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.

Comparative study of pineal clock gene and AANAT2 expression in relation to melatonin synthesis in Atlantic salmon (Salmo salar) and European seabass (Dicentrarchus labrax).

The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 &2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway.

Daily rhythms in the behavioural stress response of the zebrafish Danio rerio.

In nature, animals are exposed to stressors that occur with different likelihood throughout the day, such as risk of predation and human disturbance. Hence, the stress response is expected to vary plastically to adaptively match these challenges. Several studies have supported this hypothesis in a wide range of vertebrate species, including some teleost fish, mostly through evidence of circadian variation in physiology. However, in teleost fish, circadian variation in behavioural stress responses is less understood. Here, we investigated the daily rhythm of stress response at the behavioural level in the zebrafish Danio rerio. We exposed individuals and shoals to an open field test every 4 h over a 24 h cycle, recording three behavioural indicators of stress and anxiety levels in novel environments (thigmotaxis, activity and freezing). Thigmotaxis and activity significantly varied throughout the day with a similar pattern, in line with a stronger stress response in the night phase. The same was suggested by analysis of freezing in shoals, but not in individual fish, in which variation appeared mostly driven by a single peak in the light phase. In a control experiment, we observed a set of subjects after familiarisation with the open-field apparatus. This experiment indicated that activity and freezing might present a daily rhythmicity that is unrelated to environmental novelty, and thus to stress responses. However, the thigmotaxis was constant through the day in the control condition, suggesting that the daily variation of this indicator is mostly attributable to the stress response. Overall, this research indicates that behavioural stress response of zebrafish does follow a daily rhythm, although this may be masked using behavioural indicators other than thigmotaxis. This rhythmicity can be relevant to improve welfare in aquaculture and reliability of behavioural research in fish models.

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.

Influence of constant light and darkness, light intensity, and light spectrum on plasma melatonin rhythms in senegal sole.

Light is the most important synchronizer of melatonin rhythms in fish. This paper studies the influence of the characteristics of light on plasma melatonin rhythms in sole. The results revealed that under long-term exposure to constant light conditions (LL or DD), the total 24 h melatonin production was significantly higher than under LD, but LL and DD conditions influenced the rhythms differently. Under LL, melatonin remained at around 224 pg/ml throughout the 24 h, while under DD a significant elevation (363.6 pg/ml) was observed around the subjective evening. Exposure to 1 h light pulses at MD (mid-dark) inhibited melatonin production depending on light intensity (3.3, 5.3, 10.3, and 51.9 microW/cm(2)). The light threshold required to reduce nocturnal plasma melatonin to ML (mid-light) values was 5.3 microW/cm(2). Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (lambda>600 nm) failed to reduce plasma melatonin significantly, far violet light (lambda(max)=368 nm) decreased indoleamine's concentration to ML values. These results suggest that dim light at night (e.g., moonlight) may be perceived and hence affect melatonin rhythms, encouraging synchronization to the lunar cycle. On the other hand, deep red light does not seem to inhibit nocturnal melatonin production, and so it may be used safely during sampling at night.

Circadian rhythms of gene expression of lipid metabolism in Gilthead Sea bream liver: synchronisation to light and feeding time.

This research aimed at investigating circadian rhythm expression of key genes involved in lipid metabolism in the liver of a teleost fish (Sparus aurata), and their synchronisation to different light-dark (L-D) and feeding cycles. To this end, 90 gilthead sea bream were kept in 12:12 h (light:dark, LD, lights on at ZT0) and fed a single daily meal at mid-light (ML = ZT6), mid-darkness (MD = ZT18) and randomly (RD) at a 1.5% body weight ration. A total of 18 tanks were used, six tanks per feeding treatment with five fishes per tank; locomotor activity was recorded in each tank. After 25 days of synchronisation to these feeding regimes, fishes were fasted for one day and liver samples were taken every 4 hours during a 24 h cycle (ZT2, 6, 10, 14, 18 and 22) and stored at -80 °C until analysis. To determine whether the rhythm expression presented an endogenous control, another experiment was performed using 30 fish kept in complete darkness and fed randomly (DD/RD). Samples were taken following the same procedure as above. The results revealed that all genes investigated exhibited well defined daily rhythms. The lipolysis-related and fatty acid turnover genes (hormone-sensitive lipase (hsl) and peroxisome proliferator-activated receptor-α (pparα)) exhibited a nocturnal achrophase (Ø = ZT18:03-19:21); lipoprotein lipase (lpl) also showed the same nocturnal achrophase (Ø = ZT20:04-21:36). In contrast, lipogenesis-related gene, fatty acid synthase (fas), and of fatty acid turnover, cyclooxygenase (cox-2), showed a diurnal rhythm (Ø = ZT2:27-8:09); while pparγ was nocturnal (Ø = ZT16:16-18:05). Curiously, feeding time had little influence on the phase of these daily rhythms, since all feeding groups displayed similar achrophases. Furthermore, under constant conditions pparα and hsl showed circadian rhythmicity. These findings suggest that lipid utilisation in the liver is rhythmic and strongly synchronised to the LD cycle, regardless of feeding time, which should be taken into consideration when investigating fish nutrition and the design of feeding protocols.

Effect of lighting conditions on zebrafish growth and development.

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

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 of clock gene expression, glycaemia and digestive physiology in diurnal/nocturnal European seabass.

Seabass is a fish species with dual (diurnal/nocturnal) feeding behavior, although little is known about changes in its molecular clock, physiology and metabolism linked to this dual behavior. In the research described here possible differences in clock gene expression in central (brain) and peripheral (liver) oscillators, and in physiology (blood glucose and amylase activity in mid-intestine) were studied in seabass with diurnal or nocturnal self-feeding patterns under LD 12:12h (light:dark) (lights on=Zeitgeber Time (ZT) 00:00h). The results revealed that per1 expression in brain shows daily rhythmicity with the acrophase (Φ) around the lights offset (ZT 12:00h, Cosinor, p<0.01) in both diurnal and nocturnal seabass. In liver, per 1 daily levels of expression were higher in diurnal fish (univariate GML, p<0.02). Daily blood glucose variations were observed in both groups (ANOVA I, p<0.01), with higher glucose levels occurring at night in nocturnal as well as in diurnal fish, although only diurnal seabass displayed a significant daily rhythm (Φ=ZT 16:52h, Cosinor, p<0.02). The highest values of amylase activity coincided with the feeding-phase of fish; that is, in nocturnal seabass the maximum was reached at ZT 18:00h (ANOVA I, p<0.01), whereas in diurnal seabass the Φ was ZT 03:39h (Cosinor, p<0.02). In short, our findings indicated that the feeding rhythm (diurnal vs. nocturnal) strongly influenced the daily patterns of digestive function and clock gene expression in the liver (feeding-entrained clock), but not in the brain (light-entrained clock).

Exposure of larvae to daily thermocycles affects gonad development, sex ratio, and sexual steroids in Solea senegalensis, kaup.

The effect of water temperature during the development of fish larvae on sex differentiation is well known, but not so well known is the impact of the daily thermocycles. Our aim was to investigate the effect of early exposure of Senegal sole larvae to different temperature cycles on gonad development, sex ratio, and sex steroid (11-ketotestosterone (11-KT); estradiol (E(2) ); and testosterone, (T)) content in muscle extracts of juveniles. From 1 to 97 days posthatching (DPH) fish larvae and post-larvae were subjected to three temperature regimes: Thermophase-Cryophase (TC), Cryophase-Thermophase (CT), and constant temperature. In fish exposed to TC, sex determination occurred earlier, because 90% of soles were males/females at 110 DPH, whereas 45% of fish under CT were undifferentiated at that time. Fish under TC showed the highest growth rates, followed by fish under constant temperature and by fish under CT, the differences being statistically significant between the TC and CT groups. Regarding sex ratio, juveniles exposed to TC showed a higher proportion of females than fish under CT or constant temperature. Under TC, fish showed the highest concentration of E(2) , whereas 11-KT concentration was highest in fish under CT and constant temperature. Fish under constant temperature and CT showed higher T levels than those under TC. These results provide the first insights into the effect of daily thermocycles on sex differentiation in fish, and underline the key role of natural environmental cycles on the control of sex ratios during larval development, which may be applied to the manipulation of sex ratio in aquaculture.

Circadian rhythms of locomotor activity in the Nile tilapia Oreochromis niloticus.

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (tau) of 24.1+/-0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average tau of 23.9+/-0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7-10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.