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.     

Circadian Rhythms of Self-feeding and Locomotor Activity in Zebrafish (Danio Rerio)

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

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. (Author correspondence: jflopez@um.es)     

FEEDING ENTRAINMENT OF DAILY RHYTHMS OF LOCOMOTOR ACTIVITY AND CLOCK GENE EXPRESSION IN ZEBRAFISH BRAIN

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

Light Synchronization of the Daily Spawning Rhythms of Gilthead Sea bream (Sparus aurata L) Kept under Different Photoperiod and after Shifting the LD Cycle

Reproduction in most fish is typically a seasonal process, as spawning takes place usually at a given time of the year, depending on the reproduction strategy of the species, to ensure maximal survival of offspring. Nevertheless, fish reproduction cannot be considered an exclusively annual phenomenon, because spawning may also show daily rhythmicity. In this study, we investigated the existence of a daily spawning rhythm in gilthead seabream (Sparus aurata L) exposed to different light‐dark (LD) cycles and at different times of the year using an automatic and programmable egg collector. Floatability and fertilization rates were analyzed at different times throughout the 24 h. The results showed a daily spawning rhythm with spanning occurring from 14:30 to 21:30 h, with the acrophase (peak time) being 18:29 and 18:08 h in fish exposed to an artificial photoperiod of 9L∶15D in winter and in spring, respectively. Nevertheless, in fish exposed to a natural photoperiod of 12L∶12D in spring, the acrophase of the rhythm was recorded later, at 21:28 h. The average fertilization rate was 87%, and no significant differences were found between the different hours of spawning. Moreover, when the LD cycle (9L∶15D) was shifted by 12 h, the daily spawning rhythm gradually re‐synchronized, resuming a stable phase‐relationship after 4–5 transient days, which is characteristic of a endogenous circadian rhythm. Our results clearly demonstrated the existence of a 24 h period of spawning in gilthead sea bream, with a peak anticipating the forthcoming night, and its capacity to gradually re‐synchronize after a 12 h shift in the LD cycle, pointing to the endogenous nature of this rhythm. These findings will be valuable for better understanding the reproductive physiology of this species and for optimizing the protocols of egg collection and larvae production in aquaculture. (Author correspondence: javisan@um.es)     

Acute stress response in gilthead sea bream (Sparus aurata L.) is time-of-day dependent: Physiological and oxidative stress indicators

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

Daily rhythms in the somatotropic axis of Senegalese sole (Solea senegalensis): The time of day influences the response to GH administration

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.     

Circadian rhythms of self-feeding and locomotor activity in zebrafish (Danio Rerio)

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

Effects of starvation,re-feeding and timing of food supply on daily rhythm features of gut melatonin in carp (Catla catla)

Influences of starvation, re-feeding and time of food supply on daily rhythm features of melatonin (5-methoxy-N-acetyltryptamine) and its key regulator AANAT (arylalkylamine N-acetyltransferase) protein in the gut tissues were separately evaluated in carp Catla catla. The first experiment was aimed at demonstration of duration dependent effects of starvation and re-feeding after starvation on the daily profiles and rhythm features of gut melatonin and AANAT. Accordingly, juvenile carp were randomly distributed in three groups, which were (a) provided with balanced diet daily at a fixed time, that is, 10:00 clock hour or zeitgeber time (ZT) 4 (control), or (b) starved (for 2-, 4-, 6- or 8 days), or (c) initially starved for 8 days and then re-fed (for 2-, 4-, 6-, 8-, 12- or 16 days) daily with the same food and at the time (ZT4) used for control fish. The carp in each group were sampled for collection of gut tissues at six different time points at a regular interval of 4?h in a daily cycle. In another experiment, the influences of timing of food supply were separately examined in four fish groups, which were provided with a fixed amount of food once daily either at 06:00 or 12:00 or 18:00 or 24:00 clock hour corresponding to ZT0 or ZT6 or ZT12 or ZT18, respectively, for 7 days before sampling at 12 different time points with a regular interval of 2?h in a 24-h cycle. The study revealed a gradual increase in the mesor and amplitude values of melatonin and AANAT in gut with the progress of starvation till their values reached maximum at day-6 and remained steady thereafter. In contrast, re-feeding of 8-day starved fish resulted in a sharp decrease in their mesor and amplitude values after 2 days and then followed by a steady-state increase till re-attainment of their values close to control fish at the end of 16 days. The acrophase of these gut variables in each control, starved and re-fed fish was noted mostly at midday or ZT6. However, the results of another experiment demonstrated that a shift of food supply time led to a shift in their acrophase. The amount of residual food in the gut lumen in each, but not starved, fish by showing a significant positive correlation independently with the gut levels of melatonin and AANAT also indicated possible role of food as the synchronizer for their daily rhythms. Collectively, it appears reasonable to argue that daily profiles of gut melatonin and AANAT are strongly influenced by the availability of food, while their daily rhythm features seem to be dependent mostly on the time of food supply in carp.     

Entrainment of circadian rhythms of cholesterol‐LDL,corticosterone and motor activity by feeding schedules in rats

Abstract

The daily variations of locomotor activity, plasma and adrenal corticosterone levels and cholesterol‐LDL were studied in male Wistar rats with food ad libitum and feeding restricted to the first 4 hours of the light phase in LD 12:12..

Under LD 12:12 (light on from 9:00 to 21:00h) rats with food ad libitum were eating and moving during the dark period and the locomotor activity clearly showed a biphasic pattern with three harmonic components. Plasma and adrenal corticosterone levels increased during the light period and reached a maximum value just before the dark period whereas the acrophase of cholesterol‐LDL is found at the beginning of the light phase.

The acrophases of activity, plasma and adrenal corticosterone levels in the restricted feeding schedule rats occurred in the first three hours of lighting and the cholesterol‐LDL acrophase at the beginning of the dark phase.

These results confirm a previous report that the shift of feeding to the light phase seems to cause a concomitant phase‐shift in all the variables measured.     

Daily Rhythms in the Hypothalamus-Pituitary-Interrenal Axis and Acute Stress Responses in a Teleost Flatfish,Solea senegalensis

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. (Author correspondence: jflopez@um.es)     

鳜肝脏和心脏生物钟基因表达节律分析

在12h光照、12h黑暗交替(Light-Dark; LD)光制下,研究分析了褪黑素和皮质醇水平在鳜血清中的昼夜变化规律,以及13个生物钟基因(Arntl1、Clock、Cry1a、Cry3、Cry-dash、Npas2、Npas4、Nr1d1、Nr1d2、Per1、Per3、Rora和Tim)在鳜(Siniperca chuatsi)肝脏和心脏中的昼夜表达规律。试验在一昼夜内的ZT0(06:00)、ZT3(09:00),ZT6(12:00),ZT9(15:00),ZT12(18:00),ZT15(21:00),ZT18(24:00),ZT21(03:00,2nd d),ZT24(06:00,2nd d) (Zone time,ZT) 9个时间点随机抽取3尾鳜采集其血清、肝脏和心脏。经SPSS 单因素方差分析和Matlab余弦分析,结果显示: 鳜血清中褪黑素和皮质醇含量均呈现出昼夜节律性振荡,褪黑素含量白天显著降低(P0.05),夜间显著上升,皮质醇含量白天缓慢降低,夜间ZT15(21:00)-ZT18(24:00)显著升高,随后开始缓慢降低; 两种激素最低相位都为ZT15(21:00)。在13个生物钟基因中,Cry-dash、Npas4、Nr1d1、Per1和Tim 5个基因在鳜肝脏内具有明显的昼夜节律性,其中Npas4、Nr1d1、Per1、Tim的表达规律相似,皆呈现出光照阶段表达降低,黑暗阶段表达升高的趋势; 但Cry-dash则表现出光照阶段先升高后降低,黑暗阶段先降低后升高的规律。在鳜心脏中,Arntl1、Clock、Cry1a、Npas2、Nr1d1、Nr1d2、Per3、Rora和Tim 9个基因都表现出明显的昼夜节律,表达趋势分为两种: Arntl1、Clock、Nr1d2的表达量在光照阶段降低,黑暗阶段升高; 而Cry1a、Npas2、Nr1d1、Per3、Rora和Tim的表达量在ZT0(06:00)-ZT15(21:00)持续低水平,ZT15(21:00)-ZT18(24:00)表达量显著上升,ZT18(24:00)-ZT21(03:00)表达量降低。研究结果表明: 生物钟基因在鳜肝脏和心脏中所表达的昼夜节律不同。     

Does Lighting Manipulation During Incubation Affect Hatching Rhythms and Early Development of Sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD_W), blue light (LD_B; λ_peak?=?463?nm), or red light (LD_R; λ_peak?=?685?nm). Eggs exposed to LD_B had the highest hatching rate (94.5%?±?1.9%), whereas LD_R and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD_B (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD_W and LD_R (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD_B showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD_R and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish. (Author correspondence: borja@um.es)     

Does lighting manipulation during incubation affect hatching rhythms and early development of sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD(W)), blue light (LD(B); λ(peak)?=?463?nm), or red light (LD(R); λ(peak)?=?685?nm). Eggs exposed to LD(B) had the highest hatching rate (94.5%?±?1.9%), whereas LD(R) and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD(B) (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD(W) and LD(R) (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD(B) showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD(R) and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish.     

Aging and photoperiod affect the daily rhythm pattern of atrial natriuretic peptide in the rat atrium

This study investigates the release characteristics of atrial natriuretic peptide (ANP) from young (10 weeks) and old (22 months) rat atrium. Levels of ANP release from samples of atrium were studied by organ perifusion. Rats were exposed to light:dark (LD) cycles of 12:12 or 18:6 and sacrificed at different zeitgeber time (ZT) points: ZT0, ZT6, ZT8, ZT12, ZT16, and ZT19 for LD 12:12 or ZT0, ZT9, ZT16, ZT18, ZT20, and ZT 21.5 for LD 18:6. The heart was collected, and the right atrium was removed, weighed, and perifused with Krebs-bicarbonate buffer for 100 min, including a period of 50 min for stabilization of secretion rate. ANP concentrations released by atrium did not differ between the two age groups either under LD 12:12 or under LD 18:6, except at the light:dark transition under LD 12:12 conditions where ANP levels were significantly (P < 0.05) lower in young compared to old rats. ANP exhibited daily variations in concentrations under LD 12:12, with a peak during the beginning of photophase (ZT0) in young rats and a peak at the beginning of scotophase (ZT12) in old animals. These variations were strongly modified under LD 18:6, where the pattern of the release exhibited a peak during the light phase at ZT16 in both young and old rats. This strongly suggests that the atrial ANP rhythm is dependent on the environmental light:dark cycle. Moreover, the total ANP levels released by atria in old rats were significantly increased under LD 18:6 compared to standard LD 12:12. This observation strongly suggests that old animals are more sensitive to a photoperiodic change. In conclusion, our results show that ANP concentrations in the rat atrium exhibit daily variations which are significantly affected by the daylength (photoperiod) change in aged rats.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD?+?RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD?+?RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture. (Author correspondence: javisan@um.es).     

Light synchronization of the daily spawning rhythms of gilthead sea bream (Sparus aurata L) kept under different photoperiod and after shifting the LD cycle

Reproduction in most fish is typically a seasonal process, as spawning takes place usually at a given time of the year, depending on the reproduction strategy of the species, to ensure maximal survival of offspring. Nevertheless, fish reproduction cannot be considered an exclusively annual phenomenon, because spawning may also show daily rhythmicity. In this study, we investigated the existence of a daily spawning rhythm in gilthead seabream (Sparus aurata L) exposed to different light-dark (LD) cycles and at different times of the year using an automatic and programmable egg collector. Floatability and fertilization rates were analyzed at different times throughout the 24 h. The results showed a daily spawning rhythm with spanning occurring from 14:30 to 21:30 h, with the acrophase (peak time) being 18:29 and 18:08 h in fish exposed to an artificial photoperiod of 9L:15D in winter and in spring, respectively. Nevertheless, in fish exposed to a natural photoperiod of 12L:12D in spring, the acrophase of the rhythm was recorded later, at 21:28 h. The average fertilization rate was 87%, and no significant differences were found between the different hours of spawning. Moreover, when the LD cycle (9L:15D) was shifted by 12 h, the daily spawning rhythm gradually re-synchronized, resuming a stable phase-relationship after 4-5 transient days, which is characteristic of a endogenous circadian rhythm. Our results clearly demonstrated the existence of a 24 h period of spawning in gilthead sea bream, with a peak anticipating the forthcoming night, and its capacity to gradually re-synchronize after a 12 h shift in the LD cycle, pointing to the endogenous nature of this rhythm. These findings will be valuable for better understanding the reproductive physiology of this species and for optimizing the protocols of egg collection and larvae production in aquaculture.     

SYNCHRONIZATION TO LIGHT AND RESTRICTED-FEEDING SCHEDULES OF BEHAVIORAL AND HUMORAL DAILY RHYTHMS IN GILTHEAD SEA BREAM (SPARUS AURATA)

Food is not continuously available in the wild, and so most animals show a wide variety of circadian rhythms that can be entrained to feeding time. The aim of this research was to evaluate the effect of time-restricted feeding on the daily rhythms of gilthead sea bream, with food being provided during the day or night under a 12:12?h light-dark (LD) cycle or constant light (LL) conditions. Self-feeding and locomotor activity, as well as daily rhythms of cortisol, glucose, and melatonin, were evaluated. Fish synchronized their feeding behavior to the feeding phase, so that in LD they displayed 78% nocturnal feeding activity under night-feeding and 81% diurnal feeding activity under day-feeding, while under LL-feeding they displayed 72% of their daily activity during the 12?h feeding phase. In contrast, locomotor activity was mostly diurnal (66–71%), regardless of the feeding schedule, and it became arrhythmic under LL. Cortisol showed daily rhythms that peaked at different times, depending on the light and feeding schedule: one peak several hours before feeding under day-feeding and night-feeding, and two peaks under LL-feeding. Glucose displayed low-amplitude variations, with no daily rhythms being detected. Melatonin, however, showed a nocturnal rhythm, regardless of the feeding schedule, while the rhythm became attenuated under LL. Taken together, these results highlight the role of feeding on endocrine and metabolic rhythms, suggesting that feeding behavior should be considered when studying these variables. (Author correspondence: jflopez@um.es)