The occurrence of emotions in non-human animals has been the focus of debate over the years. Recently, an interest in expanding this debate to non-tetrapod vertebrates and to invertebrates has emerged. Within vertebrates, the study of emotion in teleosts is particularly interesting since they represent a divergent evolutionary radiation from that of tetrapods, and thus they provide an insight into the evolution of the biological mechanisms of emotion. We report that Sea Bream exposed to stimuli that vary according to valence (positive, negative) and salience (predictable, unpredictable) exhibit different behavioural, physiological and neuromolecular states. Since according to the dimensional theory of emotion valence and salience define a two-dimensional affective space, our data can be interpreted as evidence for the occurrence of distinctive affective states in fish corresponding to each the four quadrants of the core affective space. Moreover, the fact that the same stimuli presented in a predictable vs. unpredictable way elicited different behavioural, physiological and neuromolecular states, suggests that stimulus appraisal by the individual, rather than an intrinsic characteristic of the stimulus, has triggered the observed responses. Therefore, our data supports the occurrence of emotion-like states in fish that are regulated by the individual’s perception of environmental stimuli.

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.

The existence of daily rhythms under light/dark (LD) cycles in plasma cortisol, blood glucose and locomotor and self-feeding activities, as well as their persistence (circadian nature) under constant light (LL), was investigated in Senegalese sole (Solea senegalensis). For the cortisol and glucose rhythms study, 48 soles were equally distributed in 8 tanks and exposed to a 12:12 LD cycle and natural water temperature (experiment 1). After an acclimation period, blood was sampled every 3 h until a 24-h cycle was completed. Blood glucose levels were measured immediately after sampling, while plasma cortisol was measured later by ELISA. In experiment 2, the fish were exposed to LL for 11 days, and after this period, the same sampling procedure was repeated. For the study of locomotor and self-feeding rhythms (experiment 3), two groups of sole were used: one exposed to LD and the other to LL. Each group was distributed within 3 tanks equipped with infrared photocells for the record of locomotor activity, and self-feeders for feeding behavior characterization. The results revealed a marked oscillation in cortisol concentrations during the daily cycle under LD, with a peak (35.65 ± 3.14 ng/ml) in the afternoon (15:00 h) and very low levels during the night (5.30 ± 1.09 ng/ml). This cortisol rhythm persisted under LL conditions, with lower values (mean cortisol concentration = 7.12 ± 1.11 ng/ml) and with the peak shifted by 3 h. Both rhythms were confirmed by COSINOR analysis (p < 0.05). The synchronizing role of temperature and feeding schedule, in addition to light, is also discussed. Diel rhythms of glucose were not evident in LD or LL. As to locomotor and self-feeding activity, a very marked rhythm was observed under LD, with higher activity observed during the night, with acrophases located at 2:14 and 3:37 h, respectively. The statistical significance of daily rhythms was confirmed by COSINOR analysis. Under LL, both feeding and locomotor rhythms persisted, with an endogenous period (τ) around 22.5 h. In short, our findings described for the first time the existence of circadian cortisol and behavioral circadian rhythms in flat fish. Such results revealed the importance of taking into account the time of day when assessing stress responses and evaluating physiological indicators of stress in fish.

Background: Vitamin K (VK) is a fat-soluble vitamin better known for its essential role in blood coagulation, but its involvement in a range of other biological processes is not yet fully understood. Methods: Here we explore the potential role of VK in reproduction in a fish teleost model species, the Senegalese sole (Solea senegalensis), showing male reproductive dysfunction by means of biochemistry, transcriptomic and bioinformatics analysis. Results: Fish fed with dietary VK supplementation had increased testosterone plasma levels and lower sperm DNA fragmentation in males. Using a transcriptomic approach, small non-coding RNAs (sncRNAs) from blood plasma were sequenced and several differentially expressed sncRNAs (2 piRNAs and 7 miRNAs transcripts) were associated with nutritional status and/or better sperm quality. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed that they may be involved in blood coagulation and gonadotropin-releasing hormone pathways, among other processes. Conclusions: Present results suggest an unexpected and complex regulation of the nutritional status and reproductive performance at the level of the whole organism through circulating sncRNAs.

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.

A periodicity of 29 days was observed in spawning rhythms in Senegal sole Solea senegalensis, with an acrophase around the last quarter and the new moon. In both spring and autumn, a very marked nocturnal spawning rhythm was registered, with spawning beginning after dusk and the acrophase occurring around 2300 hours. When the photoperiod was artificially extended (from 10L:14D to 14L:10D), S. senegalensis synchronized to the new photoperiod: spawning took place after the new 'dusk', the beginning gradually shifting from 2100 to 2300 hours and the acrophase from 2325 to 0032 hours. Under continuous light conditions, fish sustained rhythmicity for 2 days, with an acrophase at 2249 hours, which suggested the existence of an endogenous pacemaker controlling the daily spawning rhythm. These findings provided new insights for better understanding the reproductive physiology of this species and for optimizing the timing protocols of egg collection and larvae production in S. senegalensis aquaculture.

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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 degrees C) and those exposed to the environmental temperature cycle (11.5-25 degrees 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 degrees 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.