No abstract

No abstract

Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed.

Significant variations in the number of white and red blood cells, haematocrit and haemoglobin were found throughout the year in sexually mature male and female tench Tinca tinca. In general, the lowest values were observed during autumn-winter and the highest during summer, with males exhibiting higher values than females. Plasma glucose, cholesterol and triglycerides were lower during the winter than during the summer-autumn seasons in both sexes. Gonadosomatic and hepato-somatic indices were inversely correlated in female tench throughout the year. Seasonal patterns in liver metabolic resources were very similar for both sexes. For males and females, liver glycogen and proteins increased during the autumn, whereas the liver stored lipid during spring. Dorsal muscle mainly deposited glycogen, whereas lipid was mainly stored in the ventral muscle. Relations between seasonal changes in environmental factors, such as feeding and temperature are discussed.

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light-darkness and feeding-fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light-darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs.

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock.

(MAX 350 WORDS)Melatonin is currently proposed to be synthesized in non-photosensitive organs of vertebrates, besides its well-known sites of synthesis, the pineal gland and the retina.However, very few studies have demonstrated gene expression of MEL synthesizing enzymes in extrapineal and extraretinal locations. In the present study, present study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, the AANAT and the HIOMT in central and peripheral locations of the goldfish we give report of the full-length cloning of the two enzymes catalyzing the final steps of melatonin biosynthesis, the arylalkylamine N-acetyltransferase (AANAT) and the hydroxyindole-Omethyltransferase (HIOMT), in a teleosts fish, the goldfish (Carassius auratus). Both enzymes showed high similarity with other teleost sequences, corresponding to the goldfish AANAT-2 and HIOMT-2. Two forms of AANATs were widely known to exist in teleosts, but this is the first time that two isoforms of HIOMT are deduced from a phylogenetic analysis.Both enzymes were detected in several peripheral locations, including liver and gut, being present results the first to find HIOMT in non-photosensitive structures of a fish species. No studies exist on transcriptional regulation of the expression of MEL biosynthesis enzymes in non photosensitive structures in fish The daily expression pattern of both genes in pineal gland, retina, liver and gut was investigated using quantitative real time RT-PCR and cosinor analysis. This is the first time that a rhythmic expression of AANAT and HIOMT are found in digestive tissues of a vertebrate species, supporting a functional melatonin synthesis pathway in liver and gut of the goldfish. Besides, the transcriptional regulation of Aanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions was investigated expression of gAANAT-2 is analyzed under different lighting conditions including continuous light and darkness, revealing, as expected, light-dependent rhythms in pineal gland and retina, but also in liver. Nevertheless, the persistence in hindgut of these Aanat-2 rhythms in constant conditions suggests that the expression of this transcript is under circadian clock and entrained by non-photic cues. Our results reinforce the existence of melatonin synthesis in gut and liver of the goldfish, while the rhythmic expression profiles reported point to a regulation of both genes in gut and liver by peripheral oscillators entrainable to non-photic cues.

The circadian system controls temporal homeostasis in all vertebrates. The light-dark (LD) cycle is the most important zeitgeber (“time giver”) of circadian system, but feeding time also acts as a potent synchronizer in the functional organization of the teleost circadian system. In mammals is well known that food intake during the rest phase promotes circadian desynchrony which has been associated with metabolic diseases. However, the impact of a misalignment of LD and feeding cycles in the entrainment of fish circadian oscillators is largely unknown. The objective of this work was to investigate how a time-lag feeding alters temporal homeostasis and if this could be considered a stressor. To this aim, goldfish maintained under a 12 h light-12 h darkness were fed at mid-photophase (SF6) or mid-scotophase (SF18). Daily rhythms of locomotor activity, clock genes expression in hypothalamus, liver, and head kidney, and circulating cortisol were studied. Results showed that SF6 fish showed daily rhythms of bmal1a and clock1a in all studied tissues, being in antiphase with rhythms of per1 genes, as expected for proper functioning clocks. The 12 h shift in scheduled feeding induced a short phase advance (4–5-h) of the clock genes daily rhythms in the hypothalamus, while in the liver the shift for clock genes expression rhythms was the same that the feeding time shift (∼12 h). In head kidney, acrophases of per genes underwent a 12-h shift in SF18 animals, but only 6 h shift for clock1a. Plasma cortisol levels showed a significant daily rhythm in animals fed at SF6, but not in SF18 fish fed, which displayed higher cortisol values throughout the 24-h. Altogether, results indicate that hypothalamus, liver, and head kidney oscillate in phase in SF6 fish, but these clocks are desynchronized in SF18 fish, which could explain cortisol alterations. These data reinforce the hypothesis that the misalignment of external cues (daily photocycle and feeding time) alters fish temporal homeostasis and it might be considered a stressor for the animals.