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.

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol.

The effects of starvation and re-feeding on metabolites and tissue composition, GH/IGF-I axis, and digestive enzyme activities in juvenile thick-lipped grey mullet (Chelon labrosus) were evaluated. Fish were divided into three feeding groups (n=72, 82.00±4.09 g initial body mass). The control group was fed 1% of their body mass once a day throughout the experiment with commercial pellets. The other two groups were deprived of feed for 21 days (starved), or re-fed for 7 days after 14 days of food deprivation (re-fed). Full-length cDNAs from pituitary GH and hepatic IGF-I were cloned by screening a cDNA library or by PCR techniques. Furthermore, changes in their mRNA expressions were assessed by real time PCR in specimens maintained under the different feeding patterns. Results showed a negative growth in starved and re-feeding groups. Starvation induced a significant increase in plasma triglycerides as well as a decrease in liver glucose and glycogen. Re-feeding increased plasma glucose, lactate and protein, as well as liver glucose and glycogen. In addition, starvation significantly increased pituitary GH expression, while re-feeding down-regulated it. No significant changes were observed in hepatic IGF-I expression in any dietary treatment. Digestive enzyme activities were not significantly affected either by starvation or by re-feeding. The results of the present work suggest that juveniles of the thick-lipped grey mullet may easily adjust their metabolism under situations characterized by a short-term starvation and re-feeding.

Since fish show daily rhythms in most physiological functions, it should not be 27 surprising that stressors may have different effects depending on the timing of exposure. 28Here we investigated the influence of time of day on the stress responses, at both 29 physiological and cellular levels, in gilthead sea bream (Sparus aurata L.) submitted to 30 air exposure for 30 s and then returned to their tank. One hour after air exposure, blood, 31 hypothalamus and liver samples were taken. Six fish per experimental group (control 32 and stressed) were sampled every 4 h during a 24-h cycle. Fish were fed in the middle 33 of the light cycle (ML) and locomotor activity rhythms were recorded using infrared 34 photocells to determine their daily activity pattern of behavior, which showed a peak 35 around feeding time in all fish. In the control group cortisol levels did not show daily 36 rhythmicity whereas in the stressed fish a daily rhythm of plasma cortisol was observed, 37 being the average values higher than in the control group, with increased differences 38 during the dark phase. Blood glucose showed daily rhythmicity in the control group but 39 not in the stressed one which also showed higher values at all sampling points. In the 40 hypothalamus of control fish a daily rhythm of corticotropin-releasing hormone (crh) 41 gene expression was observed, with the acrophase at the beginning of the light phase. 42However, in the stressed fish, this rhythm was abolished. The expression of 43 corticotropin-releasing hormone binding protein (crhbp) showed a peak at the end of 44 the dark phase in the control group, whereas in the stressed sea bream this peak was 45 found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: i) 46 cytochrome c oxidase 4 (coxIV) showed daily rhythmicity in both control and stressed 47 fish, with the acrophases located around ML, ii) peroxiredoxin 3 (prdx3) and 5 (prdx5) 48 only presented daily rhythmicity of expression in the stressed fish, with the acrophase 49 located at the beginning of the light cycle, and iii) uncoupling protein 1 (ucp1) showed 50 3 significant differences between sampling points only in the control group, with 51 significantly higher expression at the beginning of the dark phase. Taken Fish in the wild and in aquaculture facilities face a variety of challenges, such as 77 attacks from predators, food competition, disturbance and exposure to poor water 78 quality, which seriously compromise fish welfare (Huntingford et al., 2006). The 79 specimens react to these adverse conditions through cellular, neuroendocrine and 80 behavioral adjustments, although the circadian mechanisms controlling these responses 81are not fully understood (Kulkckzykowska & Sánchez-Vázquez, 2010). 82The primary physiological response to stress in fish involves two major 83 neuroendocrine pathways: i) the hypothalamic sympathetic chromaffin cells (HSC) axis, 84and ii) the hypothalamic-pituitary-interrenal (HPI) axis (Wendelaar Bonga, 1997). In 85 the HPI cascade, corticotropin-...

Activities of pepsin, trypsin, chymotrypsin, alphaamylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick-lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 AE 3.0 g), Group 2 (180.9 AE 4.2 g), and Group 3 (328.5 AE 43.3 g). SDS-PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha-amylase and no lipase activity recorded in pre-adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.