Circadian rhythm of preferred temperature in fish: Behavioural thermoregulation linked to daily photocycles in zebrafish and Nile tilapia.

Ectothermic vertebrates, e.g. fish, maintain their body temperature within a specific physiological range mainly through behavioural thermoregulation. Here, we characterise the presence of daily rhythms of thermal preference in two phylogenetically distant and well-studied fish species: the zebrafish (Danio rerio), an experimental model, and the Nile tilapia (Oreochromis niloticus), an aquaculture species. We created a non-continuous temperature gradient using multichambered tanks according to the natural environmental range for each species. Each species was allowed to freely choose their preferred temperature during the 24h cycle over a long-term period. Both species displayed strikingly consistent temporal daily rhythms of thermal preference with higher temperatures being selected during the second half of the light phase and lower temperatures at the end of the dark phase, with mean acrophases at Zeitgeber Time (ZT) 5.37 h (zebrafish) and ZT 12.5 h (tilapia). Interestingly, when moved to the experimental tank, only tilapia displayed consistent preference for higher temperatures and took longer time to establish the thermal rhythms. Our findings highlight the importance of integrating both light-driven daily rhythm and thermal choice to refine our understanding of fish biology and improve the management and welfare of the diversity of fish species used in research and food production.

Behavioural response to toxic elements, detoxification and organ accumulation are time-of-day-dependent in zebrafish.

Toxic elements, such as mercury (Hg) and arsenic (As), are major pollutants in aquatic environments, posing ecological threats to living organisms due to their toxicity and bioaccumulation. This paper investigated whether zebrafish response to Hg and As displayed day/night differences. Fish were exposed to either 35 µg/L of mercury chloride for 6 h or 65 mg/L of sodium arsenate for 4 h, at two different times of the day: mid-light (day; ML) and mid-darkness (night; MD). Fish were video-recorded to investigate their behavioural response and at the end of each trial, gills and liver samples were collected for gene expression measurement. Gills, liver and brain samples were also obtained to determine Hg and As concentration. A control group (non-exposed) was video-recorded and sampled too. The effect of Hg and As on zebrafish swimming activity and the expression of antioxidant and metallothionein genes was time-of-day-dependent, with a stronger response being observed during the day than at night. However, the neurobehavioural effect of Hg was more affected by the time of exposure than the effect of As. In addition, Hg concentration in the gills was significantly higher in zebrafish exposed at ML than at MD. Altogether, these findings suggest that zebrafish response to Hg and As is time-of-day-dependent and remark the importance of considering toxicity rhythms when using this fish species as a model in toxicological research.

Administration time-dependent effects of poly (I:C) on antioxidant and immune responses along the diurnal time scale in zebrafish.

The circadian clock of vertebrates regulates many biological processes, including the immune system. This paper investigated whether responsiveness to poly (I:C), a synthetic analog of double-stranded RNA used as an immunostimulant, exhibits day/night differences in zebrafish. Fish were intraperitoneally (IP) injected with either phosphate-buffered saline (PBS) or poly (I:C) at two different time points: "Zeitgeber Time" (ZT) 4 (day) and ZT16 (night). Then, 6 h later, fish were euthanized, and tissue samples (skin, liver and kidney) were collected. A control group (intact fish) was also sampled at the same time points. The effect of poly (I:C) on the expression of antioxidant and immune genes was time-of-day-dependent, and the response was stronger following poly (I:C) administration in the day than at night. Time-dependent differences were observed for some genes in the PBS and control groups. However, these differences were tissue-specific. In liver, almost all the genes were affected by time of day. In kidney, poly (I:C) affected the expression of all the gene markers regardless of administration time. These findings highlight the importance of considering the time to administer poly (I:C) when evaluating the fish immune response.

Ethanol toxicity differs depending on the time of day.

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.

A new approach to feed frequency studies and protein intake regulation in juvenile pirarucu

ABSTRACT 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.

A new approach to feed frequency studies and protein intake regulation in juvenile pirarucu.

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.

Circadian rhythms of clock gene expression in Nile tilapia (Oreochromis niloticus) central and peripheral tissues: influence of different lighting and feeding conditions.

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

The Light Wavelength Affects the Ontogeny of Clock Gene Expression and Activity Rhythms in Zebrafish Larvae.

Light plays a key role in synchronizing rhythms and setting the phase of early development. However, to date, little is known about the impact of light wavelengths during the ontogeny of the molecular clock and the behavioural rhythmicity. The aim of this research was to determine the effect of light of different wavelengths (white, blue and red) on the onset of locomotor activity and clock gene (per1b, per2, clock1, bmal1 and dbp) expression rhythms. For this purpose, 4 groups of zebrafish embryo/larvae were raised from 0 to 7 days post-fertilization (dpf) under the following lighting conditions: three groups maintained under light:dark (LD) cycles with white (full visible spectrum, LDW), blue (LDB), or red light (LDR), and one group raised under constant darkness (DD). The results showed that lighting conditions influenced activity rhythms. Larvae were arrhythmic under DD, while under LD cycles they developed wavelength-dependent daily activity rhythms which appeared earlier under LDB (4 dpf) than under LDW or LDR (5 dpf). The results also revealed that development and lighting conditions influenced clock gene expression. While clock1 rhythmic expression appeared in all lighting conditions at 7 dpf, per1b, per2 and dbp showed daily variations already at 3 dpf. Curiously, bmal1 showed consistent rhythmic expression from embryonic stage (0 dpf). Summarizing, the data revealed that daily rhythms appeared earlier in the larvae reared under LDB than in those reared under LDW and LDR. These results emphasize the importance of lighting conditions and wavelengths during early development for the ontogeny of daily rhythms of gene expression and how these rhythms are reflected on the behavioural rhythmicity of zebrafish larvae.

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio).

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.

Feeding entrainment of food-anticipatory activity and per1 expression in the brain and liver of zebrafish under different lighting and feeding conditions.

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

Light and temperature cycles as zeitgebers of zebrafish (Danio rerio) circadian activity rhythms.

Light and temperature cycles are the most important synchronizers of biological rhythms in nature. However, the relative importance of each, especially when they are not in phase, has been poorly studied. The aim of this study was to analyze the entrainment of daily locomotor activity to light and/or temperature cycles in zebrafish. Under two constant temperatures (20 degrees C and 26 degrees C) and 12:12 light-dark (LD) cycles, zebrafish were most active during the day (light) time and showed higher total activity at the warmer temperature, while diurnalism was higher at 20 degrees C than at 26 degrees C (87% and 77%, respectively). Under thermocycles (12:12 LD, 26:20 degrees C thermophase:chryophase or TC), zebrafish daily activity synchronized to the light phase, both when the thermophase and light phase were in phase (LD/TC) or in antiphase (LD/CT). Under constant dim light (3 lux), nearly all zebrafish synchronized to thermocycles (tau=24 h), although activity rhythms (60% to 67% of activity occurred during the thermophase) were not as marked as those observed under the LD cycle. Under constant dim light of 3 lux and constant temperature (22.5 degrees C), 4 of 6 groups of zebrafish previously entrained to thermocycles displayed free-running rhythms (tau=22.9 to 23.6 h). These results indicate that temperature cycles alone can also entrain zebrafish locomotor activity.