Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

Daily rhythms of intestinal cholecystokinin and pancreatic proteases activity in Senegalese sole juveniles with diurnal and nocturnal feeding.

The influence of diurnal and nocturnal feeding on daily rhythms of gut levels of cholecystokinin (CCK) and the activity of two key pancreatic proteases, trypsin and chymotrypsin, were examined in juveniles of Senegalese sole (Solea senegalensis), a species with nocturnal habits. Four feeding protocols were performed: P1) One morning meal; P2) Six meals during the light period; P3) Six meals during the dark period; and P4) 12 meals during 24 h. Daily activity patterns of both proteases were remarkably similar and showed a high correlation in all the experimental protocols. In P1, daily patterns of CCK and digestive enzymes showed a single maximum. In P2, CCK levels exhibited two peaks. Digestive enzymes activities showed slightly delayed peaks compared to CCK, although their daily fluctuations were not significant. In P3, intestinal CCK concentration exhibited two peaks at the end of light and dark periods, but only the second one was significant. The first maximum level of chymotrypsin activity occurred 4 h after the first CCK peak, while the second one coincided with the second CCK peak. Fluctuations of trypsin activity were not significant. In P4, CCK concentration showed three small peaks. Digestive enzymes daily fluctuations were not significant, although they showed an inverted trend with respect to CCK. The daily pattern of the gut CCK content in our study is in agreement with the anorexigenic function of this hormone. Our results support the existence of a negative feedback regulatory loop between CCK and pancreatic proteolytic enzymes in Senegalese sole juveniles.

Daily rhythms in endocrine factors of the somatotropic axis and their receptors in gilthead sea bream (Sparus aurata) larvae.

Living organisms have adapted to environmental oscillations in light and temperature through evolving biological clocks. Biological rhythms are pervasive at all levels of the endocrine system, including the somatotropic (growth) axis. The objective of the present research was to study the existence of daily rhythms on the somatotropic axis of a marine teleost species, specifically, the gilthead sea bream (Sparus aurata). Larvae of S. aurata at 30 dph (days post hatching), kept under a 9 L:15D (light-dark) photoperiod, were collected every 3 h throughout a 36 h cycle. The expression of the following somatotropic axis genes was analyzed by quantitative PCR: pituitary adenylate cyclase-activating polypeptide 1 (adcyap1), prepro-somatostatin-1 (pss1), growth hormone (gh), growth hormone receptor types 1 and 2 (ghr1 and ghr2, respectively), insulin-like growth factor 1 (igf1) and igf1 receptor a (igf1ra). All genes displayed significant differences among time points and, with the exception of adcyap1, all showed statistically significant daily rhythms. The acrophases of gh, ghr1, ghr2, igf1 and igf1ra were located around the end of the dark phase, between ZT19:44 and ZT0:48 h, whereas the highest expression levels of adcyap1 occurred at ZT18 h. On the other hand, the acrophase of pss1, an inhibitor of Gh secretion, was located at ZT10:16 h, hence it was shifted by several hours with respect to the other genes. The present results provide the first thorough description of somatotropic axis rhythms in gilthead sea bream. Such knowledge provides insights into the role of rhythmic regulation of the Gh/Igf1 axis system in larval growth and metabolism, and it can also improve the implementation of more species-specific feeding regimes.

Ontogeny of Expression and Activity of Digestive Enzymes and Establishment of gh/igf1 Axis in the Omnivorous Fish Chelon labrosus.

Thick-lipped grey mullet (Chelon labrosus) is a candidate for sustainable aquaculture due to its omnivorous/detritivorous feeding habit. This work aimed to evaluate its digestive and growth potentials from larval to early juvenile stages. To attain these objectives the activity of key digestive enzymes was measured from three until 90 days post hatch (dph). Expression of genes involved in digestion of proteins (try2, ctr, pga2, and atp4a), carbohydrates (amy2a), and lipids (cel and pla2g1b), together with two somatotropic factors (gh and igf1) were also quantified. No chymotrypsin or pepsin activities were detected. While specific activity of trypsin and lipase were high during the first 30 dph and declined afterward, amylase activity was low until 57 dph and increased significantly beyond that point. Expression of try2, ctr, amy2a, and cel increased continuously along development, and showed a peak at the end of metamorphosis. Expression of pla2g1b, pga2 and atp4a increased until the middle of metamorphosis and decreased afterwars. Most of these trends contrast the usual patterns in carnivorous species and highlight the transition from larvae, with high protein requirements, to post-larvae/juvenile stages, with omnivorous/detritivorous feeding preferences. Somatotropic genes, gh and igf1, showed approximately inverse expression patterns, suggesting the establishment of the Gh/Igf1 axis from 50 dph.

Molecular basis of the digestive functionality in developing Persian sturgeon (Acipenser persicus) larvae: additional clues for its phylogenetic status.

Persian sturgeon (Acipenser persicus) is a critically endangered species, mainly due to overexploitation for its caviar. The permanence of populations of this species in the Caspian Sea is fully dependent on restocking programs. Accordingly, it is considered as an interesting target for aquaculture for both restocking and commercial purposes. In addition, as a Chondrostei, it exhibits one of the slowest rates of molecular evolution among vertebrates and is propounded as an excellent candidate for phylogenetic analysis and evolutionary biology. In this study, the early ontogeny of some key digestive enzymes precursors was determined at molecular level, aiming to obtain basic knowledge on the acquisition of digestive capacity of this species, and at the same time, to advance in its phylogenetic status from the point of view of digestion. For this purpose, A. persicus cDNAs for ß-actin (actb; used as an internal reference gene), bile-salt activated lipase (cel), trypsinogen 1 (try1), pepsinogen (pga), and gastric proton pump (atp4a) were amplified and cloned, and their subsequent expressions were measured by quantitative real-time PCR during the first 34 days post hatch (dph). Two isoforms for pga and at least six for try1 were obtained in this study, probably due to the additional genome duplication which sturgeons suffered along evolution. Phylogenetic analysis of the deduced amino acids sequences from the studied genes demonstrated that this species has a close evolutionary distance to Holostei, coelacanths, and tetrapods, including amphibians, reptiles, birds and mammals. According to our results, expression of all the genes increased gradually over time and reached maximum levels around 18 dph. This pattern, which was comparable to length and weight data, could indicate that around 3 weeks after hatching, the digestive capacity of the Persian sturgeon changes from larval to juvenile mode.

The role of dietary methionine concentrations on growth, metabolism and N-retention in cobia (Rachycentron canadum) at elevated water temperatures.

This study determined impacts of dietary methionine concentrations at two temperatures on growth, feeding efficiency and N-metabolites in juvenile cobia. Methionine concentrations of the experimental diets were deficient (M9; 9 g/kg), sufficient (M12; 12 g/kg) and surplus (M16, 16 g/kg). Water temperature was normal (30°C) or elevated (34°C). Twenty cobia in triplicate tanks were fed the experimental diets for 6 weeks. Both methionine and temperature affected cobia's growth and feeding efficiency. Cobia fed M9 performed lower than the fish fed M12 and M16 diets. Additionally, cobia reared at 34°C performed poorer than at 30°C, probably due to lower voluntary feed intake in the fish reared at 34°C. Protein efficiency ratio and protein productive value in cobia fed M9 diet were less than M12 or M16 diets. This was confirmed with the improved retentions of indispensable amino acids (AAs). No interactions between methionine and temperature were observed in growth and protein accretion. At 30°C, CF improved, while HSI and VSI declined upon methionine supplementation levels. Of which an interaction between temperature and methionine was present. Plasma, muscle and liver free AA and N-metabolites were affected by methionine and temperature. Furthermore, temperature affected cobia's lipid class composition, resulting in increased phospholipids and cholesterol at 34°C.

Modelling digestive hydrolysis of nutrients in fish using factorial designs and desirability function.

Models simulating the in vitro digestive hydrolysis of nutrients by different animal species are frequently used to obtain a better understanding of factors affecting this process. Optimization algorithm of a model may be used to prospect the more favourable combination of selected factors resulting in the higher performance. This study was conducted to determine the combination of factors (pH, enzyme:substrate ratio, and reaction time) leading to highest bioavailability of proteins and carbohydrates in the gilthead seabream gastrointestinal tract. Besides, a novel multi-objective algorithm, desirability function, was introduced for optimization of the digestive hydrolysis of nutrients within the simulated gut of the species, using models based on the Response Surface Methodology. Design of experiment was defined based on the physiology and culture conditions of the species, and in vitro assays were performed in a two-phase (stomach ad intestine) digestion process, using the species-specific enzyme extract. According to results, intestinal phase of digestion makes the major contribution to the total protein hydrolysis, being the efficiency of the process directly correlated to all the three studied factors. In contrast, the efficiency of carbohydrate hydrolysis was directly correlated to the amount of substrate and inversely to the pH, while reaction time did not exert a significant effect. The physiological range of the factors studied in the assays favoured the hydrolysis of proteins over carbohydrates, a similar scenario to that observed in the live fish. Results from the mathematical models and their simultaneous optimization obtained from this work may have practical applications in design of feeds for this species.

Involvement of cholecystokinin (CCK) in the daily pattern of gastrointestinal regulation of Senegalese sole (Solea senegalensis) larvae reared under different feeding regimes.

Cholecystokinin (CCK) is an important regulator of pancreatic enzyme secretion in adult mammals and teleosteans. Although some studies have focused on the interaction between CCK and trypsin in marine fish larvae, little is known about the circadian patterns of the regulatory mechanism involving these two digestive components. In this study, we took advantage of the characteristic change from a diurnal to a nocturnal feeding habit that occurs in Senegalese sole (Solea senegalensis) post-larvae, to conduct an experiment where larvae and postlarvae were submitted to three different feeding regimes from mouth opening: continuous feeding, diurnal feeding and nocturnal feeding. The aim was to establish different daily feeding scenarios to uncover the operating mechanisms of CCK and tryptic enzyme activity over the 24-hourcycle to better understand the regulation of digestion in developing fish larvae. Results show a prevalence of simultaneous and opposing trends of CCK level and tryptic activity as a function of the postprandial time. This finding supports the existence of a regulatory loop between these two digestive components in pre- and post-metamorphic Senegal sole larvae. In addition, CCK level was also modulated by the gut content, tending to be lower when the gut is full and higher when is being emptied. Furthermore, larvae were able to synchronize digestive functions to very different feeding regimes, although it seems to be important having a diurnal feeding phase during pre-metamorphic stages for a proper development.

Effects of soybean meal on digestive enzymes activity, expression of inflammation-related genes, and chromatin modifications in marine fish (Sparus aurata L.) larvae.

The effects of soybean meal (SBM) in early diet of Sparus aurata larvae at two developmental windows were assessed. Prolonged (beyond 14 days post-hatch, dph) feeding with SBM decreased the activity of pancreatic enzymes of larvae. In the absence of SBM these larvae later resumed enzyme activities, but exhibited a significant delay in development. Larvae response to SBM involved up-regulation of extracellular matrix remodeling enzymes and pro-inflammatory cytokines, coupled with a drop in putative intestinal enzymes. Larvae receiving SBM at first feeding appear later to have lower expression of inflammation-related genes, especially those fed SBM until 14 dph. Multivariate analysis confirmed that the duration of the SBM early feeding period drives the physiology of larvae in different directions. Feeding larvae with SBM increased global histone H3 acetylation, whereas upon removal of SBM the process was reverted. A more in deep analysis revealed a dynamic interplay among several reversible histone modifications such as H3K14ac and H3K27m3. Finally, we showed that SBM feeding of larvae results in global hypomethylation that persist after SBM removal. This study is the first demonstrating an effect of diet on marine fish epigenetics. It is concluded that there are limitations for extending SBM feeding of S. aurata larvae beyond 14 dph even under co-feeding with live feed, affecting key physiological processes and normal growth. However, up to 14 dph, SBM does not affect normal development, and produces apparently lasting effects on some key enzymes, genes, and chromatin modifications.

Ghrelin in Senegalese sole (Solea senegalensis) post-larvae: Paracrine effects on food intake.

Successful food consumption and digestion depend on specifics anatomical and behavioral characteristics and corresponding physiological functions that should be ready to work at the appropriate time. The physiological regulation of appetite and ingestion involves a complex integration of peripheral and central signals by the brain. Ghrelin is a peptide hormone involved in the control of energy homeostasis and increases food intake in mammals, however ghrelin has species-specific actions on food intake in fish. The aim of this study was to investigate whether this peptide has an orexigenic or anorexigenic role in Senegalese sole (Solea senegalensis) in order to improve the knowledge of the physiological basis underlying feeding activity. Feed intake was measured at several sampling points to determine the overall action time of the peptide and its effect in Senegalese sole food intake. Artemia protein digestibility and retention were determined in order to analyze the ghrelin effect in fed and fasted Senegalese sole post-larvae. Results suggested that ghrelin acts as orexigenic hormone in Senegalese sole, with a response time around 25min. Results indicated that Senegalese sole post-larvae are able to maintain absorption and retention capacities independently of feeding rate and nutritional status. Furthermore, the present study gives insight for the first time of the fate of the retained amino acids, being mainly used for protein accretion (86.79% of retained amino acids recovered in protein and FAA fractions).

Daily feeding and protein metabolism rhythms in Senegalese sole post-larvae.

Fish hatcheries must adapt larval feeding protocols to feeding behavior and metabolism patterns to obtain more efficient feed utilization. Fish larvae exhibit daily ingesting rhythms rather than ingesting food continuously throughout the day. The aim of this study was to determine the daily patterns of feed intake, protein digestibility, protein retention and catabolism in Senegalese sole post-larvae (Solea senegalensis; 33 days post-hatching) using 14C-labeled Artemia protein and incubation in metabolic chambers. Sole post-larvae were fed at 09:00, 15:00, 21:00, 03:00 and 09:00+1 day; and those fed at 09:00, 21:00, 03:00 and 09:00+1 day showed significantly higher feed intake than post-larvae fed at 15:00 h (P=0.000). Digestibility and evacuation rate of ingested protein did not change during the whole cycle (P=0.114); however, post-larvae fed at 21:00 and 03:00 h showed the significantly highest protein retention efficiency and lowest catabolism (P=0.002). Therefore, results confirm the existence of daily rhythmicity in feeding activity and in the utilization of the ingested nutrients in Senegalese sole post-larvae.

Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny.

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes.

Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (Sparus aurata L.) exposed to different environmental salinities during larvae to post-larvae stages.

The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.

Unraveling the Tissue-Specific Gene Signatures of Gilthead Sea Bream (Sparus aurata L.) after Hyper- and Hypo-Osmotic Challenges.

A custom microarray was used for the transcriptomic profiling of liver, gills and hypothalamus in response to hypo- (38‰ → 5‰) or hyper- (38‰ → 55‰) osmotic challenges (7 days after salinity transfer) in gilthead sea bream (Sparus aurata) juveniles. The total number of differentially expressed genes was 777. Among them, 341 and 310 were differentially expressed in liver after hypo- and hyper-osmotic challenges, respectively. The magnitude of changes was lower in gills and hypothalamus with around 131 and 160 responsive genes in at least one osmotic stress condition, respectively. Regardless of tissue, a number of genes were equally regulated in either hypo- and hyper-osmotic challenges: 127 out of 524 in liver, 11 out of 131 in gills and 19 out of 160 in hypothalamus. In liver and gills, functional analysis of differentially expressed genes recognized two major clusters of overlapping canonical pathways that were mostly related to "Energy Metabolism" and "Oxidative Stress". The later cluster was represented in all the analyzed tissues, including the hypothalamus, where differentially expressed genes related to "Cell and tissue architecture" were also over-represented. Overall the response for "Energy Metabolism" was the up-regulation, whereas for oxidative stress-related genes the type of response was highly dependent of tissue. These results support common and different osmoregulatory responses in the three analyzed tissues, helping to load new allostatic conditions or even to return to basal levels after hypo- or hyper-osmotic challenges according to the different physiological role of each tissue.

Cloning and molecular ontogeny of digestive enzymes in fed and food-deprived developing gilthead seabream (Sparus aurata) larvae.

We have determined the expression pattern of key pancreatic enzymes precursors (trypsinogen, try; chymotrypsinogen, ctrb; phospholipase A2, pla2; bile salt-activated lipase, cel; and α-amylase, amy2a) during the larval stage of gilthead seabream (Sparus aurata) up to 60days after hatching (dph). Previously, complete sequences of try, cel, and amy2a were cloned and phylogenetically analyzed. One new isoform was found for cel transcript (cel1b). Expression of all enzyme precursors was detected before the mouth opening. Expression of try and ctrb increased during the first days of development and then maintained high values with some fluctuations during the whole larval stage. The prolipases pla2 and cel1b increased from first-feeding with irregular fluctuation until the end of the experiment. Contrarily, cel1a maintained low expression values during most of the larval stage increasing at the end of the period. Nevertheless, cel1a expression was negligible as compared with cel1b. The expression of amy2a sharply increased during the first week followed by a gradual decrease. In addition, a food-deprivation experiment was performed to find the differences in relation to presence/absence of gut content after the opening of the mouth. The food-deprived larvae died at 10dph. The expression levels of all digestive enzymes increased up to 7dph, declining sharply afterwards. This expression pattern up to 7dph was the same observed in fed larvae, confirming the genetic programming during the early development. Main digestive enzymes in gilthead seabream larvae exhibited the same expression profiles than other marine fish with carnivorous preferences in their juvenile stages.

Vitellogenin expression in wild cyprinid Petroleuciscus esfahani as a biomarker of endocrine disruption along the Zayandeh Roud River, Iran.

Aquatic environments are the ultimate sink for most of anthropogenic pollutants. The Zayandeh Roud River is the most important river in the central Iranian Plateau, supplying water to a large population. In order to determine the potential occurrence and in vivo effects of Endocrine Disrupting Chemicals (EDCs) with estrogenic or anti-androgenic properties we analyzed the wild populations of an extensively distributed endemic fish species, Petroleuciscus esfahani. For this purpose, specimens were caught from two sites upstream and two sites downstream of the expected major anthropogenic pollution sources. P. esfahani full-length cDNAs for vitellogenin (vtg), with 4177 base pairs (bp) encoding a 1339 amino acids (aa), and for ß-actin (actb), with 1776 bp encoding a 375 aa, were amplified and cloned. Hepatic vtg mRNA expression levels were measured by quantitative real-time PCR. Condition factor, gonadosomatic index and sex ratio were calculated and compared with vtg expression. Gonad histology was performed to study the possible presence of intersex condition. Detection of vtg transcripts in male individuals from the two downstream sampling sites supports the hypothesis of exposure to EDCs in these regions. Higher vtg expression in male individuals, together with reduced gonad size and condition factor, in specimens from the site located downstream of the major steel mill plant suggest a major endocrine disruption in this area.

Soybean Meal and Soy Protein Concentrate in Early Diet Elicit Different Nutritional Programming Effects on Juvenile Zebrafish.

There is now strong evidence that early nutrition plays an important role in shaping later physiology. We assessed here whether soy protein concentrate (SPC) or soybean meal (SBM) in early diet would modify zebrafish responses to these products in later life. We fed zebrafish larvae with SPC-, SBM-, or a control-diet for the first 3 days of feeding and then grew all larvae on the control diet up to juveniles. Finally, we assessed the expression in juveniles of genes involved in inflammation/immunity, the breakdown of extracellular matrix, luminal digestion, and intestinal nutrient absorption/trafficking. First feeding SBM had wider, stronger, and more persistent effects on gene expression with respect to SPC. Juveniles fed with SPC at first feeding were more prone to inflammation after refeeding with SPC than fish that never experienced SPC before. Conversely, zebrafish that faced SBM at first feeding were later less responsive to refeeding with SBM through inflammation and had higher expression of markers of peptide absorption and fatty acid transport. Results indicate that some features of inflammation/remodeling, presumably at the intestine, and nutrient absorption/transport in fish can be programmed by early nutrition. These findings sustain the rationale of using zebrafish for depicting molecular mechanisms involved in nutritional programming.

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream, Sparus aurata.

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL + LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24 h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL + LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL + LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.

Effect of feeding frequency on the daily rhythms of acidic digestion in a teleost fish (gilthead seabream).

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.

Effects of dietary arachidonic acid on cortisol production and gene expression in stress response in Senegalese sole (Solea senegalensis) post-larvae.

Dietary fatty acids, particularly arachidonic acid (ARA), affect cortisol and may influence the expression of genes involved in stress response in fish. The involvement of ARA on stress, lipid, and eicosanoid metabolism genes, in Senegalese sole, was tested. Post-larvae were fed Artemia presenting graded ARA levels (0.1, 0.4, 0.8, 1.7, and 2.3%, dry matter basis), from 22 to 35 days after hatch. Whole-body cortisol levels were determined, before and 3 h after a 2 min air exposure, as well as the expression of phospholipase A2 (PLA 2 ), cyclooxygenase-2 (COX-2), steroidogenic acute regulatory protein (StAR), glucocorticoid receptors (GRs), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferator-activated receptor alpha (PPARα). Relative growth rate (6.0-7.8% day(-1)) and survival at the end of the experiment (91-96%) and after stress (100%) were unaffected. Fish reflected dietary ARA content and post-stress cortisol increased with ARA supply up to 1.7%, whereas 2.3% ARA seemed to enhance basal cortisol slightly and alter the response to stress. Results suggested that elevating StAR transcription might not be necessary for a short-term response to acute stress. Basal cortisol and PLA 2 expression were strongly correlated, indicating a potential role for this enzyme in steroidogenesis. Under basal conditions, larval ARA was associated with GR1 expression, whereas the glucocorticoid responsive gene PEPCK was strongly related with cortisol but not GR1 mRNA levels, suggesting the latter might not reflect the amount of GR1 protein in sole. Furthermore, a possible role for PPARα in the expression of PEPCK following acute stress is proposed.