Luisa María Vera

Feeding entrainment of locomotor activity rhythms, digestive enzymes and neuroendocrine factors in goldfish.

The existence of food anticipatory activity (FAA) in animals subjected to daily feeding schedules seems to be mediated by a feeding-entrainable oscillator (FEO). Such an FEO may help in anticipating meal time and so optimizing food acquisition and nutrient utilization. In this study we investigated the existence of FAA and whether digestive enzymes, plasma cortisol, hypothalamic NPY and gastrointestinal tract (GIT) and plasma melatonin were entrained by periodic feeding in goldfish. We observed that periodically fed goldfish showed FAA in locomotor activity as well as in amylase and NPY. Alkaline protease and GIT melatonin were higher after feeding, whereas plasma cortisol levels were reduced. Plasma melatonin remained unmodified before and after meal time. These results suggested that scheduled feeding entrained both behavioral and certain physiological patterns in goldfish, FAA being of adaptive value to anticipate a meal and prepare the digestive physiology of fish.

Daily locomotor activity and melatonin rhythms in Senegal sole (Solea senegalensis).

The daily locomotor and melatonin rhythms of the Senegal sole, a benthonic species of increasing interest in aquaculture, are still unknown, despite the fact that such knowledge is of prime importance for optimising its production. The aim of the present research was therefore to investigate the daily rhythms of locomotor activity and melatonin in the Senegal sole. For this purpose, the individual locomotor activity rhythms of fish were registered using a photocell. Plasma and ocular melatonin rhythms were studied in animals reared in circular tanks placed in earth under an LD 12:12 light regime and 16-18 degrees C temperature range (spring equinox). Blood and eye samples were taken every 3 h during a complete 24-h cycle. The impact of a light pulse in the middle of the dark period (MD) on plasma melatonin was also studied. Locomotor activity was mainly nocturnal, with 84.3% of the total activity occurring during darkness. The levels of plasma melatonin were higher at night (55 pg/ml) than during the day (2 pg/ml), while ocular melatonin levels appeared to be arrhythmic. Both weight and melatonin content were found to be significantly higher in the left eye in relation to the right eye. A light pulse in MD provoked a significant decrease in plasma melatonin levels. In summary, photoperiod is a key factor in synchronizing locomotor activity and melatonin rhythms in the Senegal sole, whose nocturnal habits should be taken into account for their rearing by aquaculture.

Seasonal and daily plasma melatonin rhythms and reproduction in Senegal sole kept under natural photoperiod and natural or controlled water temperature.

Abstract:  The melatonin daily rhythm provides the organism with photoperiod‐related information and represents a mechanism to transduce information concerning time of day. In addition, the duration and amplitude of the nocturnal elevation gives information about duration and thus the time of year. In this study, we investigate the existence of an annual rhythm of plasma melatonin in the Senegal sole. Differences in plasma melatonin levels between fish kept at a controlled temperature (17–20°C) and those exposed to the environmental temperature cycle (11.5–25°C) were also examined throughout the year. Spawning was registered in both groups to determine the time of year in which reproductive rhythms occurred. Our results pointed to the existence of an annual rhythm of plasma melatonin at mid‐darkness (MD), with the highest levels (203 ± 44 pg/mL) observed when water temperature reached 25°C. Water temperature influenced nocturnal, but not diurnal melatonin. Daily melatonin rhythms showed seasonal differences, with higher mean nocturnal levels during the summer solstice (138 ± 19 pg/mL) and autumn equinox (149 ± 49 pg/mL). When animals were kept at a constant temperature throughout the year, plasma melatonin levels differed from those observed in fish exposed to the environmental temperature cycle. Regarding the reproductive rhythms, spawning was observed at the end of spring in sole kept under natural temperature conditions, whereas no spawning at all was registered in sole reared at a constant temperature. In short, both photoperiod and temperature affected melatonin production in the Senegal sole, transducing seasonal information and controlling annual reproductive rhythms.

Differential light intensity and spectral sensitivities of Atlantic salmon, European sea bass and Atlantic cod pineal glands ex vivo

Photoperiod is perceived by pineal photoreceptors and transduced into rhythmic melatonin signals. These rhythms can be influenced by light intensity and spectral content. In this study we compared the light sensitivity of Atlantic salmon, European sea bass and Atlantic cod by testing ex vivo the effect of different intensities and narrow bandwidth lights on nocturnal melatonin suppression by isolated pineal glands in a flow-through culture system. Using combinations of neutral density and bandpass interference filters we tested a range of light intensities (ranging from 1.22x10(13) to 3.85x10(6) photons s(-1) cm(-2)) and three wavelengths of 80 nm width (472, 555 and 661 nm corresponding to blue, green and red, respectively). Results showed clear species specific light intensity and spectral sensitivities, with cod being from 100 to 1000 times more sensitive than sea bass and salmon. Regarding the influence of spectrum, red light was less efficient on suppressing melatonin than blue and green in salmon but results were not as clear in the two other species studied. Finally, the first evidence of relative photoreception in teleosts was obtained in cod suggesting that the definition of illuminance thresholds (day/night perception) would depend on the day intensity. Indeed, a single order of magnitude increase or decrease in day intensity was shown to elicit a significant shift in the intensity response curve of night-time melatonin suppression. Taken together, this study demonstrated species specific light intensity and spectral sensitivities within temperate teleosts.

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 µW/cm2). The light threshold required to reduce nocturnal plasma melatonin to ML (mid‐light) values was 5.3 µW/cm2. Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (λ>600 nm) failed to reduce plasma melatonin significantly, far violet light (λmax=368 nm) decreased indoleamines 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.

Influence of light intensity on plasma melatonin and locomotor activity rhythms in tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm2), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm2) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm2 range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm2 light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).

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).

Impact of Daily Thermocycles on Hatching Rhythms, Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.

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 (τ) 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 τ 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.

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.