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

Comparative Biochemistry and Physiology, Part A 204 (2017) 85–92

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Ghrelin in Senegalese sole (Solea senegalensis) post-larvae: Paracrine effects on food intake Carmen Navarro-Guillén a,⁎, Manuel Yúfera a, Sofia Engrola b a b

Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Apartado Oficial, 11519 Puerto Real, Cádiz, Spain Centro de Ciências do Mar (CCMAR), Edifício 7, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

a r t i c l e

i n f o

Article history: Received 8 August 2016 Received in revised form 13 October 2016 Accepted 8 November 2016 Available online 11 November 2016 Keywords: Artemia protein Retained amino acids Food intake regulation Ghrelin Protein metabolism Solea senegalensis

a b s t r a c t 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 25 min. 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). © 2016 Elsevier Inc. All rights reserved.

1. Introduction Digestion is a complex process controlled by several factors that transforms the ingested food into nutrients that allow animals to maintain homeostasis and daily growth. Fish larvae exhibit very high growth rate during the first weeks after hatching, fact that is only possible with a high food intake. Furthermore, efficient digestive and absorptive functions are crucial for a proper fish larval development. 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 (Holt, 2011; Rønnestad et al., 2013; Jobling, 2016). In vertebrates, the physiological regulation of appetite and ingestion involves a complex integration of peripheral and central signals by the brain. The hypothalamus produces key factors that either stimulate (orexigenic) or inhibit (anorexigenic) food intake. Nevertheless, information regarding regulation of appetite in fish, particularly in larval stages, has only recently begun to be known (Rønnestad et al., 2007; Cerdá-Reverter and Canosa, 2009; Kortner et al., 2011; Rønnestad et al., 2013; Tinoco et al., 2014; Gomes et al., 2015; Blanco et al., ⁎ Corresponding author. E-mail address: [email protected] (C. Navarro-Guillén).

http://dx.doi.org/10.1016/j.cbpa.2016.11.004 1095-6433/© 2016 Elsevier Inc. All rights reserved.

2016a; Blanco et al., 2016b; Bonacic et al., 2016; Le et al., 2016; Moguel-Hernández et al., 2016; Velasco et al., 2016). The study of the control of appetite and ingestion is essential to understand nutritional characteristics and requirements of different species, as food intake is closely associated with gut transit time and the digestive and absorptive efficiency of dietary nutrients (Conceição et al., 2007a). Ghrelin, the only known orexigenic gut hormone, is an acylated peptide that was described for the first time in rat and human stomach as an endogenous ligand specific for growth-hormone secretagogue-receptor (GHS-R) of 28 amino acids (Kojima et al., 1999). Currently, ghrelin has been identified in several vertebrate species. Specifically in fish, ghrelin mature peptide has been identified in goldfish, Carassius auratus (Unniappan et al., 2002), Japanese eel, Anguilla japonica (Kaiya et al., 2003a), Mozambique tilapia, Oreochromis mossambicus (Kaiya et al., 2003b), Nile tilapia, Oreochromis niloticus (Parhar et al., 2003), rainbow trout, Oncorhynchus mykiss (Kaiya et al., 2003c), channel catfish, Ictalurus punctatus (Kaiya et al., 2005), seabream, Sparus aurata (Yeung et al., 2006), European sea bass, Dicentrarchus labrax (Terova et al., 2008) and zebrafish, Danio rerio (Amole and Unniappan, 2009), and has different lengths from 12- to 23-amino acids. Comparison of ghrelin sequences displays high sequence homology within the seven amino acids at the N-terminal region, essential for receptor binding (Matsumoto et al., 2001; Kaiya et al., 2008). Ghrelin is predominantly

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produced in endocrine cells in the stomach (or in the intestine in species that lack a proper stomach) as well as in many other organs and tissues to a lesser extent (Jönsson, 2013). Ghrelin mRNA is highly expressed in fish stomach/gut but moderate expression levels have also been detected in the brain of goldfish (Unniappan et al., 2002). In sea bass, ghrelin is highly expressed in the stomach with lower levels of expression in the proximal intestine and brain (Terova et al., 2008). In zebrafish, preproghrelin mRNA expression has been identified in several tissues including the brain, gut, ovary, testis, heart and gill (Amole and Unniappan, 2009). In Atlantic halibut (Hippoglossus hippoglossus) ghrelin was found mainly in gastrointestinal tract and muscle, and lower levels were found in brain, eye and gills (Einarsdóttir et al., 2011; Gomes et al., 2015). Ghrelin-producing cells in the gastrointestinal mucosa has been described as being both open- (in contact with the lumen) and closed-type in rats (Sakata et al., 2002; Zhao and Sakai, 2008) and also in the rainbow trout (Sakata et al., 2004) and the Atlantic halibut (Einarsdóttir et al., 2011). The distinct distribution of openedand closed-type cells in the gastrointestinal tract suggest that the ghrelin cells may be modulated by different stimulators and may play different physiological roles in various regions of the gastrointestinal tract (Zhao and Sakai, 2008; Sakata and Sakai, 2010). Regarding to ghrelin receptors in fish, GHS-R are located mainly in the hypothalamus, followed by brain and peripheral tissues as gastrointestine, liver and gonads. Peripheral receptor distribution varies among species (Jönsson, 2013; Kaiya et al., 2013). Co-expression of ghrelin and GHS-R in the same tissues, as gills or gastrointestinal tract, indicates that ghrelin can exert both endocrine and paracrine actions in developing Atlantic halibut (Einarsdóttir et al., 2011). Ghrelin has species-specific actions on food intake in fish. An orexigenic role for ghrelin was suggested in zebrafish, due to an increase in the expression of preproghrelin mRNA during fasting, and its decrease following refeeding (Amole and Unniappan, 2009). In goldfish, ghrelin injections also stimulate food intake as well as the release of growth hormone (GH) and gonadotropins from the pituitary gland (Volkoff et al., 2005; Miura et al., 2009). Peaks of circulating ghrelin have been found in Atlantic salmon (Salmo salar) before anticipated meal times, suggesting an orexigenic effect (Vikeså et al., 2015). On the other hand, in rainbow trout, intracerebral ghrelin injections decreased food intake in comparison with individuals injected physiological saline, suggesting that in this species ghrelin may act as an anorexigenic hormone (Jönsson et al., 2010). Nonetheless, a recent study suggested a new model of hypothalamic integration of fatty acid and ghrelin levels on the metabolic regulation of food intake in rainbow trout (Velasco et al., 2016). This model suggests that the orexigenic actions of ghrelin are associated with changes in fatty acid metabolism in hypothalamus and an inhibition of fatty acid-sensing mechanisms, which lead to changes in the expression of peptides resulting in increased orexigenic potential and food intake. In tilapia, ghrelin does not appear to play an important role in meal initiation, although it may act as a long-term indicator of negative energy balance since plasma ghrelin levels were elevated significantly after 2 and 4 weeks of fasting (Fox et al., 2009). Senegalese sole is a species of great interest for the Mediterranean aquaculture due to its high price, market demand and high growth potential (Morais et al., 2016; Bjørndal et al., 2016). As a flatfish, the species has a complex metamorphosis with a strong anatomical transformation. During metamorphosis, the change from the pelagic to benthic habitat implies important changes in food habits and in digestive physiology (Fernández-Díaz et al., 2001; Engrola et al., 2009b; Navarro-Guillén et al., 2015). Senegalese sole larvae presents a high digestive capacity for digesting live prey from the onset of exogenous feeding, reflected in a high growth potential (Martínez et al., 1999; Parra and Yúfera, 2001; Conceição et al., 2007b; Engrola et al., 2009b). In spite of the profuse research on feeding and nutrition in this species, the information regarding neural control of appetite is still scarce, particularly during larval and postlarval stages. Rodríguez-Gómez et al. (2001) described the

distribution of Neuropeptide Y-Like immunoreactivity in the brain of Senegalese sole adults, and suggested a possible role of this peptide in feeding regulation. Navarro-Guillén et al. (2017) analyzed the involvement of cholecystokinin (CCK) in gastrointestinal regulation of sole larvae. Results supported the existence of a regulatory loop between CCK and tryptic activity in pre- and post-metamorphic larvae. In addition, CCK level was also modulated by the gut content, tending to be lower when the gut being filled and higher when is empty. Considering the importance of ghrelin and its contradictory effects reported in regulating the hypothalamic-stomach axis in fish, the objective of this study was to investigate whether this peptide has an orexigenic or anorexigenic role in Senegalese sole in order to improve the knowledge of the physiological basis underlying feeding activity. Two trials were carried out. The first trial aimed to determine the overall action time of the peptide. The second trial aimed to analyze the effect of tube-feeding ghrelin in food intake and metabolism of proteins in relation to the nutritional status of the post-larvae (fed/fasted). 2. Materials and methods The aim of the present study was to determine response time to ghrelin (time to an observed effect) and its effect on Senegalese sole postlarvae. Due to the difficulty to make either intravenous or intracerebral injections in fish larvae, the ghrelin solution was injected directly in the stomach of the post-larvae using tube-feeding technique (Rønnestad et al., 2001; Morais et al., 2005). Food intake and protein metabolism was measured using radiolabeled Artemia protein (Morais et al., 2004a). The experiments were carried out in compliance with the Guidelines of the European Union Council (2010/63/EU) and Spanish legislation for the use of laboratory animals, with approval of the Bioethics Committee of the Spanish National Research Council for project RIDIGEST (AGL2011-23722). CCMAR facilities and their staff are certified to house and conduct experiments with live animals (‘group-1’ license by the ‘Direção Geral de Veterinaria’, Ministry of Agriculture, Rural Development and Fisheries of Portugal). 2.1. Food intake and protein metabolism trials 2.1.1. Trial 1: ghrelin response time This trial was designed to assess intrastomach ghrelin effect on Senegalese sole post-larval feeding activity and post-larval response time to ghrelin. Trial was conducted in fish at 23 days post-hatching (dph). On the evening prior to the trial, sole post-larvae were transferred to 1 L containers at the nutrient flux laboratory (20 ± 1 °C), acclimated overnight and deprived of food for 16 h. At the nutrient flux laboratory, post-larvae were divided in three treatments: control (post-larvae that were not tube-fed, CTR), tube-fed control (post-larvae tube-fed Ringer solution without ghrelin, RS) and ghrelin treatment (post-larvae tube-fed Ringer solution with ghrelin, GHR). Rat ghrelin (G8903, Sigma-Aldrich, USA) was intrastomachly administrated to sole post-larvae, which has been shown to be equally active as homologous ghrelins in a number of teleosts (Riley et al., 2002; Kaiya et al., 2003a, 2003c; Ran et al., 2004; Shepherd et al., 2007). Ghrelin was dissolved as manufacturer instructions and diluted in Ringer solution for seawater teleost (Young, 1933), to a final concentration of 0.06 ng mg body weight−1. Concentration was determined according to previous studies (Matsuda et al., 2006b; Miura et al., 2006). Larvae were first sedated with tricaine methanesulfonate 33 μM (MS-222, Sigma-Aldrich, USA) and then tubefed 18.4 nL of corresponding solution (Ringer or ghrelin solution). Each larva was then individually placed in 15 mL transparent plates with 300 radiolabeled Artemia each. In the case of CTR treatment, after sedation, post-larvae were directly placed into the transparent plates with 300 radiolabeled Artemia each. Larvae were allowed to eat radiolabeled Artemia during 5, 10, 15, 20, 25 or 30 min (minutes). Eight post-larvae from each control treatment (CTR and RS) and 15 post-larvae from GHR treatment were sampled after 5, 10, 15, 20, 25 or 30 min of being placed

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in the plates. To determine quantity of ingested Artemia, whole larvae were dissolved in 0.75 mL of aqueous based solubilizer (Solvable™, PerkinElmer, USA) at 50 °C for 24 h. 2.1.2. Trial 2: ghrelin regulation The objective of this trial was to evaluate how tube-fed ghrelin could modulate food intake and protein metabolism in fed and fasted postlarvae. Thus, larvae food intake was analyzed based on nutritional status (fed and fasted). Trial was conducted in fish at 27 dph. On the evening prior to the metabolic trial, sole post-larvae were transferred to 1 L containers at the nutrient flux laboratory (20 ± 1 °C), acclimated overnight and deprived of food for 16 h. At the nutrient flux laboratory, postlarvae were divided in two groups: Fed and Fasted, simultaneously, post-larvae from each group were assigned in two treatments: tubefed control (post-larvae tube-fed Ringer solution without ghrelin, RS) and tube-fed ghrelin treatment (post-larvae tube-fed ghrelin solution, GHR). Post-larvae from Fed group were allowed to eat unlabeled Artemia (cold) for 15 min prior to being tube-fed. Ghrelin (G8903, Sigma-Aldrich, USA) was dissolved as manufacturer instructions and diluted in Ringer solution for seawater teleost (Young, 1933), to a final concentration of 0.06 ng mg body weight−1. Fish were sedated with tricaine methanesulfonate 33 μM (MS-222, Sigma-Aldrich, USA) and tube-fed 27.6 nL of corresponding solution (Ringer (RS) or ghrelin (GHR)). Each larva was then individually placed in 15 mL transparent plates with 350 radiolabeled Artemia each. Eight post-larvae from control treatment and 15 post-larvae from ghrelin treatment were sampled after 25 min of being placed in the plates. After that period, post-larvae were rinsed twice in clean seawater and individually incubated for 18 h in vials containing 6 mL of seawater in a sealed system, linked up to a CO2 metabolic trap (5 mL 0.5 mol/L KOH) (Rønnestad et al., 2001). After acidification (with 1 mL 0.1 M HCl) of incubation water, the fraction of the label that was catabolized by sole and became entrapped in seawater by conversion to HCO− 3, was recovered in the metabolic trap as 14CO2 that diffused out of the water. Finally, the label remaining in the water corresponds to Artemia protein that was evacuated/undigested. Larval bodies were fractionated in lipids, protein, FAA and other metabolites and each fraction was separately counted for radioactivity (disintegrations per minute; DPM). 2.2. Senegalese sole larval rearing Larvae were obtained from Sea8 (Póvoa de Varzim, Portugal) at 20 dph, with an average dry weight (DW) of 1.51 ± 0.10 mg. Larvae were maintained in a recirculation system at the Centre of Marine Sciences (University of Algarve, Faro, Portugal) in 3-litre flat bottom trays until metabolism assays were performed. A 12/12 h light/dark photoperiod cycle, with lights turned on and off at 9:00 and 21:00, was maintained and the light intensity was 400 lx at water surface, following CCMAR's standard Senegalese sole rearing conditions. Salinity was 34 ± 1.3 g L−1 and temperature 19.6 ± 0.92 °C. Until 33 dph, larvae were fed to satiation four times daily Artemia metanauplii enriched with DHA Selco (INVE Aquaculture, Belgium). 2.3. Artemia [U-14C] labeling The procedure for Artemia labeling was the same for both trials. Artemia cysts were incubated during 24 h in a plastic cylindricalconical flask, under standard conditions (Van Stappen, 1996). The newly hatched Artemia nauplii were harvested and washed on 150μm plankton net and transferred to clean seawater. Artemia concentration was determined by counting the nauplii under a binocular microscope. For Artemia metanauplii [U-14C] labeling, newly hatched nauplii (200 nauplii mL−1) were stocked in a single cylindrical glass container with 150 mL of seawater vigorously aerated and at 28 °C. Artemia nauplii were kept for 6 h, which is approximately the time needed to reach their first feeding stage (Van Stappen, 1996). After that period,

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Artemia nauplii were enriched with a [U-14C] uniformly labeled protein hydrolysate (3.7 MBq mL−1; American Radiolabeled Chemicals, Inc., Saint Louis, U.S.A.) during a 9 h period in a sealed incubation system at 28 °C, and with a dose of 1.6 μL of the [U-14C] protein hydrolysate per mL of seawater (Morais et al., 2004a). The incubation system consisted of an aquarium with controlled temperature and an incubation bottle connected to a KOH trap to capture radiolabelled 14CO2 (Morais et al., 2004a). To evaluate the amount of label incorporated by Artemia, Artemia metanauplii were washed several times and counted. Samples (n = 4, 3 mL per sample) were taken to measure the incorporated radiolabel, as described in Morais et al. (2004a). Seawater from the beaker containing the radiolabeled Artemia was also sampled in order to correct the 14C present in the incubation seawater (n = 4, 3 mL each sample). Artemia samples were dissolved in aqueous based solubilizer (1 mL; Solvable™, PerkinElmer, U.S.A.) at 50 °C for 24 h. 2.4. Radiolabel measurements Samples from incubation seawater, KOH-CO2 traps, larval bodies and Artemia metanauplii, collected during the two metabolic trials, were counted for radioactivity (DPM, disintegrations per minute) by adding Ultima Gold XR scintillation cocktail (Perkin Elmer, USA). All samples were counted in a liquid scintillation counter (Tri-Carb 2910TR, Perkin Elmer, USA). The metabolic budgets were calculated after subtraction of blanks for quench and lumex correction. Results for each component (evacuation, catabolism, retention in whole larvae and retention in several fractions of organic compounds) were expressed as a percentage of total 14C-label (the sum of DPM in all compartments of metabolic chambers and fish). Larvae food intake (% DW) was calculated as described by Conceição et al. (1998): Trial 1 : FI ¼ ½ðRfish =SRArtemia Þ= DWfish   100 Trial 2 : FI ¼ ½ðRtotal =SRArtemia Þ= DWfish   100 where, Rfish is the radioactivity in post-larva (DPM), Rtotal is the sum of the radioactivity in the incubation water, in the CO2 trap and in fish (DPM), SRArtemia is the specific radioactivity in Artemia samples (DPM/ mg Artemia DW), and DWfish is the fish dry weight (mg). Protein utilization was determined based on protein digestibility, evacuation, retention efficiency and catabolism fraction. These estimates were determined as follows: Digestibility ¼





 Rbody þRCO2 trap = Rbody þRCO2 trap þRwater  100;


 Evacuation ¼ Rwater = Rbody þRCO2 trap þRwater  100; 
 Retention ¼ Rbody = Rbody þRCO2 trap  100; 
 Catabolism ¼ RCO2 trap = Rbody þRCO2 trap  100 where Rbody is the total radioactivity in fish body (DPM), RCO2 trap is the total radioactivity per CO2 trap (DPM), and Rwater is the total radioactivity in the incubation seawater (DPM). Additionally, it was determined the relative retention in lipid fraction (rRLip, %), protein fraction (rRProt, %), FAA fraction (rRFAA, %) and other metabolites fraction where glycogen was included (rROther, %) (Rocha et al., 2016). The several relative retentions were determined as:   rRLip ¼ RLipids =Rbody  100;   rRprot ¼ RProtein =Rbody  100;   rRFAA ¼ RFAA =Rbody  100;

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  rROther ¼ ROther =Rbody  100 where Rbody is the sum of all fractions of compounds extracted from body (DPM), RLipids is the total radioactivity in chloroform fraction (DPM), RProtein is the total radioactivity in protein precipitate (DPM), RFAA is the total radioactivity in TCA soluble fraction (DPM) and ROther is the total radioactivity in the washes performed during protein extraction (DPM). 2.5. Statistical analysis For trial 1, after data analysis the CTR treatment (post-larvae that were not tube-fed) was excluded from further analysis since results showed a statistical effect of tube-feeding technique on ingestion, therefore, it was only comparable the effect of ghrelin between tube-fed postlarvae (post-larvae tube-fed Ringer solution without (RS) and with ghrelin (GHR)). Two-way ANOVA was used to evaluate differences in food intake due to the interaction between time and treatments. Differences between sampling points were compared using a one-way ANOVA after assessing equality of variances by a Levene's test. Post hoc multiple comparisons were carried out using Tukey's test. To determine the response time to ghrelin, food intake between GHR and RS post-larvae was compared at each sampling point by means of an unpaired two-tailed Student's t-test. For trial 2, all percentage data were arcsine square root-transformed prior to analysis. Two-way ANOVA was performed to analyze differences in food intake and protein metabolism due to the interaction between treatments and nutritional status. Feed intake and protein metabolism between GHR and RS post-larvae from each nutritional status were tested by means of an unpaired two-tailed Student's t-test. Differences in relative retention between fractions were also tested using a one-way ANOVA after assessing equality of variances by a Levene's test. Post hoc multiple comparisons were carried out using Tukey's test. Differences were considerate significant at P b 0.05. All statistical analyses were performed with SPSS® 15.0 software (IBM, New York, U.S.A.) and the results are given as means and standard deviation (SD). 3. Results 3.1. Trial 1: ghrelin response time Results showed a statistical effect of tube-feeding in the feeding activity of post-larvae (Fig. 1). Post-larvae from CTR treatment (postlarvae that were not tube-fed) showed an increasing food intake, reaching maximum ingestion 20 min after Artemia were offered (4.9 ± 2.74% BW). Two-way ANOVA did not reveal interaction between treatment and time post-tube-feeding (p = 0.897). Post-larvae from both tube-fed treatments showed similar patterns of feeding activity. None tube-fed larvae showed feeding activity until 10 min after-tubefeeding, although GHR post-larvae showed progressively higher ingestion than RS post-larvae. Food intake between tube-fed treatments was statistically different at 25 min after tube-feeding (2.27 ± 0.62 and 1.22 ± 0.31% BW, GHR and RS, respectively, p b 0.05). GHR post-larvae showed significant higher food intake at 20 and 30 min (3.34 ± 2.07 and 3.34 ± 1.60% BW, respectively) than after 5 min post-injection (0 ± 0.37% BW), however, RS post-larvae did not show statistical differences along time.

Fig. 1. Feed intake ([(Rfish / SRArtemia) / DWfish]×100) of 23 dph Senegalese sole post-larvae along 30 min after been tube-fed. CTR (control, n = 8 per sampling point), RS (Ringer solution treatment, n = 8 per sampling point) and GHR (ghrelin treatment, n = 15 per sampling point). Results are represented as mean ± SD. Letters mean significant differences between each sampling point for each treatment. Asterisk indicates statistical differences between RS and GHR treatments at each sampling point.

No statistical differences were found in food intake in fed post-larvae tube-fed ghrelin or Ringer solution (p = 0.534). Regarding to the involvement of the nutritional status on ghrelin effect, food intake was statistically higher in fasted than in fed post-larvae in the ghrelin treatment (p = 0.037), whereas no differences were recorded in food intake between fasted and fed larvae in the RS treatment (p = 0.549). Digestibility in fasted larvae was around 55% of total label (54.60 ± 2.88 and 56.01 ± 1.4, RS and GHR, respectively), in fed larvae it was 54.9 ± 1.79 and 52.78 ± 3.08% of total label, RS and GHR, respectively (Fig. 3; p N 0.05). Fasted RS and GHR post-larvae showed retention capacities of 63.10 ± 3.2 and 64.46 ± 2.17% of absorbed label, respectively. Fed post-larvae showed retention capacities of 68.33 ± 1.78 and 62.73 ± 2.56% of absorbed label, RS and GHR, respectively (Fig. 4; p N 0.05). Absorbed amino acids were preferentially used for protein synthesis in all treatments (Fig. 5). No differences were found for the different fractions between RS and GHR treatments, neither in fasted or in fed post-larvae. Of the retained 14C in fasted RS post-larvae, 69.94 ± 5.29% was found in the protein fraction, 15.08 ± 5.32% in the FAA, 9.99 ± 2.93 in the lipids and 4.99 ± 1.86% in the other metabolites fraction.

3.2. Trial 2: ghrelin regulation Overall, fasted post-larvae showed a higher food intake than fed post-larvae (p = 0.049) (Fig. 2). Two-way ANOVA did not reveal interaction between treatment and nutritional status (p = 0.238). Regarding to ghrelin effect on food intake, in fasted post-larvae, GHR food intake was closer to be statistically higher than RS food intake (p = 0.069).

Fig. 2. Food intake ([(Rtotal / SRArtemia) / DWfish]×100) of 27 dph Senegalese sole post-larvae tube-fed Ringer solution (RS, n = 8) or ghrelin (GHR, n = 15). Results are represented as mean ± SD. Asterisk indicates statistical differences between GHR-fasted and GHR-fed post-larvae.

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Fig. 3. Artemia protein digestibility (gray bars; % of radiolabel in the sole body and CO2 trap in relation to total radiolabel fed) and Artemia protein evacuation (white bars; % of radiolabel in sea water in relation to total radiolabel fed) in 27 dph Senegalese sole postlarvae tube-fed Ringer solution (RS, n = 8) or ghrelin (GHR, n = 15) and after 18 h of incubation. Values are means ± SD.

Fig. 5. Proportion (%) of relative retention of radiolabel in each fraction of post-larval body (protein, FAA, lipids and other metabolites) in relation to total retained label, after 18 h of incubation, in 27 dph Senegalese sole post-larvae tube-fed Ringer solution (RS, n = 8) or ghrelin (GHR, n = 15). Values are means ± SD. Different letters and numbers mean statistical differences in relative retention between fractions for each treatment.

In fasted GHR post-larvae, 73.04 ± 5.98% was found in the protein fraction, 13.75 ± 2.25% in the FAA, 8.72 ± 2.72% in the lipids and 4.50 ± 1.82% in the other metabolites fraction. Regarding post-larvae from fed treatments, in fed RS post-larvae, 74.24 ± 3.20% of 14C was retained in proteins, 13.23 ± 2.51% in the FAA, 7.33 ± 0.51% in the lipids and 5.20 ± 1.27% in the other metabolites fraction. While in fed GHR postlarvae, 70.05 ± 9.93, 17.85 ± 10.73, 7.65 ± 1.83 and 4.45 ± 1.62% of 14C was retained in proteins, FAA, lipids and other metabolites, respectively.

CTR sole post-larvae confirmed a statistical effect of tube-feeding technique on food intake, since non tube-fed post-larvae displayed feeding activity after 5 min, and from there onwards, showing statistically higher feed intake than tube-fed larvae. On the other hand, tubefed post-larvae showed feeding activity only from 10 min post-tubefeeding onwards. These results suggest the need to recover after tubefeeding, since the effect of anesthesia was taken into account in all treatments. CTR results described the gut filling curve in Senegalese sole post-larvae, reaching the maximum after 20 min of feeding. In tubefed larvae, similar ingestion patterns were recorded in both treatments, with the maximum value also reached at 20 min after food was supplied, with a trend to decrease and increase again at 30 min post-tube-feeding. This second increase in food intake might be due to post-larvae experience a feeling of fullness due to volume of tube-feeding, which is quickly absorbed, being ready to ingest again after 30 min. Considerable research effort has been centered to elucidate what are the optimal rearing or nutritional conditions during these early life stages that could improve juvenile quality. In this context, early cofeeding with microdiet has been found to improve starting diets acceptability and survival at weaning (Engrola et al., 2009b; Mai et al., 2009; Engrola et al., 2010). Several studies have focused on Senegalese sole digestive capacity by examining the activity of the main digestive enzymes (Martínez et al., 1999; Ribeiro et al., 1999; Engrola et al., 2007; Engrola et al., 2009a; Navarro-Guillén et al., 2015) or by evaluating nutrient metabolism (Morais et al., 2004b, 2006; Engrola et al., 2009b; Engrola et al., 2010; Navarro-Guillén et al., 2014). Results from those studies confirmed that sole larvae are ready to digest from the first feeding, discarding a low digestive capacity as a possible reason causing nutritional problems in Senegalese sole larvae. Results from the present study reveal a high individual dispersion in food intake, in accordance with previous results that also measured individual food consumption in Senegalese sole larvae using 14C (Engrola et al., 2009b; Mai et al., 2009; Engrola et al., 2010). This high dispersion in food intake in Senegalese sole larvae was also reflected in Navarro-Guillén (in revision) results, where food intake at 33 dph under the same feeding conditions ranged from 49 to 187 Artemia/larva (3.6 to 13.7% BW). Therefore, results suggest that further studies on mechanisms regulating food consumption might be necessary to obtain lower feeding variability and to get better results in larval rearing. Our results also suggest that ghrelin has an orexigenic effect in this species and indicate an almost immediate response to ghrelin after been secreted in the stomach, though at least 25 min would be necessary for a statistically effective response in Senegalese sole post-larvae.

4. Discussion The objectives of the present study were to advance in the understanding of the regulation of feeding activity in Senegalese sole and to establish the involvement of ghrelin in this process. Results from the first trial gave information about the kinetics of gastrointestinal filling, the effect of ghrelin in food intake and post-larval response time to ghrelin. On the other hand, results from the second trial allowed to compare the effect of tube-feeding ghrelin on food intake and protein metabolism between fed and unfed post-larvae, and also gave information about the fate of retained amino acids.

Fig. 4. Artemia protein retention (gray bars; % of radiolabel in the sole in relation to digested label), and catabolism (white bars; % of radiolabel in the metabolic trap in relation to digested label) in 27 dph Senegalese sole post-larvae tube-fed Ringer solution (RS, n = 8) or ghrelin (GHR, n = 15) and after 18 h of incubation. Values are means ± SD.

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Although physiological mechanism actions of ghrelin have been described for several fish species showing either orexigenic effects (Matsuda et al., 2006a; Jönsson, 2013) or anorexigenic effects (Jönsson et al., 2007; Jönsson et al., 2010; Jönsson, 2013), to the best of our knowledge, this is the first report about response time to ghrelin. In both cases, the mechanisms for ghrelin effects on food intake may involve species-specific actions on appetite-regulating pathways in the brain. In goldfish, studies suggest that ghrelin's central action to produce increased food consumption is mediated through the stimulation of the neuropeptide Y (NPY) and orexin neurons, as well as a potential feedback of orexin on ghrelin expression (Miura et al., 2006, 2007; Jönsson, 2013). In rainbow trout ghrelin appears to act through the anorexigenic CRH neurons to suppress food intake (Jönsson et al., 2010; Jönsson, 2013). Nevertheless, a recent study about ghrelin in rainbow trout described an orexigenic effect of the peptide associated with changes in fatty acid metabolism in hypothalamus (Velasco et al., 2016). Endocrine and paracrine actions have been described for hormone ghrelin. Hormone ghrelin acts at gastrointestinal level, where openedtype ghrelin cells and GHS-Rs have been demonstrated in humans, rats, but also in fish (Broglio et al., 2003; Sakata et al., 2004; Zhao and Sakai, 2008; Sakata and Sakai, 2010; Einarsdóttir et al., 2011; Jönsson, 2013; Kaiya et al., 2013). Du et al. (2007) provided the first evidence that ghrelin could act locally without involving the central nervous system since a direct exposure to ghrelin increased H+-K+-ATPase activity of gastric mucosal cells in vitro. Similarly, in zebrafish, direct exposition of intestine sections to rat ghrelin solution resulted in an increase of intestinal motility (Olsson et al., 2008). In the present study, paracrine effect of ghrelin in food intake was analyzed. When compared with RS post-larvae, ghrelin supply did not lead to a statistical effect in food intake, independently of nutritional status (fasted/fed). However, in fasted post-larvae from GHR-treatment food intake was closer to be statistically higher than RS larvae. This lack of statistical differences is mainly due to the high dispersion of the results, as explained above, as well as, probably, to saturation of gastrointestinal GHS-Rs derived of high endogenous ghrelin levels in fasted post-larvae. Changes in ghrelin production and secretion during fasting/feeding vary among fishes. In zebrafish, it was described a marked and rapid down-regulation of ghrelin mRNA levels after refeeding (Amole and Unniappan, 2009). Similar results were found in goldfish, where plasma ghrelin levels and preproghrelin mRNA expression in the hypothalamus and gut were both lower after feeding (Unniappan et al., 2004). This postprandial regulation of ghrelin production may be in part explained by the regulatory loop described between hormone cholecystokinin (CCK) and ghrelin. Kobelt et al. (2005) suggested that CCK abolishes ghrelininduced food intake by dampening increased ARC neuronal activity. Food intake results from trial 2 in the present study revealed statistical differences in food intake between fasted and fed post-larvae due to ghrelin, since there were no differences in food intake between fasted and fed larvae from RS treatment. Recently, Navarro-Guillén et al. (2017) described the involvement of CCK in the gastrointestinal regulation of Senegalese sole larvae. Results indicated a modulation of CCK levels by gut content, with increasing levels of CCK after gut filling. Results lead to think that after the first feeding (cold Artemia) ghrelin down-regulation pathways started to work, probably including CCK involvement. These results are consistent with the suggested role of ghrelin as an orexigenic hormone in Senegalese sole. Digestibility of ingested Artemia protein in the present study ranged from 52.78 to 56.01%. Values are slightly lower than those reported in 22 dph sole post-larvae (Engrola et al., 2009b, 2010). In our study, no differences were recorded in relative digestibility and therefore in the relative evacuation of ingested label among treatments. It would be expected that either, feeding rate or feeding preconditioning (fasting/ feeding) had some impact on the digestive capacity. Higher food intake may involve a faster gut transit and consequently, a shorter time available for efficient digestion and absorption of proteins. Engrola et al. (2009b) assessed if protein digestibility and retention efficiency in

Senegalese sole larvae are influenced by a second meal. Results showed that sole larvae that were re-fed had a slightly, though significant, higher protein digestibility than sole fed only a single meal. However, in other species, as Pacific herring (Clupea harengus pallasi), re-fed larvae showed a decrease in digestibility when a second meal was provided (Boehlert and Yoklavich, 1984). Nevertheless, in our study, digestive capacity was not influenced by nutritional status. Likewise, we did not found differences in protein retention and catabolism between treatments, ranging relative retention between 62.74 (fed GHR tube-fed post-larvae) and 68.34% of absorbed label (fed RS tube-fed post-larvae). The use of 14C to evaluate the larval digestive capacity allowed us to analyze the compartmentalization of the fraction retained in the sole post-larvae. These are novel results in fish larvae, with only previous results in seabream (Sparus aurata) larvae, using 14C glucose (Rocha et al., 2016). The analysis of the retention of protein-derived 14C in the postlarval body fractions showed no statistical differences between treatments. Similar retention profile was reported for seabream larvae tube-fed 14C-glucose; protein and FAA fractions represented almost 77% of all retained, while the conversion into lipids and glycogen was much lower than expected (Rocha et al., 2016). These results together with those presented in this study indicate that larvae use retained nutrients preferably for growth, as expected at this stage of development. Ghrelin is involved in long-term regulation of energy metabolism, mainly related to glucose and lipid metabolism, promoting adipogenesis and glucose metabolism (Jönsson, 2013). The present study helps to get a better knowledge about the preference of sole post-larvae to use retained amino acids. Dietary amino acids can be used for protein deposition and growth or diverted from this pathway to serve as carbon substrates for gluconeogenesis, lipogenesis, ketogenesis or energy production. Approximately, 12% of total retained protein was recovered in the lipids and other metabolites (in which glycogen is included) fractions (on average, 7 and 5%, respectively). These results demonstrate the capacity of Senegalese sole post-larvae to use amino acids as precursor for lipids and glucose accretion (energy storage). It has been described a limited capacity in Senegalese sole, as in other flatfish, to store fat, supported by a low activity of fatty acid synthase (FAS), suggesting a marginal contribution of the de novo synthesis of lipids for the increment of body lipid content. However, in the present study, fasted post-larvae showed a trend of a higher conversion of amino acids into lipids, probably as consequence of a higher consumption of lipid reserves as energy fuel during fasting. In addition, FAS activity was found to be highly sensitive to the composition of nutrient intake, being more active with low-fat/high-protein diets, as is the case of Artemia metanauplii (Conceição et al., 2010). Overall, approximately 5% of total retained 14C was recovered as glycogen. Similar results have been reported for gilthead seabream larvae (Rocha et al., 2016). Since glucose is an essential fuel for a number of tissues and also as energy fuel, it is particularly important to maintain glycogen reserves. In nature, Senegalese sole fed on worms, clams and small crabs (Cabral, 2000; Sá et al., 2003) being considered as a species with carnivorous feeding habits with limited ability to use carbohydrates. Nevertheless, recently, several studies suggested that sole may be able to use them efficiently (Parma et al., 2013; Borges et al., 2014; Conde-Sieira et al., 2015). This fact and the present results support the capacity for omnivorous feeding although with carnivorous preference suggested for S. senegalensis and S. solea (Yúfera and Darias, 2007; Parma et al., 2013). Even so, further studies on sole carbohydrates metabolism and its interaction with other metabolic pathways are needed to get a better knowledge about global energy metabolism and improve the design of artificial diets for these species. In conclusion, this study contributes to advance in the knowledge of regulation mechanisms for food intake in Senegalese sole post-larvae. Results suggest that ghrelin acts as orexigenic hormone in Senegalese sole, indicating a response time to the peptide of 25 min, although further studies using intravenous or intracerebral administration of ghrelin in Senegalese sole juveniles or adults are needed to confirm this hypothesis.

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In accordance with the suggested role of ghrelin as orexigenic factor in Senegalese sole post-larvae, results showed a postprandial regulation of ghrelin effects, with statistically lower food intake in fed than in fasted post-larvae tube-fed ghrelin. Results indicated that Senegalese sole larvae are able to maintain their absorption and retention capacities independently of feeding rate, since no differences were found in protein metabolism between treatments. 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 were recovered in protein and FAA fractions). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This work received national funds through FCT - Foundation for Science and Technology through project CCMAR/Multi/04326/2013 (Portugal), project SOLEAWIN (310305/FEP/71) partially supported by PROMAR Program (Portugal) with FEDER funds, and also through the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution granted to M. Yúfera (Spain). Carmen Navarro-Guillén was supported by a doctoral fellowship (BES-2012-051956) from MINECO (Spain). Sofia Engrola acknowledges a FCT investigator grant IF/00482/2014/ CP1217/CT0005 funded by the European Social Fund, the Operational Programme Human Potential and the Foundation for Science and Technology of Portugal (FCT – Portugal). References Amole, N., Unniappan, S., 2009. Fasting induces preproghrelin mRNA expression in the brain and gut of zebrafish, Danio rerio. Gen. Comp. Endocrinol. 161, 133–137. Bjørndal, T., Guillen, J., Imsland, A., 2016. The potential of aquaculture sole production in Europe: production costs and markets. Aquac. Econ. Manag. 20, 109–129. Blanco, A.M., Gómez-Boronat, M., Redondo, I., Valenciano, A.I., Delgado, M.J., 2016a. Periprandial changes and effects of short- and long-term fasting on ghrelin, GOAT, and ghrelin receptors in goldfish (Carassius auratus). J. Comp. Physiol. B. 186, 727–738. Blanco, A.M., Sánchez-Bretaño, A., Delgado, M.J., Valenciano, A.I., 2016b. Brain mapping of ghrelin O-acyltransferase in goldfish (Carassius auratus): novel roles for the ghrelinergic system in fish? Anat. Rec. 299, 748–758. Boehlert, G.W., Yoklavich, M.M., 1984. Carbon assimilation as a function of ingestion rate in larval pacific herring, Clupea harengus pallasi Valenciennes. J. Exp. Mar. Biol. Ecol. 79, 251–262. Bonacic, K., Campoverde, C., Gómez-Arbonés, J., Gisbert, E., Estevez, A., Morais, S., 2016. Dietary fatty acid composition affects food intake and gut-brain satiety signaling in Senegalese sole (Solea senegalensis, Kaup 1858) larvae and post-larvae. Gen. Comp. Endocrinol. 228, 79–94. Borges, P., Valente, L.M.P., Véron, V., Dias, K., Panserat, S., Médale, F., 2014. High dietary lipid level is associated with persistent hyperglycaemia and downregulation of muscle Akt-mTOR pathway in Senegalese sole (Solea senegalensis). PLoS One 9, e102196. Broglio, F., Gottero, C., Arvat, E., Ghigo, E., 2003. Endocrine and non-endocrine actions of ghrelin. Horm. Res. Paediatr. 59, 109–117. Cabral, H.N., 2000. Comparative feeding ecology sympatric Solea solea and S. senegalensis, within the nursery areas of the Tagus estuary, Portugal. J. Fish Biol. 57, 1550–1562. Cerdá-Reverter, J.M., Canosa, L.F., 2009. Neuroendocrine systems of the fish brain. Fish Physiol. 28, 3–74. Conceição, L.E.C., Dersjant-Li, Y., Verreth, J.A.J., 1998. Cost of growth in larval and juvenile African catfish (Clarias gariepinus) in relation to growth rate, food intake and oxygen consumption. Aquaculture 161, 95–106. Conceição, L.E.C., Morais, S., Rønnestad, I., 2007a. Tracers in fish larvae nutrition: a review of methods and applications. Aquaculture 267, 62–75. Conceição, L.E.C., Ribeiro, L., Engrola, S., Aragão, C., Morais, S., Lacuisse, M., Soares, F., Dinis, M.T., 2007b. Nutritional physiology during development of Senegalese sole (Solea senegalensis). Aquaculture 268, 64–81. Conceição, L.E.C., Yúfera, M., Makridis, P., Morais, S., Dinis, M.T., 2010. Live feeds for early stages of fish rearing. Aquac. Res. 41, 613–640. Conde-Sieira, M., Soengas, J.L., Valente, L.M.P., 2015. Potential capacity of Senegalese sole (Solea senegalensis) to use carbohydrates: metabolic responses to hypo- and hyperglycaemia. Aquaculture 438, 59–67. Du, G.M., Liu, M.J., Shi, Z.M., Zhang, L., Wei, X.H., Zhao, R.Q., 2007. In vitro effects of ghrelin on gastric H+-K+-ATPase and pepsin activity and mRNA expression of gastrin, somatostatin, receptors for GH and IGF-1 in cultured gastric mucosal cells of weanling piglets. Anim. Sci. 82, 823–828.

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