Catarina Oliveira

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.

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)

Monthly day/night changes and seasonal daily rhythms of sexual steroids in Senegal sole (Solea senegalensis) under natural fluctuating or controlled environmental conditions

In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.

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)

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36 per thousand), isotonic water (15 per thousand), brackish water (4 per thousand) and freshwater (0 per thousand). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.

Melatonin Binding Sites in Senegal Sole: Day/Night Changes in Density and Location in Different Regions of the Brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (Bmax) at mid‐light (ZT06) and mid‐dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2‐[125I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid‐light (189.5±46 pg/ml) than mid‐dark (455.5±163 pg/ml). Values of Bmax were statistically significantly higher in the optic tectum (5.6±0.6 and 12.3±1 fmol/mg prot, at mid‐light and mid‐dark, respectively) and in the cerebellum (7.7±1.1 and 10.6±1.3 fmol/mg prot, at mid‐light and mid‐dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down‐regulation.

Circadian Rhythms of Embryonic Development and Hatching in Fish: A Comparative Study of Zebrafish (Diurnal), Senegalese Sole (Nocturnal), and Somalian Cavefish (Blind)

During early development, most organisms display rhythmic physiological processes that are shaped by daily changes in their surrounding environment (i.e., light and temperature cycles). In fish, the effects of daily photocycles and their interaction with temperature during early developmental stages remain largely unexplored. We investigated the existence of circadian rhythms in embryonic development and hatching of three teleost species with different daily patterns of behavior: diurnal (zebrafish), nocturnal (Senegalese sole), and blind, not entrained by light (Somalian cavefish). To this end, fertilized eggs were exposed to three light regimes: 12 h of light: 12 h of darkness cycle (LD), continuous light (LL), or continuous darkness (DD); and three species-appropriate temperature treatments: 24°C, 28°C, or 32°C for zebrafish and cavefish and 18°C, 21°C, or 24°C for sole. The results pointed to the existence of daily rhythms of embryonic development and hatching synchronized to the LD cycle, with different acrophases, depending on the species: zebrafish embryos advanced their developmental stage during the light phase, whereas sole did so during the dark phase. In cavefish, embryogenesis occurred within 24 h post fertilization (hpf) at the same pace during day or night. The hatching rhythms appeared to be controlled by a clock mechanism that restricted or “gated” hatching to a particular time of day/night (window), so that embryos that reached a certain developmental state by that time hatch, whereas those that have not wait until the next available window. Under LL and DD conditions, hatching rhythms and the gating phenomenon persisted in cavefish, in zebrafish they split into ultradian bouts of hatching occurring at 12–18-h intervals, whereas in sole DD and LL produced a 24-h delay and advance, respectively. Hatching rates were best under the LD cycle and the reported optimal temperature for each species (95.2 ± 2.7% of the zebrafish and 83.3 ± 0.1% of the cavefish embryos hatched at 28°C, and 93.1 ± 2.9% of the sole embryos hatched at 21°C). In summary, these results revealed that hatching rhythms in fish are endogenously driven by a time-keeping mechanism, so that the day and time of hatching are determined by the interplay between the developmental state (temperature-sensitive) and the circadian clock (temperature-compensated), with the particular phasing being determined by the diurnal/nocturnal behavior of the species. (Author correspondence: javisan@um.es)

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

This research aimed to investigate melatonin rhythms in the pineal organ of two nocturnal fish species, sole and tench, which show high sensitivity to light. Pineal organs were cultured in vitro under an LD (12 h light:12 h dark) cycle to study the daily rhythmicity of melatonin release. In addition, the in vitro culture was performed under conditions of constant darkness (DD) to study the endogenous control of the rhythm. In the pineal organs cultured under an LD cycle, rhythmic melatonin release was evident in both species, with low values observed during the photophase (15.6+/-7.2 and 22.6+/-2.6 pg/mL for sole and tench, respectively) and high values coinciding with the scotophase (74.0+/-8.2 and 82.1+/-9.1 pg/mL, for sole and tench, respectively). Under LD, the rhythm had a period of 24 h (p<0.001) and presented similar acrophases for both species, located around 9-10 h after lights off (2 and 3 h before the end of the dark phase). When the pineal organs were cultured under DD, the results differed between the species studied. A marked circadian rhythm in melatonin release by the pineal was registered in tench, with lower values during the subjective day, i.e. the period that was previously day (6.2+/-1.6 pg/mL) and higher values during the subjective night, i.e. the period that was previously night (20.4+/-5.5 pg/mL). The rhythm had a mean tau of 24.1 h (p<0.01) and the acrophase was located around 12 h after lights off (the beginning of the subjective day), slightly later than that registered under LD conditions. In contrast, melatonin values in sole remained high during darkness (around 92.0+/-6.9 pg/mL) for four consecutive days, including subjective day periods. In short, these findings revealed that the rhythm of melatonin release in tench is under endogenous control by a circadian oscillator within the pineal organ, while no such pacemaker was evident in sole, which melatonin rhythm appeared to be exclusively light-driven.