Ontogeny of daily rhythms in the expression of metabolic factors in Nile tilapia (Oreochromis niloticus) kept at two different temperature regimes: Thermocycle and constant temperature.

The daily variations of temperature are one of the main synchronizers of the circadian rhythms. In addition, water temperature influences the embryonic and larval development of fish and directly affects their metabolic processes. The application of thermocycles to fish larvae has been reported to improve growth and the maturation of the digestive system, but their effects on metabolism are poorly understood. The aim of the present study was to evaluate the effect of two different temperature regimes, cycling versus constant, on the daily rhythms of metabolic factors of Nile tilapia (Oreochromis niloticus) larvae. For this purpose, fertilized eggs were divided into two groups: one reared in a 31 °C:25 °C day:night thermocycle (TCY) and another group maintained in a constant 28 °C temperature (CTE). The photoperiod was set to a 12:12 h light/dark cycle. Samples were collected every 4 h during a 24-h cycle on days 4, 8 and 13 post fertilization (dpf). The expression levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), malic enzyme, glucose-6-phosphate dehydrogenase (g6pd), phosphofructokinase (pfk) and pyruvate kinase (pk) were analyzed by qPCR. Results showed that, in 13 dpf animals, most of the genes analyzed (alt, ast, malic, g6pd and pfk) showed daily rhythms in TCY, but not in the group kept at constant temperature, with most acrophases detected during the feeding period. An increase in nutrient metabolism around feeding time can improve food utilization and thus increase larval performance. Therefore, the use of thermocycles is recommended for tilapia larviculture.

Does exposure to moonlight affect day/night changes in melatonin and metabolic parameters in Amazonian fish?

Lunar cycle modulates the rhythmic activity patterns of many animals, including fish. The effect of the moonlight cycle on daily melatonin and metabolic parameters was evaluated in matrinxã (Brycon amazonicus) subjected to external natural lighting. Eighty juvenile were distributed in 4 tanks of 1m3 (20 fish/tank) and divided into two groups. One group was exposed to the full moon and the other group to the new moon for 30 days, which corresponds to the duration of the lunar period. At the end of the lunar phase, 6 fish from each group were anesthetized to collect blood, tissue and eye samples at midday and midnight. The comparison between the light and dark periods revealed a significant increase in plasma and ocular melatonin in the last period. However, there was no significant difference for plasma melatonin between moons. Ocular melatonin presented higher concentrations during the new moon. Glucose, total proteins, cortisol, liver glutathione and gill lipid peroxidation were higher in the full moon compared to in the new moon. Plasma triglyceride was higher during the night for the full moon, and the opposite was found for the new moon. Total cholesterol values were higher at night regardless the moon phase. Glutathione in the gills and lipid peroxidation in the liver showed no significant differences. These results highlight the importance of considering both the day and lunar cycles for melatonin and metabolic parameters in species of commercial interest and susceptible to stressful situations in rearing conditions.

Combined effects of rearing temperature regime (thermocycle vs. constant temperature) during early development and thermal treatment on Nile tilapia (Oreochromis niloticus) sex differentiation.

In nature, water temperature experiences daily variations known as thermocycles. Temperature is the main environmental factor that influences sex determination in most teleost fish. The purpose of this study was to examine the effects of rearing temperature (thermocycle (TC) vs. constant (CTE)) on development and a posterior thermal shock throughout the period of sex differentiation of Nile tilapia (Oreochromis niloticus). Embryos and larvae were kept under two temperature regimes: TC of 31 °C:25 °C day:night vs. CTE of 28 °C from 0 to 11 dpf. After this period, the larvae from each group were subjected to either heat treatment (HT, 36 °C for 12 days) or kept under the same rearing temperatures until 23 dpf (Control, C). Then all the groups remained at constant temperature until 270 dpf, when blood and gonads were collected. Larval samples were used to examine the expression of genes related to male (amh, ara, sox9a, dmrt1a) and female (cyp19a1a, foxl2, era) sexual differentiation. In juveniles, sex was characterized by histology, the gonadal expression of the genes involved in the sex steroid synthesis was analyzed by qPCR, and plasma testosterone (T) and estradiol (E2) levels were analyzed by ELISA. In larvae, daily TCs increased the survival rate against HT and up-regulated the expression of ovarian differentiation genes. In juveniles, TC + C induced a higher proportion of females and higher cyp19a1a expression compared to CTE + C. HT induced changes in the CTE group by up-regulating testicular differentiation genes and down-regulating female promoting genes, which did not occur in the TC group. Juveniles from TC + C group presented a higher proportion of females with higher E2 and cyp19a1a than CTE + HT. Fish from the CTE + HT group showed a higher percentage of males with highest T and amh. These findings indicate that daily TCs during larval development promote ovarian differentiation and diminish the masculinizing effects of HT.

Size-selective mortality induces evolutionary changes in group risk-taking behaviour and the circadian system in a fish.

Intensive and trait-selective mortality of fish and wildlife can cause evolutionary changes in a range of life-history and behavioural traits. These changes might in turn alter the circadian system due to co-evolutionary mechanisms or correlated selection responses both at behavioural and molecular levels, with knock-on effects on daily physiological processes and behavioural outputs. We examined the evolutionary impact of size-selective mortality on group risk-taking behaviour and the circadian system in a model fish species. We exposed zebrafish Danio rerio to either large or small size-selective harvesting relative to a control over five generations, followed by eight generations during which harvesting was halted to remove maternal effects. Size-selective mortality affected fine-scale timing of behaviours. In particular, small size-selective mortality, typical of specialized fisheries and gape-limited predators targeting smaller size classes, increased group risk-taking behaviuor during feeding and after simulated predator attacks. Moreover, small size-selective mortality increased early peaks of daily activity as well as extended self-feeding daily activity to the photophase compared to controls. By contrast large size-selective mortality, typical of most wild capture fisheries, only showed an almost significant effect of decreasing group risk-taking behaviour during the habituation phase and no clear changes in fine-scale timing of daily behavioural rhythms compared to controls. We also found changes in the molecular circadian core clockwork in response to both size-selective mortality treatments. These changes disappeared in the clock output pathway because both size-selected lines showed similar transcription profiles. This switch downstream to the molecular circadian core clockwork also resulted in similar overall behavioural rhythms (diurnal swimming and self-feeding in the last hours of darkness) independent of the underlying molecular clock. To conclude, our experimental harvest left an asymmetrical evolutionary legacy in group risk-taking behaviour and in fine-scale daily behavioural rhythms. Yet, the overall timing of activity showed evolutionary resistance probably maintained by a molecular switch. Our experimental findings suggest that size-selective mortality can have consequences for behaviour and physiological processes.

Rearing temperature conditions (constant vs. thermocycle) affect daily rhythms of thermal tolerance and sensing in zebrafish.

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.

Daily rhythms of expression in reproductive genes along the brain-pituitary-gonad axis and liver of zebrafish.

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 (fshß, lhß, 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, 20ßhsd, lhr, fshr, cyp19a1a, foxl2, amh, dmrt1 and 11ßhsd) revealed statistically significant daily rhythms with nocturnal acrophases, except for female cyp17a1a (ZT06:21 h) and 20ßhsd (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.

Coping with exposure to hypoxia: modifications in stress parameters in gilthead seabream (Sparus aurata) fed spirulina (Arthrospira platensis) and brewer's yeast (Saccharomyces cerevisiae).

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.

Effects of pinealectomy on the neuroendocrine reproductive system and locomotor activity in male European sea bass, Dicentrarchus labrax.

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.

Synchronization to light and mealtime of daily rhythms of locomotor activity, plasma glucose and digestive enzymes in the Nile tilapia (Oreochromis niloticus).

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±0.6g) were distributed in 12 tanks (10 fish per tank) and divided into two groups. One group was fed once a day at 11:00h (zeitgeber time, ZT6) (ML group) and the other group was fed at 23:00h (ZT18) (MD group). The fish were anesthetized to collect samples of blood, stomach and midgut at 4-hour intervals over a period of 24h. 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 12h 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.

Synchronization to light and mealtime of the circadian rhythms of self-feeding behavior and locomotor activity of white shrimps (Litopenaeus vannamei).

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.

A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception.

The circadian clock is synchronized with the day-night cycle primarily by light. Fish represent fascinating models for deciphering the light input pathway to the vertebrate clock since fish cell clocks are regulated by direct light exposure. Here we have performed a comparative, functional analysis of the circadian clock involving the zebrafish that is normally exposed to the day-night cycle and a cavefish species that has evolved in perpetual darkness. Our results reveal that the cavefish retains a food-entrainable clock that oscillates with an infradian period. Importantly, however, this clock is not regulated by light. This comparative study pinpoints the two extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin as essential upstream elements of the peripheral clock light input pathway.

Sporadic feeding regulates robust food entrainable circadian clocks in blind cavefish.

The circadian clock represents a key timing system entrained by various periodic signals that ensure synchronization with the environment. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle and the other set by feeding time. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and experience food deprivation. Here, we have investigated feeding regulated clocks in two cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus, in comparison with the surface-dwelling zebrafish Danio rerio. Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide insight into the connections between feeding and circadian clocks.

Combined blue light and daily thermocycles enhance zebrafish growth and development.

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.

Long photoperiod impairs learning in male but not female medaka.

Day length in conjunction with seasonal cycles affects many aspects of animal biology. We have studied photoperiod-dependent alterations of complex behavior in the teleost, medaka (Oryzias latipes), a photoperiodic breeder, in a learning paradigm whereby fish have to activate a sensor to obtain a food reward. Medaka were tested under a long (14:10 LD) and short (10:14 LD) photoperiod in three different groups: mixed-sex, all-males, and all-females. Under long photoperiod, medaka mixed-sex groups learned rapidly with a stable response. Unexpectedly, males-only groups showed a strong learning deficit, whereas females-only groups performed efficiently. In mixed-sex groups, female individuals drove group learning, whereas males apparently prioritized mating over feeding behavior resulting in strongly reduced learning performance. Under short photoperiod, where medaka do not mate, male performance improved to a level similar to that of females. Thus, photoperiod has sex-specific effects on the learning performance of a seasonal vertebrate.

Daily rhythms in the morphometric parameters of hepatocytes and intestine of the European sea bass (Dicentrarchus labrax): influence of feeding time and hepatic zonation.

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.

Daily and circadian melatonin release in vitro by the pineal organ of two nocturnal teleost species: Senegal sole (Solea senegalensis) and tench (Tinca tinca).

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 (p<0.001) 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 (p<0.01) 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.

Environmental Cycles, Melatonin, and Circadian Control of Stress Response in Fish.

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.

Daily rhythms of swimming activity, synchronization to different feeding times and effects on anesthesia practice in an Amazon fish species (Colossoma macropomum).

This study aimed to investigate the existence of day-night differences in the time for anesthesia and recovery in tambaqui exposed to the anesthetic eugenol and the influence of feeding time. Thus, we evaluated: (1) swimming activity; (2) food anticipatory activity (FAA) as a synchronizer of swimming activity and change to susceptibility to anesthetic; and (3) the effects of diurnal/nocturnal anesthesia exposure of fish feeding in the mid-light phase: 12:00 h (ML) and fish feeding in the mid-dark phase: 00:00 h (MD). Our findings revealed strictly nocturnal activity for tambaqui (94.2%), known as diurnal fish to date. Moreover, FAA was observed in tambaqui fed at MD, which showed a sustained increase in activity that began 2 h before feeding time and lasted until feeding. In contrast, no FAA was observed in fish fed at ML. Regarding anesthesia by day or night, the tambaqui treated with eugenol exhibited no difference in induction time. However, differences were observed in recovery times, with fish anesthetized at day recovering in 1-2 min and fish anesthetized at night recovering in 5-7 min. In short, our findings revealed for the first time the nocturnal behavior of tambaqui. These results indicated that recovery by day/night by eugenol in tambaqui has a strong dependence of behavioral patterns and the time of day.

Circadian expression of DNA methylation and demethylation genes in zebrafish gonads.

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.

Daily rhythms after vaccination on specific and non-specific responses in Nile tilapia (Oreochromis niloticus).

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 × 107 CFU mL-1) following the same ZT schedule to stimulate the immune response without leading to widespread infection and mortality. On day 45, blood and head kidney samples were collected during the same ZT schedule. The variations in time of the following parameters within each group were evaluated: hematology, peroxidase activity, IgM, tnf-α3, tgf-ß1, il-1ß and il-12 gene expression. No significant mortality was observed for the groups or the ZT schedule (p > 0.05). Daily rhythms with diurnal acrophases were found in T2 for il12, tnf-α3 and tgf-ß1 expression gene, while the acrophases of the peroxidase level, hematocrit and thrombocytes were at nighttime (p < 0.05). In contrast, most of the parameters in the vaccinated tilapia showed no daily rhythms (p > 0.05), except IgM. For all the parameters, the interaction effect between time and treatment (vaccinated and non-vaccinated groups) depended on ZT. Our results reveal that the humoral and non-specific immune system displayed a circadian rhythm based on the light-dark cycle, which could be affected by the vaccination procedure in tilapia.