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In the wild, the light/temperature environment cyclically oscillates insofar as the temperature rises after dawn and drops after dusk. In the underwater photo-environment, light is filtered through the water column so that blue photons reach greater depths. This paper investigates the combined effects of both factors with two temperature regimes (constant temperature = 26°C, CTE vs. daily thermocycle = 28°C day:24°C night, TC) and three light wavelengths (white-W, blue-B, red-R) on Danio rerio embryos and larvae from fertilization to 30 days post-fertilization (dpf). It studied hatching rate, larval survival, growth, and food intake (gut content). It analyzed the expression of the genes involved in stress (crh), somatic growth (gh, ifg1a, igf2a), and food intake control (npy, agrp, ghrelin, orexin, mch1, mch2, grp, cck8) at 10 and 30 dpf. The results revealed that the lowest hatching rate was in R regardless of the temperature regime. The highest growth rate was for the larvae reared with B + TC, which was consistent with the highest expression values of the growth factors. The highest feeding and expression levels of the genes involved in food intake were for the larvae in B (regardless of the temperature regime) and W + TC. Conversely, the R + CTE combination obtained the worst growth and feeding results. These findings indicate that the best larval performance can be achieved with combinations of blue wavelengths and cyclic temperature regimes that come closer to those in the natural environment. These results should be considered when optimizing rearing protocols to improve the growth and welfare of the fish larvae.

Control of reproduction is one of the key issues to extend tilapia aquaculture. In most fish species, reproduction is a rhythmic process which ensures that reproduction timing matches the most favorable environmental conditions to maximize the survival of offspring. Although the existence of daily spawning rhythms has been reported for a number of fish species, the daily rhythms in neuroendocrine factors along the BPG (brain-pituitary-gonadal) axis remains mostly unknown. The objective of this paper was to investigate the existence of daily rhythms in BPG gene expression and plasma sex steroids in tilapia (Oreochromis niloticus). To this end, 36 males and 36 females were kept in separate tanks at 29 ± 0.5 °C and in a 12 h:12 h light/dark cycle. After 4 weeks of acclimation, male and female tilapia were mixed into six groups at a female:male ratio of 6:6. Two weeks later, fish were anesthetized and sampled every 4 h during a 24-hour cycle, and blood, brain, pituitary, gonad and eggs were collected for later analyses. The expression levels of the genes from the brain (gnrh1, gnrh2, gnrh3, kiss2, gnih), pituitary (fshβ and lhβ), gonad (star, cyp11a, cyp17, cyp19a1a and amh) and eggs (zp3b and fyna) were analyzed by qPCR. ELISA was used to analyze testosterone and estradiol in male and female plasma, respectively, and maturation-inducing hormone (MIH) in plasma in both sexes. The results revealed timely-coordinated daily rhythms along the BPG axis of tilapia. The expression of the brain genes showed that their acrophase was distributed throughout the LD cycle. However, a coordinated and harmonious pattern of daily rhythms appeared on the following levels of the BPG axis. The peaks in expression of the pituitary genes were reached at night, while the gonadal genes and plasma sex steroids peaked in the light phase. These findings can be useful for improving breeding protocols for tilapia aquaculture and for defining optimum timing for reproduction in captivity.

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 digestive system presents daily rhythms at both physiological and histological levels. Although cell morphology rhythms in mammals have been reported, they have scarcely been investigated in fish. The aim of the present research was to investigate the existence of daily rhythms in the morphology of cells in the liver and intestine of a teleost fish, the European sea bass (Dicentrarchus labrax), and how feeding time influences them. Regarding liver, we also focused on differences between the two metabolic zones: perivenous and periportal. For this purpose, fish were divided into two groups: fish fed once a day in the mid-light phase (ML) or the mid-dark phase (MD). After 1 month under each feeding regime, liver and intestine samples were collected every 4 h during a 24-h cycle, and different parameters were studied by light microscopy and image analysis. Daily rhythms occurred in most of the parameters evaluated in the liver. The effect of feeding time depended on the metabolic zone: the rhythms in the periportal zone were synchronized mainly by the light/dark cycle regardless of feeding time, whereas in the perivenous zone, rhythms were influenced more by feeding time. In the intestine, a daily rhythm in villi height was found with acrophases coinciding with feeding time in each group. These findings show for the first time the existence of cellular morphological rhythms in fish liver and intestine, and highlight the interactions between light and feeding cycles in the different metabolic zones of the liver.

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01~ZT22:17 h), except for dnmt7 in males (ZT2:25 h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26~ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.

The aim of this study was to investigate the ability of tambaqui (Colossoma macropomum) to balance the energy (E) and protein (P) ratio by encapsulated diets (without sensory properties). Thirty fish (71.37 ± 2.58 g) were distributed in six 250-litre tanks. Protein (P), carbohydrate (C) and fat (F) were encapsulated separately to ensure the same sensory properties of each macronutrient. Diets varied in terms of macronutrients to allow the maximal/minimal possibility of ingesting each nutrient following a geometric approach. P, C and F capsules were provided for 40 days (phase 1). After this procedure, fish were subjected to fixed low and poor protein challenges (200 g/kg), and freely to C + F, to evaluate whether they maintained a given intake target (phase 2). As a result, tambaqui showed an intake target at 300 g/kg of crude protein and 16.74 MJ per kg. Give the possibility of freely feeding protein, fish regulated their feed intake and showed a constant E/P ratio pattern (5.024-5.861 MJ/kg). When protein was restricted and fixed at 200 g/kg, fish maintained similar feed consumption until the end of phase 1, probably to keep a minimal protein amount according to their nutritional requirements.

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.

The aim of this work was to determine whether juvenile and adult European seabass (Dicentrarchus labrax L.), fed ad libitum manually and with self-feeding demand feeders, respectively, were able to discriminate between a fish oil-based diet and a series of diets made with vegetable oils (soybean, linseed, rapeseed and sunflower oil), as well as a fat-free diet. Adult D. labrax (443.6 ± 108.8 g) selected diets made from soybean (SYO) and rapeseed (RO) in proportions similar to fish oil (FO). Demand levels for the other two diets, sunflower (SUO) and linseed (LO) oils, were significantly lower than FO, whilst juveniles (56.4 ± 11.2 g) displayed a preference for SYO and FO. The lowest demand levels were associated with a fat-free diet (FF). Selection percentages of the diets for adult and juvenile fish were, respectively, FF—22.96% and 39.76%, SYO—43.02% and 53.26%, LO—36.48% and 36.19%, SUO—39.87% and 37.32%, and RO—46.38% and 40.94%. The animals’ size and feeding method did not significantly affect dietary selection. However, a statistical difference in specific growth rate (1.37% ± 0.26% day−1) for juveniles was only observed for FO+FF combination. For juveniles, no differences were observed in the conversion index. The juvenile group (FO + FF) had the highest food intake (2.00 ± 0.35 g 100 g−1 body weight). Moreover, the groups fed FO + SUO and FO + RO consumed the least. In conclusion, this study shows D. labrax can discriminate between diets where the only difference is the lipid source. These findings should be used to understand the acceptance or rejection of different source oils, which are increasingly being used in aquaculture such as aquafeeds for European seabass.

This study aimed to investigate the stress response of Sparus aurata specimens fed with nutraceutical aquafeed brewer’s yeast (Saccharomyces cerevisiae) and spirulina (Arthrospira platensis). For that purpose, 96 (169.0 ± 2.8 g) animals were distributed randomly in 12 tanks (8 fish per tank, 4 replicates) and divided in 3 groups (D1, casein/gelatin, control; D2, brewer’s yeast; D3, spirulina) and fed for 30 days. At the end of this period, fish from two replicates of each experimental diet were submitted to air exposure for 60 s while the fish from the other two replicates were maintained undisturbed (control). Afterwards, samples of blood, skin mucus, and head kidney were collected. The results revealed that after air exposure, cortisol, and glucose levels increased in the groups fed D1 (18.5 ± 2.6 mg/mL; 7.3 ± 0.6 mmol/L, respectively) and D2 (20.0 ± 6.2 mg/mL; 7.7 ± 0.6 mmol/L), but glucose not increased in fish fed D3 (13.7 ± 2.6 mg/mL; 5.5 ± 0.3 mmol/L). Lactate levels increased in all stressed groups, but in D1, its levels were significantly higher. After stress procedure, immunoglobulin M (IgM) levels in mucus increased only in fish fed D3 (0.1901 ± 0.0126 U/mL). Furthermore, there was a reduction in the expression of some genes involved in stress response (coxIV, prdx3, csfl-r, ucp1, and sod in fish fed D2 and D3). csf1 decreased only in stressed fish fed D2. However, cat increased in fish fed with D3. In summary, these findings points to the beneficial effects of spirulina and brewer’s yeast to improve stress resistance in aquaculture practices of gilthead seabream.

Daily feeding rhythms and the ability of pirarucu ( Arapaima gigas ) to compose a balanced diet through macronutrient self-selection were evaluated. Twelve fish (1573.3 ± 74.4 g) were distributed in six tanks of 250 l, two fish per tank. First, three experimental diets were prepared using an incomplete mixture of macronutrients (75%protein/25%carbohydrate-PC, 75%protein/25%fat-PF and 10% protein/45%fat/45%carbohydrate-PFC). These diets were provided to fish through a self-feeding system connected to a computer to record feeding activity. After this procedure, fish were challenged with a 50% protein dilution (diets: PC50 and PF50). The results showed that pirarucu exhibited a strict diurnal feeding pattern with 95.4% of daily feeding activity observed in the daytime. Fish selected 56.3% P, 24.2% C, 19.5% F, and consumed 150–151 kJ/kg BW/day of energy. After protein dilution, fish sustained energy intake by increasing the consumption of PC50 and PF50 to maintain the target protein intake of the previous stage. When fish were protein-restricted, they failed to sustain previous energy intake. These findings can be used to design feeding regimes and for formulating aquafeed for pirarucu. Statement of relevance The use of an automatic feeder system activated by the fish could allow the animal to feed at their preferred time and hence reduces waste and improve the intake. Studies that consider the fish preference as a guide in designing diets can provide data on mechanisms of nutrient intake regulation for the development of aquafeeds for new species with potential for aquaculture.

We report feeding studies on hybrid fish (carnivorous × omnivorous) to determine the interactions between protein sources during diet selection trials. We also examined behavioural rhythms (feeding and locomotor activity) using a self-feeding system mounted with a feeder connected to an infrared photocell. Twenty fish (28.20 ± 0.05 g) were distributed in four aquariums (50 L each). First, the system was tested to evaluate the adaptation of catfish (Pseudoplatystoma reticulatum × Leiarius marmoratus) to self-feeding (step I). During this 28-day period, feeding and locomotor activity rhythms were recorded. In the second step, to analyse protein self-selection, a group of 20 fish (19.10 ± 0.05 g) was released in the aquaria. The hybrid catfish were given free access to diets (soya bean meal protein (SP) vs. bovine liver protein (BP)) through two self-feeders. The same procedure was used for (SP) vs. fish meal protein (FP). The catfish learned to activate the feeders to demand a daily feed amount equivalent to 1.8% of their body weight. The fish performed most of their actions (89.0% feeding activity and 94.1 0% locomotor activity) in the night. The diet choice by the catfish became clearer with a significantly lower intake of BP (0.37 g per 100 g body weight, 24.6%) vs. SP (1.06 g per 100 g body weight, 75.4%) and SP (0.61 g per 100 g body weight, 38.9%) vs. FP (0.91 g per 100 g body weight, 61.1%). These findings should be considered when discussing feeding behaviour, nutritional targets and protein sources for future aquaculture feeds.

Environmental factors such as light and temperature present cyclic variations during the day. High temperatures occur during the light phase, whereas low temperatures take place at night (i.e. a daily thermocycle). Although applying thermocycles to fish larvae improves growth in some species, nothing is known about the impact during the early development of the digestive system. The aim of the present research was to investigate the effect of different temperature regimes, cycling versus constant, on the daily rhythms of digestive factors and growth of Nile tilapia (Oreochromis niloticus) larvae. For this purpose, fertilized eggs (stage 1) were divided into two groups: one under a thermocycle (TC) of 31 °C:25 °C day:night and another group at constant temperature of 28 °C (CTE). Photoperiod was set at 12:12 h light/dark cycle. Larvae length was measured on 4, 8 and 13 days post-fertilization (dpf) and samples were collected every 4 h during a 24-h cycle on these days. The expression levels of pepsinogen, chymotrypsinogen, trypsinogen, lipase, maltase, isomaltase, npy and cck were analyzed by qPCR. The results showed that growth was greater when larvae were reared at TC than at CTE. Moreover, on 13 dpf, most analyzed genes (chymotrypsinogen, lipase, maltase, isomaltase, npy and cck) displayed daily rhythms in the TC group but not in CTE, with most acrophases located around mealtime. These rhythms might explain the higher growth rate observed in the TC larvae due to improved synchronization of feed digestion and utilization. These findings could be useful for improving rearing protocols used in tilapia larviculture and enhancing production performance.

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.

We evaluated the daily changes in immunological and hematological factors in tilapia (Oreochromis niloticus) after an immunization period with a subsequent challenge. Experiments were divided into two phases: Phase 1 (immunization): 144 fish were distributed into two groups with 72 fish in six tanks. One group (T1) was immunized, comprising six vaccination time points (ZT schedule = ZT2 h, ZT6 h, ZT10 h, ZT14 h, ZT18 h, ZT22 h). The same schedule was applied to the other group, but with saline solution (non-vaccinated: T2). Both groups remained in the laboratory for 30 days (considered the immunization period). Phase 2 (challenge): on day 30, both vaccinated and non-vaccinated groups were challenged with Streptococcus agalactiae (2.0 × 10

The principles of modern aquaculture encourage the development of fish feeds containing low fish meal content and several types of plant ingredients plus nutrients to avoid depleting global fish stocks and to reduce costs. However, food constituents can affect animal nutrition and feeding behavior, so the effect of different diets on fish behavior and growth needs to be understood to optimize the use of nutrients and to improve fish welfare. The development of multiple-choice self-feeding systems led to a new perspective for investigating these issues in aquaculture species. Our purpose with this review is to summarize the information that has been published to date on this topic and to identify gaps in knowledge where research is needed. Key subjects are assessed under the following major headings: How do we study dietary selection in fish? What food signals do fish use to choose the right diet? and How do fish respond to food challenges? The present review will provide a picture of the main results obtained to date in these studies in aquaculture fish species, as well as perspectives for future research in the field.

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.

Ethanol is one of the most commonly abused drugs and consequently its toxic and psychoactive effect has been widely investigated, although little is known about the time-dependent effects of this drug. In the present research zebrafish was used to assess daily rhythms in ethanol toxicity and behavioural effects, as well as the temporal pattern of expression of key genes involved in ethanol detoxification in the liver (adh8a, adh5, aldh2.1 and aldh2.2). Our results showed marked differences in the mortality rate of zebrafish larvae depending on the time of day of the exposure to 5% ethanol for 1h (82% and 6% mortality in the morning and at night, respectively). A significant daily rhythm was detected with the acrophase located at "zeitgeber" time (ZT) = 04:22 h. Behavioural tests exposing zebrafish to 1% ethanol provoked a major decrease in swimming activity (68-84.2% reduction) at ZT2, ZT6 and ZT10. In contrast, exposure at ZT18 stimulated swimming activity (27% increase). During the day fish moved towards the bottom of the tank during ethanol exposure, whereas at night zebrafish increased their activity levels right after the exposure to ethanol. Genes involved in ethanol detoxification failed to show significant daily rhythms in LD, although all of them exhibited circadian regulation in constant darkness (DD) with acrophases in phase and located at the end of the subjective night. Taken altogether, this research revealed the importance of considering the time of day when designing and carrying out toxicological and behavioural tests to investigate the effects of ethanol, as the adverse effects of this drug were more marked when fish were exposed in the morning than at night.

The environment in which aquatic organisms inhabit is not constant and many factors such as light and temperature display cyclic and predictable variations. Animals present biological rhythms in many of their physiological variables to adapt to these cyclic changes, timing their functions to occur when the possibility of success is greater. Besides, the underwater photo-environment is complex since light characteristics (i.e. intensity and spectrum) are variable and depend on the absorbance properties of the water column. Fish development is hence highly influenced by all of these factors, affecting processes such as survival, growth, hatching, sex determination/differentiation and occurrence of malformations. The aim of this chapter is to review how fish development and performance is shaped by environmental factors, paying special attention to the effects of environmental cycles of light and temperature, the characteristics of illumination and the role of the developing biological clock.

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.

In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light–12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth.

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.

This study aimed to investigate pirarucu's (Arapaima gigas) ability to trigger a self-feeding system to regulate protein intake between two standard diets that contained 39% and 49% of crude protein. The same system allowed the evaluation of daily feeding and locomotor activity rhythms. Eighteen fish (654.44±26.85g) were distributed into six 250 L tanks (3 fish/tank). Fish had free access to both diets (39% vs. 49% protein) by feeders (2 per tank), adapted to be activated by fish themselves. This system was connected to a computer system. After an adaptation period, fish learned to activate feeders and the mean food intake recorded was 2.14% of their body weight on a daily basis. Fish showed feeding (72.48%) and locomotor (72.49%) activity predominantly during the daytime, and daily variations of choice between diets, but fixed a protein intake feeding target at 44.53%. These results should be considered when discussing feeding behavior, feeding schedules and diet intake regulations.

The role of light and feeding cycles in synchronizing self-feeding and locomotor activity rhythms was studied in white shrimps using a new self-feeding system activated by photocell trigger. In experiment 1, shrimps maintained under a 12:12h light/dark (LD) photoperiod were allowed to self-feed using feeders connected to a photoelectric cell, while locomotor activity was recorded with a second photocell. On day 30, animals were subjected to constant darkness (DD) for 12days to check the existence of endogenous circadian rhythms. In the experiment 2, shrimps were exposed to both a 12:12h LD photoperiod and a fixed meal schedule in the middle of the dark period (MD, 01:00h). On day 20, shrimps were exposed to DD conditions and the same fixed feeding. On day 30, they were maintained under DD and fasted for 7days. The results revealed that under LD, shrimps showed a clear nocturnal feeding pattern and locomotor activity (81.9% and 67.7% of total daily food-demands and locomotor activity, respectively, at nighttime). Both feeding and locomotor rhythms were endogenously driven and persisted under DD with an average period length (τ) close to 24h (circadian) (τ=24.18±0.13 and 23.87±0.14h for locomotor and feeding, respectively). Moreover, Shrimp showed a daily food intake under LD condition (1.1±0.2gday(-1) in the night phase vs. 0.2±0.1gday(-1) in the light phase). Our findings might be relevant for some important shrimp aquaculture aspects, such as developing suitable feeding management on shrimp farms.

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.

Sea bass change their feeding rhythms from diurnal to nocturnal in winter, returning to diurnal feeding in spring. Despite behavioral data, the physiological changes that take place during such changes remain unexplored. In this paper, blood glucose rhythms of European sea bass with diurnal/nocturnal self-feeding rhythms were investigated during phase inversions of their feeding behavior (in winter and spring) when both diurnal and nocturnal fish coexist. Blood glucose showed daily variations in both seasons (ANOVA, p

Senegalese sole ( Solea senegalensis ) failed to reproduce and spawn viable eggs when bred and reared in captivity (first generation G1 cultured broodstock). Cultured sole were also shown to have significantly lower levels of arachidonic fatty acid (20:4n−6, ARA) in reproductive tissues compared to wild counterparts. However, to date no information about ARA requirements of Senegalese sole broodstock (wild or cultured) has been available. The present study was designed to determine dietary ARA preferences of adult G1 Senegalese sole by the use of automated demand feeders. For this purpose, three groups of 16 (eight females and eight males) G1 fish were offered to select between two diets, one with high (6.0% of total fatty acids, TFA) and another with low (0.7% TFA) ARA content. The experiment was conducted for 16 consecutive months. The results revealed that Senegalese sole changed the preference for ARA during the year, in correlation with the seasonal water temperature (r 2 = 0.7), and with periods of high and low demand of total food ingested. Thus, a high ARA (3.9% TFA) selection was observed in summer and early-autumn, whereas low ARA (2.2% TFA) selection occurred in winter. The average ARA demanded during the whole year was 3.0% TFA, with a resultant high level of ARA in the tissues of the fish; especially in male testis (8.8% TFA). These results should be considered when formulating seasonal diets to suit sole broodstock preferences in ARA in intensive aquaculture.

We investigated dietary self-selection in gilthead sea bream using a multiple-choice self-feeding system. To this end, five groups of 15 fish (154 g BW) were distributed among 500-l tanks and allowed to self-feed from 3 self-feeders, each of which distributed feeds composed of combinations of macronutrients. Three different selection ranges of protein (P), carbohydrate (C) and fat (F) (expressed as percentage of total macronutrients) were tested: in phase 1, PF (75%–25%, respectively), PC (75%–25%) and CF (85%–15%); in phase 2, PF (60%–40%), PC (75%–25%) and CF (60%–40%); and in phase 3, PF (60%–40%), PCF (20%–50%–30%) and PC (90%–10%). In addition, the effect of P deprivation on energy intake was tested. The results revealed that, despite the different selection ranges in each phase, the macronutrient composition of the selected diets did not differ significantly during the experiments, being on average 63.7 ± 0.6% P, 15.5 ± 1.7% F and 20.6 ± 1.15% C. When sea bream were restricted to self-feeding on a protein-free diet for 10 days, and were only given the CF (60%–40%) diet, the fish sustained their average energy intake (260.0 ± 37.7 kJ/kg BW/day), which did not differ statistically from the average energy intake during the previous three experimental phases (284.6 ± 26.9 kJ/kg BW/day). In conclusion, sea bream were able to compose and defend a balanced diet by selecting from three incomplete diets, and the fish were also able to maintain their energy intake for a short period of time when allowed to self-feed on a protein-free diet.

Fish have an internal biological clock that drives their daily rhythms, including those of feeding and reproduction. The European sea bass has been reported to exhibit a phase inversion of its diel feeding pattern: nocturnal in winter (coinciding with the spawning period) and diurnal during the remainder of the year. In this study, we investigated the locomotor activity of sea bass during the spawning and resting periods as well as the possible existence of a daily spawning rhythm by calculating the number of eggs (and their viability) released every hour. The results showed that sea bass locomotor activity was clearly nocturnal (68 ± 4.3% of the daily activity displayed at night) during the spawning season and mostly diurnal (65.5 ± 7.5% of the activity displayed during the day) out of the reproductive period. Spawning occurred during the dark phase with two peaks, 6 and 11 h after lights off. Egg viability was highest in the batch released near the spawning peaks. These findings could be useful for optimizing egg production and to improve hatchery practices, which should take into account the reproduction rhythms of sea bass.

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.

Light plays a key role in the development of biological rhythms in fish. Recent research in Senegal sole has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions. However, the effect of light on the daily patterns of behavior remains unexplored. Therefore, the aim of this study was to investigate the impact of different photoperiod regimes and white, blue, and red light on the activity rhythms and foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To this end, eggs were collected immediately after spawning during the night and exposed to continuous white light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white (LDW), blue (LDB, λpeak = 463 nm), or red light (LDR, λpeak = 685 nm). A filming scenario was designed to video record activity rhythms during day and night times using infrared lights. The results revealed that activity rhythms in LDB and LDW changed from diurnal to nocturnal on days 9 to 10 DPH, coinciding with the onset of metamorphosis. In LDR, sole larvae remained nocturnal throughout the experimental period, while under LL and DD, larvae failed to show any rhythm. In addition, larvae exposed to LDB and LDW had the highest prey capture success rate (LDB = 82.6% ± 2.0%; LDW = 75.1% ± 1.3%) and attack rate (LDB = 54.3% ± 1.9%; LDW = 46.9% ± 3.0%) during the light phase (ML) until 9 DPH. During metamorphosis, the attack and capture success rates in these light conditions were higher during the dark phase (MD), when they showed the same nocturnal behavioral pattern as under LDR conditions. These results revealed that the development of sole larvae is tightly controlled by light characteristics, underlining the importance of the natural underwater photoenvironment (LD cycles of blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae during early ontogenesis.

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.

Animals do not eat whatever food item they encounter, but choose different foods that best match their requirements. Fish exhibit such "nutritional wisdom" and adapt their feeding behaviour and food intake according to their needs and the nutritional properties of diets. In this paper, we tested the ability of Nile tilapia to select between diets with a balanced or unbalanced composition of essential amino acids. To this end, three different diets were prepared: a gelatine based diet (D(1)), a gelatine diet supplemented with three essential amino acids (EAA, l-tryptophane, l-methionine, l-threonine) (D(2)), and a diet containing only cellulose and the three crystalline EAA (D(3)). In addition, the putative role of both orosensorial factors (using pellets vs capsules) and social interactions (single vs groups of ten fish) was investigated. To this end, a total of 68 male tilapia of about 141±48 g (mean±S.D.) were challenged, individually or in groups, to select between D(1)vs D(2) using pellets dispensed by self-feeders (exp. 1). In another experiment (exp. 2), 11 individual fish were challenged to select encapsulated diets with non flavour or smell proprieties (D(1)vs D(2)), and in exp. 3 fish were challenged to self-supplementation in EAA (D(1)vs D(3)). The results showed the ability of tilapia to avoid the EAA-deficient diet, choosing 82.2% D(2) in the case of individual fish, and 80.8% D(2) in the case of fish groups. Dietary selection was not directly driven by the orosensorial characteristics of food, since tilapia sustained a higher preference for D(2) when fed with encapsulated diets. Finally, in exp. 3 tilapia self-supplemented the EAA deficiency by selecting a synchronised combination of D(1) and D(3) that matched their nutritional requirements. These findings highlighted the capacity of fish to make dietary selection based on the EAA content, which should be considered when discussing food intake regulation mechanisms, and diet formulation and supplementation with EAA.

Our objective was to evaluate the capacity of Nile tilapia (Oreochrmis niloticus) to self-select the most nutritious foods when offered with other foods. The fish were offered pairs of macronutrients (protein + carbohydrate, PC; protein + fat, PF; carbohydrate + fat, CF) in three self-feeders and subjected to different challenges: i) the relative position of the self-feeders was changed, ii) the protein content was reduced by 50%, and iii) protein was withdrawn. In the experiment, 90 males (142.0 ± 18.0 g) were kept in 6 tanks (150 l each) and their daily macronutrient intake was calculated. Regardless of the self-feeder position, fish composed a diet containing 41.7% P, 34.8% C and 23.5% F, and a relatively stable energy content of around 187.0 kJ/kg BW/day. When the protein-containing diets (PC and PF) were diluted by 50%, fish increased their food intake to compensate for the energy dilution and thus sustain the dietary composition (42.6% P, 33.3% C and 23.2% F). Finally, when PC and PF were removed, fish maintained the energy intake at around 178.2 kJ/kg BW/day. These results indicate that tilapia can adapt to protein dilution/removal to sustain its energy intake, making it a useful model for dietary selection and nutritional studies.