.

27 páginas, 5 figuras., Feeding cycles entrain biological rhythms, which enables animals to anticipate feeding times and so maximizes food utilization. In this article the effect of mealtime on locomotor activity, blood glucose, gastric pH and digestive enzymes was studied in two groups of seabream (Sparus aurata): one group received a single daily meal at random times either during the light or the dark (random feeding, RF), whereas the other group received the meal during the light period every day at the same time (periodic feeding, PF). PF fish showed strong synchronisation of locomotor activity to the light phase (97.9 ± 0.2% of their total daily activity during daytime). In addition, the locomotor activity rhythm of PF fish showed a statistically significant daily rhythm (p < 0.05) for a period of 24 h, whereas RF fish did not display a statistically significant rhythm. Blood glucose levels were higher in RF fish during the 8 h following feeding. Gastric pH showed a postprandial decrease in both groups, but RF fish showed a lower daily average value (4.31 ± 0.21 compared with 5.52 ± 0.20). Amylase and alkaline protease activity increased some hours before mealtime in PF fish, whereas amylase activity increased 1 h after feeding and alkaline protease showed no statistically significant differences in RF fish. Acid protease activity showed no statistically significant differences in any group. Taken together, these results demonstrate that altering the feeding time affects the physiology and behaviour of seabream, which have the capacity to prepare themselves for a forthcoming meal., The present research was funded by projects from the Spanish Ministry of Science and Innovation (MICINN) (project AGL2007-66507-C02-02), Fundación Séneca (project 05690/PI/07) and AQUAGENOMICS 28502 (Consolider-Ingenio Program) granted to F. J. Sánchez-Vázquez; and by a project from MICINN (Plan Nacional de Investigación Desarrollo e Innovación, project number AGL2007-64450-C02-01) granted to M. Yúfera.

International audience; With the exception of modern mammals, most vertebrate species possess two GnRH genes, GnRH-1 and GnRH-2. In addition, in many teleost fish, there is a third gene called GnRH-3. If the main function of GnRH-1 is unambiguously to stimulate gonadotropin release, the other two GnRH forms still lack clear functions. This is particularly true for the highly conserved GnRH-2 that encodes chicken GnRH-II. This GnRH variant is consistently expressed in neurons of the dorsal synencephalon in most vertebrate groups but still has no clear functions supported by anatomical, pharmacological, and physiological data. In this study performed on a perciform fish, the European sea bass, we show for the first time that the pineal organ receives GnRH-2-immunoreactive fibers originating from the synencephalic GnRH-2 neurons. This was shown through a combination of retrograde tracing and immunohistochemistry, using highly specific antibodies. Supporting the presence of GnRH-2 functional targets, RT-PCR data together with the in situ hybridization studies showed that the sea bass pineal gland strongly expressed a GnRH receptor (dlGnRHR-II-2b) with clear selectivity for GnRH-2 and, to a lesser extent, the dlGnRHR-II-1a subtype. Finally, in vitro and in vivo experiments demonstrate stimulatory effects of GnRH-2 on nocturnal melatonin secretion by the sea bass pineal organ. Altogether, these data provide, for the first time in a vertebrate species, converging evidence supporting a role of GnRH-2 in the modulation of fish pineal functions.

24 p., il., y bibliografía, The effect of the moon light cycle on plasma melatonin rhythms was examined in Senegal sole (Solea senegalensis) exposed to natural outdoor or artificial indoor lighting conditions. Furthermore, in a second experiment, the effect of the lunar cycle on vitellogenin and sex steroids (Testosterone, T; Estradiol, E2; 11-ketotestosterone, 11kt) was studied using mature individuals during reproductive season. In the first experiment, during full moon, plasma melatonin peaked at night in covered tanks (deprived of night illumination) from both outdoor (133.2 ± 12.8 pg ml-1) and indoor (190.6 ± 41.5 pg ml-1) groups. However, for fish in the open tanks, exposed to approximately 0.3 lux of illumination, nocturnal plasma melatonin was significantly reduced (p, This work has been financed by the Spanish Ministry of Education and Science (MEC) through project Reprosolea AGL2004-07984-CO2-01/ACU to FJSV and project AGL2006-13777-C03-01 to EM. CO received an FPI fellowship from the same institution.

Food availability is far from constant but tends to be cyclic, and fish therefore show a variety of circadian rhythms which can be entrained to feeding time. The aim of this study was to investigate the synchronization to mealtimes of behavioral (locomotor activity), metabolic (glucose) and endocrine (cortisol and thyroid hormones) daily rhythms in gilthead seabream. To this end, fish were reared under a 12:12 LD cycle and fed 1% of their body weight once a day either at mid-light (ML) or at mid-dark (MD) of the LD cycle. Fish synchronized their locomotor activity to the phase in which food was delivered, ML and MD fish displaying 86 ± 3% and 81 ± 1% of their total daily activity during daytime and nighttime, respectively. Daily variations of blood glucose were strongly synchronized to feeding time in both experimental groups, peaking 8 h after the meal. A postprandial cortisol peak was observed in both groups. In fish fed at MD the cortisol values were high during the 8 h following feeding, whereas in fish fed at ML cortisol levels returned to basal values within 4 h. In addition, MD fish showed a higher average daily cortisol value (24.75 ± 2.65 ng/ml) than ML fish (10.30 ± 2.18 ng/ml). Feeding time affected the magnitude of daily variations in the thyroid hormones. When the time of feeding was delayed by 12 h, a shift of the same magnitude could be observed in the glucose daily rhythm and a dramatic change in the cortisol levels of the ML-fed group compared with results mentioned above. In summary, gilthead seabream synchronized their locomotor activity to the phase when food was provided and showed different degrees of synchronization of their behavioral, metabolic and endocrine rhythms to feeding time. Since these parameters are used to evaluate stress responses and welfare in seabream, their daily rhythm and synchronization to light and feeding time should be taken into account.

Light and feeding cycles strongly synchronize daily rhythms in animals, which may, as a consequence, develop food anticipatory activity (FAA). However, the light/food entraining mechanisms of the central circadian oscillator remain unknown. In this study, we investigate the existence of FAA in seven groups of zebrafish subjected to a light/dark (LD) cycle or constant light (LL) and different feeding regimes (random, fasting, and feeding in the middle of the light phase or dark phase). The aim was to ascertain whether the daily rhythm of behavior and clock gene (per1 and cry1) expression in the zebrafish brain was entrained by the light and feeding regime. The results revealed that FAA developed in zebrafish fed daily at a fixed time, under LD and under LL. Zebrafish displayed locomotor activity mostly during the daytime, although the percentage of activity during the light phase varied depending on feeding time (ranging from 93.2% to 63.1% in the mid-light and mid-dark fed groups, respectively). However, the different feeding regimes failed to modify the daily rhythm of per1 and cry1 expression in the zebrafish brain under LD (approximate acrophases [peak times] at ZT22 and ZT4, respectively; lights-on = ZT0). Under LL, per1 and cry1 expression did not show significant daily rhythmicity, regardless of the feeding regime. These findings indicate that, although schedule-fed zebrafish developed FAA as regards locomotor activity, feeding had little effect on clock gene expression in whole brain homogenates, suggesting the feeding-entrainable oscillator may be located elsewhere or at specific brain sites.

In addition to light cycles, temperature cycles are among the most important synchronizers in nature. Indeed, both clock gene expression and circadian activity rhythms entrain to thermocycles. This study aimed to extend our knowledge of the relative strength of light and temperature as zeitgebers for zebrafish locomotor activity rhythms. When the capacity of a 24:20 degrees C (thermophase:cryophase, referred to as TC) thermocycle to synchronize activity rhythms under LL was evaluated, it was found that most groups (78%) synchronized to these conditions. Under LD, when zebrafish were allowed to select the water temperature (24 degrees C vs. 20 degrees C), most fish selected the higher temperature and showed diurnal activity, while a small (25%) percentage of fish that preferred the lower temperature displayed nocturnal activity. Under conflicting LD and TC cycles, fish showed diurnal activity when the zeitgebers were in phase or in antiphase, with a high percentage of activity displayed around dawn and dusk (22% and 34% of the total activity for LD/TC and LD/CT, respectively). Finally, to test the relative strength of each zeitgeber, fish were subjected to ahemeral cycles of light (T=25 h) and temperature (T=23 h). Zebrafish synchronized mostly to the light cycle, although they displayed relative coordination, as their locomotor activity increased when light and thermophase coincided. These findings show that although light is a stronger synchronizer than temperature, TC cycles alone can entrain circadian rhythms and interfere in their light synchronization, suggesting the existence of both light- and temperature-entrainable oscillators that are weakly coupled.

This research aimed to investigate melatonin rhythms in the pineal organ of two nocturnal fish species, sole and tench, which show high sensitivity to light. Pineal organs were cultured in vitro under an LD (12 h light:12 h dark) cycle to study the daily rhythmicity of melatonin release. In addition, the in vitro culture was performed under conditions of constant darkness (DD) to study the endogenous control of the rhythm. In the pineal organs cultured under an LD cycle, rhythmic melatonin release was evident in both species, with low values observed during the photophase (15.6+/-7.2 and 22.6+/-2.6 pg/mL for sole and tench, respectively) and high values coinciding with the scotophase (74.0+/-8.2 and 82.1+/-9.1 pg/mL, for sole and tench, respectively). Under LD, the rhythm had a period of 24 h (p0.001) and presented similar acrophases for both species, located around 9-10 h after lights off (2 and 3 h before the end of the dark phase). When the pineal organs were cultured under DD, the results differed between the species studied. A marked circadian rhythm in melatonin release by the pineal was registered in tench, with lower values during the subjective day, i.e. the period that was previously day (6.2+/-1.6 pg/mL) and higher values during the subjective night, i.e. the period that was previously night (20.4+/-5.5 pg/mL). The rhythm had a mean tau of 24.1 h (p0.01) and the acrophase was located around 12 h after lights off (the beginning of the subjective day), slightly later than that registered under LD conditions. In contrast, melatonin values in sole remained high during darkness (around 92.0+/-6.9 pg/mL) for four consecutive days, including subjective day periods. In short, these findings revealed that the rhythm of melatonin release in tench is under endogenous control by a circadian oscillator within the pineal organ, while no such pacemaker was evident in sole, which melatonin rhythm appeared to be exclusively light-driven.

Daily rhythms in glucose tolerance have been reported for several species of mammals, which seem to be linked to factors such as daily rhythms of insulin resistance and feeding habits. In this paper, we studied entrainment to a daily scheduled meal of blood glucose after carbohydrate intake (dextrin or glucose). After a meal containing dextrin, blood glucose showed different maximum concentrations (Cmax) and time to peak (Tmax) depending on mealtime, the greatest differences in Cmax being found 2 h after feeding. The highest Cmax (8.22 mmol/l) was obtained when mealtime was set in the middle of the light phase (ML), and the lowest Cmax (3.46 mmol/l) when goldfish were fed in the middle of the dark phase (MD). Cosinor analysis revealed a significant daily rhythm in dextrin tolerance with its acrophase around ML, amplitude of 1.99 mmol/l, and a mesor of 5.49 mmol/l. However, when the carbohydrate source in the meal was glucose, an inverse pattern was observed: higher blood glucose in goldfish fed at MD compared with ML (6.98 vs 4.32 mmol/l, respectively). Similar results were obtained when glucose was administered intraperitoneally, with higher values at MD than at ML (13.82 vs 9.54 mmol/l, respectively). Finally, no differences in amylase activity were observed in the gut after dextrin load at ML and MD (522 vs 446 U/mg protein), discarding the effect of digestive factors in the daily rhythm of tolerance. In conclusion, our results described for the first time a daily rhythm in tolerance to carbohydrate in a teleost fish, highlighting the impact of the time of day in glucose metabolism.

The use of a single diet with a well defined composition to feed fish throughout their life cycle is an oversimplification that probably does not respond to their metabolic requirements. For example, the seasonal reproduction that characterizes most fish species demands changes in nutritional requirements. Bearing this in mind, the macronutrient selection pattern was studied from January to August in twelve individually housed sea bass exposed to a constant photoperiod (12L:12D h) and temperature (23 ± 0.5 °C). The endogenous “seasonal” effect on food and energy intake regulation and macronutrient selection was determined, using protein (P), carbohydrate (CH), and fat (F) packaged separately into gelatine capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Energy intake changed monthly, the highest values being recorded in May and June and the lowest values in March and April. The preliminary results illustrated “seasonal” changes in the sea bass macronutrient selection pattern with, which showed a predominantly proteinic selection during April (53% P, 21% CH, 25% F) and lipidic in July (35% P, 19% CH, 42% F); the increase in fat selection from May to July being statistically significant. This is the first evidence supporting the existence of an endogenous rhythm in the “seasonal” energy regulation and macronutrient selection in fish through post-ingestive mechanisms and probably involving chemosensory detection in the gut and/or post-absorptive mechanisms, although the exact mechanisms involved have yet to be clarified., Sí

Teleost fish are able to adjust their energy intake when fed on pure macronutrient sources, although the exact mechanisms regulating macronutrient selection remain unknown. Since cholecystokinin (CCK) has been reported to modify macronutrient selection patterns in mammals, we explored the effect of CCK administered orally to European sea bass on the selection of separately encapsulated macronutrients. CCK doses of 0.05, 0.15 and 0.25 mg/kg BW administered in gelatine capsules for 5 consecutive days produced a significant inhibition of total food intake (21, 28 and 51%, respectively) at highest doses, evenly reducing the quantity of all the macronutrients ingested and, without affecting their relative proportions in the diet. Oral administration of proglumide, a non-specific CCK receptor antagonist, at doses of 5, 15 and 25 mg/kg BW, induced a quantitative total food intake increase of 2, 18 and 44%, respectively, and an increase of 52% in CH and 43% in P quantity ingested at highest dose. Co-administration of proglumide (25 mg/kg BW) and CCK (0.25 mg/kg BW) in a single preload capsule blocked the effects observed with CCK alone. In conclusion, orally administered CCK induced an anorexigenic effect on both total food and single macronutrient intake, an effect that is counteracted by the CCK antagonist proglumide.

The existence of food anticipatory activity (FAA) in animals subjected to daily feeding schedules seems to be mediated by a feeding-entrainable oscillator (FEO). Such an FEO may help in anticipating meal time and so optimizing food acquisition and nutrient utilization. In this study we investigated the existence of FAA and whether digestive enzymes, plasma cortisol, hypothalamic NPY and gastrointestinal tract (GIT) and plasma melatonin were entrained by periodic feeding in goldfish. We observed that periodically fed goldfish showed FAA in locomotor activity as well as in amylase and NPY. Alkaline protease and GIT melatonin were higher after feeding, whereas plasma cortisol levels were reduced. Plasma melatonin remained unmodified before and after meal time. These results suggested that scheduled feeding entrained both behavioral and certain physiological patterns in goldfish, FAA being of adaptive value to anticipate a meal and prepare the digestive physiology of fish.

Teleost fish are able to regulate their energy intake selecting from pure macronutrients sources, but the regulatory mechanisms involved in macronutrients selection remain unknown. Serotonin (5-HT) reduces food intake in mammals and fish and modifies the macronutrients selection pattern in mammals; however, no information is available about its role on macronutrients selection in fish. The aim was to determine the effect of orally administered 5-HT (0.1, 0.5 and 2.5 mg kg BW(-)(1)) into gelatine capsules on the subsequent macronutrient selection of sea bass, using for this purpose gelatine capsules including carbohydrates, protein, or lipids separately. The voluntary ingested 5-HT was released into the plasma of fish, reaching a level two times greater than the controls, 45 min after the ingestion of a capsule containing 2.5 mg kg BW(-1) of 5-HT. The indoleamine, at doses of 0.1, 0.5 and 2.5 mg kg BW(-1), produced a reduction in total food intake of 31%, 49% and 37%, respectively, compared to the baseline, modifying the macronutrient selection pattern. The percentage of fat selected was significantly reduced whereas the percentage of protein significantly increased after administration of highest dose, but no changes were observed in the proportion of carbohydrate for any 5-HT doses. In conclusion, oral administration of 5-HT affected both amount of food intake and pattern of macronutrients selected. This is the first evidence supporting a role of 5-HT as a neurohumoral mediator involved in macronutrients selection in fish.

The daily locomotor and melatonin rhythms of the Senegal sole, a benthonic species of increasing interest in aquaculture, are still unknown, despite the fact that such knowledge is of prime importance for optimising its production. The aim of the present research was therefore to investigate the daily rhythms of locomotor activity and melatonin in the Senegal sole. For this purpose, the individual locomotor activity rhythms of fish were registered using a photocell. Plasma and ocular melatonin rhythms were studied in animals reared in circular tanks placed in earth under an LD 12:12 light regime and 16–18 °C temperature range (spring equinox). Blood and eye samples were taken every 3 h during a complete 24-h cycle. The impact of a light pulse in the middle of the dark period (MD) on plasma melatonin was also studied. Locomotor activity was mainly nocturnal, with 84.3% of the total activity occurring during darkness. The levels of plasma melatonin were higher at night (55 pg/ml) than during the day (2 pg/ml), while ocular melatonin levels appeared to be arrhythmic. Both weight and melatonin content were found to be significantly higher in the left eye in relation to the right eye. A light pulse in MD provoked a significant decrease in plasma melatonin levels. In summary, photoperiod is a key factor in synchronising locomotor activity and melatonin rhythms in the Senegal sole, whose nocturnal habits should be taken into account for their rearing by aquaculture.

This study investigates the role of the pineal organ and lateral eyes (the two most important sources of melatonin in vertebrate species) on daily melatonin rhythms of sea bass, a fish exhibiting reversed melatonin profiles, as well as their contribution to circulating melatonin levels. To this aim, the pineal and/or the eyes were surgically removed (Exp. 1), the optic nerve sectioned and retinal dopaminergic neurons damaged with injections of 6-hydroxydopamine (Exp. 2), and the pineal or the eyes covered with aluminium foil (Exp. 3). The results show that plasma and ocular melatonin display opposing profiles. In Experiment 1, pinealectomized fish displayed lower nightly plasma melatonin levels (66±22 pg/ml) than intact or sham-operated groups (131±14 pg/ml), as it occurred in ophthalmectomized fish (64±12 pg/ml). Fish that were both pinealectomized and ophthalmectomized showed a further decrease in plasma melatonin levels (1.4±0.4 pg/ml), which approached daytime levels. In Experiment 2, plasma melatonin levels in both optic nerve-sectioned and ophthalmectomized fish were lower than control levels, while injection of 6-hydroxydopamine did not modify plasma melatonin concentrations. In Experiment 3, covering only the pineal made melatonin drop after a light pulse at MD, and covering only the eyes had a similar effect. In conclusion, these findings suggest that even though sea bass eyes do not directly contribute to plasma melatonin, the pineal organ, which unlike that of mammals is a direct photoreceptor in fish, requires light information from the lateral eyes to normally secrete melatonin into the bloodstream.