Sea water acclimation of rainbow trout (Oncorhynchus mykiss) modulates the mucosal transcript immune response induced by Vibrio anguillarum and Aeromonas salmonicida vaccine, and prevents further transcription of stress-immune genes in response to acute stress.

Mucosal tissues appear to be more important in fish than in mammals due to living in a microbial-rich aquatic milieu, yet the complex interaction between the immune and the neuroendocrine system in these tissues remains elusive. The aim of this work was to investigate the mucosal immune response in immunized rainbow trout vaccinated with Alpha ject vaccine (bivalent), kept in fresh water (FW) or transferred to seawater (SW), and to evaluate their response to acute stress (chasing). Acute stress resulted in higher levels of plasma cortisol (Sham + Stress and Vaccine + Stress). A similar response was observed in skin mucus, but it was lower in Vaccine + Stress compared with stressed fish. With a few exceptions, minimal alterations were detected in the transcriptomic profile of stress-immune gene in the skin of vaccinated and stressed fish in both FW and SW. In the gills, the stress elicited activation of key stress-immune components (gr1, mr, ß-ar, hsp70, c3, lysozyme, α-enolase, nadph oxidase, il1ß, il6, tnfα, il10 and tgfß1) in FW, but fewer immune changes were induced by the vaccine (nadph oxidase, il6, tnfα, il10 and igt) in both SW and FW. In the intestine, an array of immune genes was activated by the vaccine particularly those related with B cells (igm, igt) and T cells (cd8α) in FW with no stimulation observed in SW. Therefore, our survey on the transcriptomic mucosal response demonstrates that the immune protection conferred by the vaccine to the intestine is modulated in SW. Overall, our results showed: i) plasma and skin mucus cortisol showed no additional stress effect induced by prolonged SW acclimation, ii) the stress and immune response were different among mucosal tissues which indicates a tissue-specific response to specific antigens/stressor. Further, the results suggest that the systemic immune organs may be more implicated in infectious events in SW (as few changes were observed in the mucosal barriers of immunized fish in SW) than in FW.

High protein requirements of juvenile Atlantic wolffish, Anarhichas lupus: Effects of dietary protein levels on growth, health, and welfare.

The objective of the present study was to investigate the optimal dietary protein requirement and the effect of varying protein levels on the growth and health of juvenile, wild-caught Atlantic wolffish, Anarhichas lupus, a promising candidate for cold-water aquaculture diversification. Six iso-energetic (ca. 18.3 MJ kg-1), fish meal-based experimental diets were formulated with crude protein levels ranging from 35% to 60%, with graded increments of 5% in a 12-week feeding trial in a recirculating aquaculture system (RAS). Weight gain, specific growth rate (SGR), and condition factor (K) were evaluated in response to dietary protein levels. Liver, muscle, and blood parameters were assessed for possible changes in protein and lipid metabolism and welfare. Overall growth was highly variable throughout the experiment on all diets, as expected for a wild population. The feed with highest in protein (60%) inclusion resulted in the highest growth rates, with an average weight gain of 37.4% ± 33.8% and an SGR of 0.31% ± 0.2% day-1. This was closely followed by feeds with 55% and 50% protein inclusion with an average weight gain of 22.9% ± 34.8% and 28.5% ± 38.3%, respectively, and an SGR of 0.18% ± 0.3% day-1 and 0.22% ± 0.3% day-1, respectively. Fish fed the high protein diets generally had increased hepatic lipid deposition (17%-18%) and reduced free fatty acid levels (3.1-6.8 µmol L-1) in the plasma relative to fish that were fed the lower protein diets (35%-45%). No effects of diet were found on plasma protein levels or muscle protein content. Furthermore, stress parameters such as plasma cortisol and glucose levels were unaffected by diet, as were plasma ghrelin levels. Overall, these results suggest that a high protein inclusion in the diet for Atlantic wolffish is required to sustain growth with a minimum protein level of 50%.

Fish processing side streams are promising ingredients in diets for rainbow trout (Oncorhynchus mykiss) - Effects on growth physiology, appetite, and intestinal health.

Due to the growth of aquaculture and the finite supply of fishmeal and oil, alternative marine protein and lipid sources are highly sought after. Particularly promising is the use of side streams from the fish processing industry, allowing for the recovery and retention of otherwise lost nutrients in the food production chain. The aim of the present study was to evaluate the potential of three fish processing side streams as fish feed ingredients. The side streams originated from different stages of the production chain, were used without further processing, and included sprat trimmings (heads, frames, viscera), marinated herring (fillets) and mackerel in tomato sauce (fillets and sauce). The three side streams contained moderate levels of protein (28-32% dry matter) and high levels of lipid (34-43%). The sprat trimmings included ca. 29% ash and 1.5% phosphorous which may add value due to the high level of essential minerals but needs to be considered in feed formulations. Three diets were formulated to include 50% of each side stream replacing all fishmeal and ca. 80% of the fish oil of the control diet, which contained 35% fishmeal and 10% fish oil. The diets were evaluated in a 12-week feeding trial using rainbow trout (Oncorhynchus mykiss). Fish fed the sprat diet displayed the highest feed intake and growth, and showed no negative effects on the intestinal health. The mackerel side stream displayed a good digestibility but resulted in lower growth rates compared to the sprat trimmings. Fish fed the herring diet, displayed the lowest performance regarding growth, feed intake and digestibility. They further exhibited a reduction in nutrient uptake in both proximal and distal intestine, likely contributing to the observed lower digestibility and growth, and a reduction in plasma ghrelin levels. As part of a circular approach to increase marine lipid and protein production for fish feed, the tested sprat and mackerel side streams are promising raw materials however additional studies using more commercial-like feed formulations are encouraged.

The role of environmental salinity on Na+-dependent intestinal amino acid uptake in rainbow trout (Oncorhynchus mykiss).

Na+/K+-ATPases (NKA) in the basolateral membrane of the intestinal enterocytes create a Na+-gradient that drives both ion-coupled fluid uptake and nutrient transport. Being dependent on the same gradient as well as on the environmental salinity, these processes have the potential to affect each other. In salmonids, L-lysine absorption has been shown to be higher in freshwater (FW) than in seawater (SW) acclimated fish. Using electrophysiology (Ussing chamber technique), the aim was to explore if the decrease in L-lysine transport was due to allocation of the Na+-gradient towards ion-driven fluid uptake in SW, at the cost of amino acid transport. Intestinal NKA activity was higher in SW compared to FW fish. Exposure to ouabain, an inhibitor of NKA, decreased L-lysine transport. However, exposure to bumetanide and hydrochlorothiazide, inhibitors of Na+, K+, 2Cl--co-transporter (NKCC) and Na+, Cl--co-transporter (NCC) respectively, did not affect the rate of intestinal L-lysine transport. In conclusion, L-lysine transport is Na+-dependent in rainbow trout and the NKA activity and thus the available Na+-gradient increases after SW acclimation. This increased Na+-gradient is most likely directed towards osmoregulation, as amino acid transport is not compromised in SW acclimated fish.

Thyroid function and immune status in perch (Perca fluviatilis) from lakes contaminated with PFASs or PCBs.

The environment contains a multitude of man-made chemicals, some of which can act as endocrine disruptors (EDCs), while others can be immunotoxic. We evaluated thyroid disruption and immunotoxic effects in wild female perch (Perca fluviatilis) collected from two contaminated areas in Sweden; one site contaminated with per- and polyfluoroalkyl substances (PFASs) and two sites contaminated with polychlorinated biphenyls (PCBs), with one reference site included for each area. The hepatic mRNA expression of thyroid receptors α and ß, and the thyroid hormone metabolising iodothyronine deiodinases (dio1, dio2 and dio3) were measured using real-time PCR, while the levels of thyroid hormone T3 in plasma was analysed using a radioimmunoassay. In addition, lymphocytes, granulocytes, and thrombocytes were counted microscopically. Our results showed lower levels of T3 as well as lower amounts of lymphocytes and granulocytes in perch collected from the PFAS-contaminated site compared to reference sites. In addition, expressions of mRNA coding for thyroid hormone metabolising enzymes (dio2 and dio3) and thyroid receptor α (thra) were significantly different in these fish compared to their reference site. For perch collected at the two PCB-contaminated sites, there were no significant differences in T3 levels or in expression levels of the thyroid-related genes, compared to the reference fish. Fish from one of the PCB-contaminated sites had higher levels of thrombocytes compared with both the second PCB lake and their reference lake; hence PCBs are unlikely to be the cause of this effect. The current study suggests that lifelong exposure to PFASs could affect both the thyroid hormone status and immune defence of perch in the wild.

Bisphenol A and Bisphenol S Induce Endocrine and Chromosomal Alterations in Brown Trout.

Bisphenol A is a widely used compound found in large amount of consumer products. As concerns have been raised about its toxicological and public health effect, the use of alternatives to bisphenol A are now increasing. Bisphenol S is one of the analogues being used as a replacement for bisphenol A despite the fact that little is known about the effects of bisphenol S on living organisms. In this study, we investigated the potential endocrine and genotoxic effects of bisphenol A and bisphenol S in juvenile brown trout (Salmo trutta). The fish were exposed to the compounds for either 2 weeks or 8 weeks via sustained-release cholesterol implants containing doses of 2 mg/kg fish or 20 mg/kg fish of the substances. The effects on the thyroid hormone levels and the estrogenic disrupting marker vitellogenin were evaluated, along with the genotoxic markers micronucleated cells and erythrocyte nuclear abnormalities. An increase in plasma vitellogenin was observed in fish exposed to the high dose of bisphenol A for 2 weeks. At this experimental time the level of the thyroid hormone triiodothyronine (T3) in plasma was elevated after bisphenol S exposure at the high concentration, and paralleled by an increase of micronucleated cells. Moreover, bisphenol A induced an increase of micronuclei frequency in fish erythrocytes after the exposure at the lowest dose tested. Taken together the results indicate that both bisphenol A and its alternative bisphenol S cause endocrine disrupting and genotoxic effects in brown trout, although suggesting two different mechanisms of damage underlying bisphenol A and bisphenol S activity.

Ghrelin and Its Receptors in Gilthead Sea Bream: Nutritional Regulation.

Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.

Appetite-Controlling Endocrine Systems in Teleosts.

Mammalian studies have shaped our understanding of the endocrine control of appetite and body weight in vertebrates and provided the basic vertebrate model that involves central (brain) and peripheral signaling pathways as well as environmental cues. The hypothalamus has a crucial function in the control of food intake, but other parts of the brain are also involved. The description of a range of key neuropeptides and hormones as well as more details of their specific roles in appetite control continues to be in progress. Endocrine signals are based on hormones that can be divided into two groups: those that induce (orexigenic), and those that inhibit (anorexigenic) appetite and food consumption. Peripheral signals originate in the gastrointestinal tract, liver, adipose tissue, and other tissues and reach the hypothalamus through both endocrine and neuroendocrine actions. While many mammalian-like endocrine appetite-controlling networks and mechanisms have been described for some key model teleosts, mainly zebrafish and goldfish, very little knowledge exists on these systems in fishes as a group. Fishes represent over 30,000 species, and there is a large variability in their ecological niches and habitats as well as life history adaptations, transitions between life stages and feeding behaviors. In the context of food intake and appetite control, common adaptations to extended periods of starvation or periods of abundant food availability are of particular interest. This review summarizes the recent findings on endocrine appetite-controlling systems in fish, highlights their impact on growth and survival, and discusses the perspectives in this research field to shed light on the intriguing adaptations that exist in fish and their underlying mechanisms.

Acute anorexigenic action of leptin in rainbow trout is mediated by the hypothalamic Pi3k pathway.

Leptin (Lep) is an anorexigenic hormone and regulates appetite-related neuropeptides in mammals. A number of neuropeptides have also been linked to appetite regulation in teleost fish, but Lep signaling activation and effects on appetite-regulating neurons are poorly elucidated in early vertebrates. This study uses cellular, tissue and organismal approaches to elucidate the acute, central Lep action in rainbow trout. The results demonstrate that Lep activates phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription 3 in rainbow trout hypothalamus-derived cells, and that the phosphatidylinositol-3-kinase (Pi3k) inhibitor LY294002 can suppress the Lep-induced Akt phosphorylation. Intracerebroventricular (ICV) Lep administration strongly suppresses food intake at the doses of 0.05 and 0.5 µg Lep fish(-1) At low dose, Lep stimulates hypothalamic transcription of anorexigenic cocaine- and amphetamine-regulated transcript (Cart) and orexigenic neuropeptide Y. At high dose, Lep stimulates hypothalamic transcription of anorexigenic proopiomelanocortin (Pomc) A1, A2, and B, while coinjection with LY294002 reverses this upregulation. The data suggest that the anorexigenic action of Lep in rainbow trout is mediated through stimulation of the anorexigenic neuropeptides Pomc and Cart. Furthermore, ICV Lep treatment increases phosphor-Akt-immunoreactive cells in the nucleus lateralis tuberis, periventricular zone along infundibulum, and lateral recess surrounded by nucleus anterior tuberis, while LY294002 inhibits this effect. Lep receptor-immunoreactive cells are also predominant in these regions. These results demonstrate that Lep activates the Pi3k-Akt pathway in the lateral tuberal hypothalamus of rainbow trout for acute appetite regulation, indicating the conservation of anorexigenic Lep action in the mediobasal hypothalamus.

Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro.

Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes, ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or ß-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, fatty acid synthase and peroxisome proliferator-activated receptor-ß, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout.

As leptin has a key role on appetite, knowledge about leptin regulation is important in order to understand the control of energy balance. We aimed to explore the modulatory effects of adiposity on plasma leptin levels in vivo and the role of potential regulators on leptin expression and secretion in rainbow trout adipocytes in vitro. Fish were fed a regular diet twice daily ad libitum or a high-energy diet once daily at two ration levels; satiation (SA group) or restricted (RE group) to 25% of satiation, for 8weeks. RE fish had significantly reduced growth (p<0.001) and adipose tissue weight (p<0.001), and higher plasma leptin levels (p=0.022) compared with SA fish. Moreover, plasma leptin levels negatively correlated with mesenteric fat index (p=0.009). Adipocytes isolated from the different fish were treated with insulin, ghrelin, leucine, eicosapentaenoic acid or left untreated (control). In adipocytes from fish fed regular diet, insulin and ghrelin increased leptin secretion dose-dependently (p=0.002; p=0.033, respectively). Leptin secretion in control adipocytes was significantly higher in RE than in SA fish (p=0.022) in agreement with the in vivo findings, indicating that adipose tissue may contribute to the circulating leptin levels. No treatment effects were observed in adipocytes from the high-energy diet groups, neither in leptin expression nor secretion, except that leptin secretion was significantly reduced by leucine in RE fish adipocytes (p=0.025). Overall, these data show that the regulation of leptin in rainbow trout adipocytes by hormones and nutrients seems to be on secretion, rather than at the transcriptional level.

Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO2 in Atlantic halibut.

As a consequence of increasing atmospheric CO2, the world's oceans are becoming warmer and more acidic. Whilst the ecological effects of these changes are poorly understood, it has been suggested that fish performance including growth will be reduced mainly as a result of limitations in oxygen transport capacity. Contrary to the predictions given by the oxygen- and capacity-limited thermal tolerance hypothesis, we show that aerobic scope and cardiac performance of Atlantic halibut (Hippoglossus hippoglossus) increase following 14-16 weeks exposure to elevated temperatures and even more so in combination with CO2-acidified seawater. However, the increase does not translate into improved growth, demonstrating that oxygen uptake is not the limiting factor for growth performance at high temperatures. Instead, long-term exposure to CO2-acidified seawater reduces growth at temperatures that are frequently encountered by this species in nature, indicating that elevated atmospheric CO2 levels may have serious implications on fish populations in the future.

Ghrelin increases food intake, swimming activity and growth in juvenile brown trout (Salmo trutta).

Several key functions of ghrelin are well conserved through vertebrate phylogeny. However, some of ghrelin's effects are contradictory and among teleosts only a limited number of species have been used in functional studies on food intake and foraging-related behaviors. Here we investigated the long-term effects of ghrelin on food intake, growth, swimming activity and aggressive contest behavior in one year old wild brown trout (Salmo trutta) using intraperitoneal implants. Food intake and swimming activity were individually recorded starting from day 1, and aggressive behavior was tested at day 11, after ghrelin implantation. Body weight and growth rate were measured from the beginning to the end of the experiment. Triglycerides and lipase activity in muscle and liver; monoaminergic activity in the telencephalon and brainstem; and neuropeptide Y (NPY) mRNA levels in the hypothalamus were analyzed. Ghrelin treatment was found to increase food intake and growth without modifying lipid deposition or lipid metabolism in liver and muscle. Ghrelin treatment led to an increased foraging activity and a trend towards a higher swimming activity. Moreover, ghrelin-treated fish showed a tendency to initiate more conflicts, but this motivation was not reflected in a higher ability to win the conflicts. No changes were observed in monoaminergic activity and NPY mRNA levels in the brain. Ghrelin is therefore suggested to act as an orexigenic hormone regulating behavior in juvenile wild brown trout. These actions are accompanied with an increased growth without the alteration of liver and muscle lipid metabolism and they do not seem to be mediated by changes in brain monoaminergic activity or hypothalamic expression of NPY.

The role of ghrelin in energy balance regulation in fish.

Knowledge about the endocrine regulation of energy balance in fish is of interest for basic as well as aquaculture research. Ghrelin is a peptide hormone that was first identified in fish 10 years ago and has important roles in the control of food intake and metabolism. Both ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), have been found in numerous fish species. Their tissue distributions support the idea that ghrelin has an integrative role in the regulation of energy balance at both the central nervous system level and systemic level. In tilapia and goldfish, ghrelin treatment appears to increase food intake and to stimulate lipogenesis and tissue fat deposition to promote a more positive energy status. In rainbow trout, on the other hand, ghrelin decreases food intake. Goldfish and rainbow trout are the fish species in which the mode of action of ghrelin on food intake has been most thoroughly investigated. The results from these studies indicate that ghrelin alters food intake by acting on well-known appetite signals, such as CRH, NPY and orexin, in the hypothalamus in a species-specific manner. In goldfish, sensory fibres of the vagus nerve convey the signal from gut-derived ghrelin to modulate appetite. The data also indicate that ghrelin may modulate foraging/swimming activity and the perception of food in fish. Results related to the effects of energy status, temperature, and stressors on plasma ghrelin/tissue ghrelin mRNA levels are occasionally inconsistent between short- and long-term studies, between the protein and mRNA, and between species. Recent data also imply a role of ghrelin in carbohydrate metabolism. More functional studies are required to understand the role of ghrelin and its mechanisms of action in the regulation of energy balance among fish.

The impact of temperature on the metabolome and endocrine metabolic signals in Atlantic salmon (Salmo salar).

The aim was to elucidate the effects of elevated temperature on growth performance, growth- and appetite-regulating hormones and metabolism in Atlantic salmon, Salmo salar. Post-smolts in seawater (average mass 175g) that had been reared at 12°C were kept at three temperatures (8, 12 and 18°C) and sampled after one and three months. After three months, the fish kept in 18°C had decreased growth rate and condition factor, and elevated plasma levels of growth hormone (GH) and leptin, compared with fish kept at the lower temperatures. Food conversion efficiency was also decreased at 18°C, while at the same time protein uptake was improved and thus was not a limiting mechanism for growth. Redistribution of energy stores in fish at the highest temperature is evident as a preference of maintaining length growth during times of limited energy availability. NMR-based metabolomics analyses of plasma revealed that several metabolites involved in energy metabolism were negatively affected by temperature in the upper temperature range of Atlantic salmon. Specifically, the high temperature induced a decline of several amino acids (glutamine, tyrosine and phenylalanine) and a shift in lipid metabolism. It appears likely that the decreased food intake at the highest temperature is linked to an anorexigenic function of leptin, but also that the decreased food intake, feed conversion efficiency and condition factor can be linked to changes in GH endocrinology.

The role of growth hormone in growth, lipid homeostasis, energy utilization and partitioning in rainbow trout: interactions with leptin, ghrelin and insulin-like growth factor I.

The growth-promoting effects of in vivo growth hormone (GH) treatment were studied in relation to size and lipid content of energy stores including liver, mesentery, white muscle and belly flap in rainbow trout. In order to elucidate endocrine interactions and links to regulation of growth, adiposity and energy metabolism, plasma levels of GH, insulin-like growth factor I (IGF-I), leptin (Lep) and ghrelin, were assessed and correlated to growth and energy status. In addition tissue-specific expression of lepa1 mRNA was examined. Juvenile rainbow trout were implanted with sustained-release bovine GH implants and terminally sub-sampled at 1, 3 and 6 weeks. GH increased specific growth rate, reduced condition factor (CF) and increased feed conversion efficiency resulting in a redistribution of energy stores. Thus, GH decreased mesenteric (MSI) and liver somatic index (LSI). Lipid content of the belly flap increased following GH-treatment while liver and muscle lipid content decreased. Independent of GH substantial growth was accompanied by an increase in muscle lipids and a decrease in belly flap lipids. The data suggest that the belly flap may function as an energy buffering tissue during episodes of feeding and lean growth. Liver and muscle lipids were positively correlated to body weight, indicating a size-dependent change in adiposity. Hepatic lepa1 mRNA positively correlated to MSI and CF and its expression decreased following GH treatment, coinciding with decreased hepatic lipid content. Plasma Lep was positively correlated to MSI and belly flap lipid content, suggesting that Lep may communicate energy status. In summary, the observed GH tissue-specific effects on lipid metabolism in rainbow trout highlight the complex physiology of the energy reserves and their endocrine control.

Occurrence of ghrelin-producing cells, the ghrelin receptor and Na+,K + -ATPase in tissues of Atlantic halibut (Hippoglossus hippoglossus) during early development.

Ghrelin is a pituitary growth hormone (GH)-secretagogue that also has metabolic, reproductive, proliferative, immunological and brain functions in mammals. Far less is known about its role in fish. We have therefore performed an immunohistochemical determination of its tissue distribution in the developing Atlantic halibut (Hippoglossus hippoglossus) to gain insights into its potential function. Ghrelin immunoreactivity was detected in first-feeding halibut larvae in the skin, urinary bladder, gastrointestinal (GI) tract and olfactory lobe of the brain. In subsequent stages up to metamorphosis, ghrelin immunoreactivity declined in the skin and became evident in the gills. When the stomach developed, ghrelin immunoreactivity declined throughout the GI tract with the exception of the stomach, which exhibited an intense signal. Immunoreactive ghrelin cells were also present in the olfactory lobe, nerve and epithelium and in occasional cells of the buccal cavity and oesophagus. Ghrelin immunoreactivity had an overlapping distribution with that for Na(+),K(+)-ATPase, colocalisation also being observed in some ionocytes of the gill. The co-expression of ghrelin and the GH-secretagogue receptor in the same tissue indicates that ghrelin can exert both endocrine and paracrine actions in the developing halibut. The presence of immunoreactive ghrelin in several osmoregulatory tissues, the GI tract and sensory tissue provides strong evidence that ghrelin has multiple functions during development and also suggests targets for future investigations.

Ghrelin decreases food intake in juvenile rainbow trout (Oncorhynchus mykiss) through the central anorexigenic corticotropin-releasing factor system.

Ghrelin stimulates pituitary growth hormone (GH) release, and has a key role in the regulation of food intake and adiposity in vertebrates. To investigate the central effect of native rainbow trout ghrelin (rtghrelin) on food intake in rainbow trout, as well as its possible mode of action, four groups of fish received a single injection into the third brain ventricle (i.c.v. injection): (1) control group (physiological saline) (2) ghrelin-treated group (2.0 ng rtghrelin g bwt(-1)), (3) group given the corticotropin-releasing hormone receptor antagonist alpha-helical CRF 9-41 (ahCRF) (4.0 ng g bwt(-1)) and (4) group receiving the same dose of both ghrelin and ahCRF. Food intake was assessed 1h after treatment. In addition, the presence of the GHS-R (the ghrelin receptor) in the rainbow trout CNS was examined with Western blot. To investigate peripheral effects of ghrelin, rainbow trout received an intraperitoneal cholesterol-based implant with or without rtghrelin, and daily food intake was measured during 14 days. Weight and length were measured at the start and termination of the experiment and specific growth rates were calculated. Mesenteric fat stores, muscle and liver lipid content were analysed after the treatment period. Central ghrelin injections decreased food intake compared with controls, and treatment with ahCRF abolished the ghrelin-effect. Western blot analysis of the GHS-R revealed a single band at around 60 kDa in pituitary, hypothalamus, brain and stomach. Long-term peripheral ghrelin treatment decreased daily food intake compared with controls. This was reflected in a ghrelin-induced decrease in weight growth rate (p<0.06). There was no effect of ghrelin on plasma GH levels or tissue fat stores. The conclusion from this study is that the GHS-R is indicated in the CNS in rainbow trout and that ghrelin may act there as an anorexigenic hormone, through a CRF-mediated pathway. Elevated peripheral ghrelin levels also seem to lead to decreased feed intake in the longer term.

Identification of genes for the ghrelin and motilin receptors and a novel related gene in fish, and stimulation of intestinal motility in zebrafish (Danio rerio) by ghrelin and motilin.

In mammals ghrelin has a diverse range of effects including stimulation of gut motility but although present in teleost fish its effects on motility have not been investigated. The present study used bioinformatics to search for fish paralogues of the ghrelin receptor and the closely related motilin receptor, and investigated the effects of ghrelin and motilin on gut motility in zebrafish, Danio rerio. Fish paralogues of the human ghrelin and motilin receptor genes were identified, including those from the zebrafish. In addition, a third gene was identified in three species of pufferfish (the only fish genome completely sequenced), which is distinct from the ghrelin and motilin receptors but more closely aligned to these receptors relative to other G-protein coupled receptors. Immunohistochemistry demonstrated strong ghrelin receptor-like reactivity in the muscle of the zebrafish intestine. In isolated intestinal bulb and mid/distal intestine preparations, ghrelin, motilin, and the motilin receptor agonist erythromycin all evoked contraction; these responses ranged between 9% and 51% of the contractions evoked by carbachol (10(-6) M). There were some variations in the concentrations found to be active in the different tissues, e.g., whereas motilin and rat ghrelin caused contraction of the intestinal bulb circular muscle at concentrations as low as 10(-8) M, human ghrelin (10(-8) to 10(-6) M) was without activity. Neither ghrelin (10(-7) M) nor erythromycin (10(-5) M) affected the contractions evoked by electrical field stimulation. The results suggest that both ghrelin and motilin can regulate intestinal motility in zebrafish and most likely other teleosts, and are discussed in relation to the evolution of these regulatory peptides.