Parental selection for growth and early-life low stocking density increase the female-to-male ratio in European sea bass.

In European sea bass (Dicentrarchus labrax), as in many other fish species, temperature is known to influence the sex of individuals, with more males produced at relatively high temperatures. It is however unclear to what extent growth or stress are involved in such a process, since temperature is known to influence both growth rate and cortisol production. Here, we designed an experiment aiming at reducing stress and affecting early growth rate. We exposed larvae and juveniles originating from both captive and wild parents to three different treatments: low stocking density, food supplemented with tryptophan and a control. Low stocking density and tryptophan treatment respectively increased and decreased early growth rate. Each treatment influenced the stress response depending on the developmental stage, although no clear pattern regarding the whole-body cortisol concentration was found. During sex differentiation, fish in the low-density treatment exhibited lower expression of gr1, gr2, mr, and crf in the hypothalamus when compared to the control group. Fish fed tryptophan displayed lower crf in the hypothalamus and higher level of serotonin in the telencephalon compared to controls. Overall, fish kept at low density produced significantly more females than both control and fish fed tryptophan. Parents that have been selected for growth for three generations also produced significantly more females than parents of wild origin. Our findings did not allow to detect a clear effect of stress at the group level and rather point out a key role of early sexually dimorphic growth rate in sex determination.

Dietary amino acid supplementation affects temporal expression of amino acid transporters and metabolic genes in selected and commercial strains of rainbow trout (Oncorhynchus mykiss).

Replacement of fishmeal as the major protein source in feeds is critical for continued growth and sustainability of the aquaculture industry. However, numerous studies have shown suboptimal fish growth performance and reduced protein retention efficiency when carnivorous fish species are fed low fishmeal-high plant protein feeds. A study was conducted using a commercial strain and a genetically improved strain of rainbow trout selected for improved performance when fed an all plant protein diet to identify physiological differences associated with growth performance in the selected trout strain. Fifty individuals per strain (average weight ~ 580 g) were force-fed a plant-protein blend with and without amino acid supplementation (lysine, methionine and threonine) at 0.5% body weight and sampled at intervals over 24 h. Samples from intestine and liver were analyzed for specific gene expression analysis related to amino acid transporters, digestive process control, protein degradation and amino acid metabolism. The results showed that expression levels of various intestinal amino acid transporters (SLC1A1, SLC7A9, SLC15A, SLC1A5 SLC6A19 and SLC36A1) were affected by strain, diet and time. Moreover, significant interactions were found regarding the temporal expression levels of cholecystokinin (CCK-L), Krüppel-like factor 15 (KLF15) and aspartate aminotransferase (GOT) transcripts in the examined tissues. The results provide evidence that improved growth and protein retention of the selected strain fed an all-plant protein diet is a result of nutritional adaptation and an overall change in physiological homeostatic control.

Effects of short-term exercise on food intake and the expression of appetite-regulating factors in goldfish.

In mammals, growing evidence indicates that exercise affects food intake, metabolism and the expression and blood levels of appetite regulators. In this study, we examined the effects of short-term (30 min, at low and high water flow) exercise on food intake, glucose levels and the expressions of appetite regulators in goldfish hypothalamus (irisin, orexin, CART, leptin), intestine (CCK, PYY, proglucagon/GLP-1), muscle (irisin) and liver (leptin), of brain-derived neurotrophic factor (BDNF) in brain, interleukin-6 (IL6) in muscle and hypothalamus, and major metabolic enzymes, the glycolytic enzyme glucokinase (GCK) and its regulatory protein (GCKR) in liver, the lipolytic enzyme lipoprotein lipase in intestine and muscle, and trypsin in intestine. Fish submitted to high flow exercise had a lower post-exercise food intake compared to control fish but no differences were seen in glucose levels between groups. Exercise induced an increase in hypothalamic expression levels of CART, IL6 and BDNF, but not orexin, irisin, CRF, leptin and NPY. High flow exercise induced an increase in intestine CCK, PYY and GLP-1, and muscle irisin and IL-6 expression levels. Exercise had no effects on expression levels of hepatic leptin or any of the metabolic enzymes examined. Our results suggest that, in goldfish, short-term exercise might decrease feeding in part by affecting the expressions of myokines and peripheral, but not central appetite regulators or metabolic enzyme/hormones.

Photoperiod regulate gonad development via kisspeptin/kissr in hypothalamus and saccus vasculosus of Atlantic salmon (Salmo salar).

Atlantic salmon exhibit seasonal reproduction. However, the mechanisms governing this are still unclear. Generally speaking, kisspeptin has been recognized as a regulator of reproduction. Here, we report a relationship between kisspeptin, GnRH and photoperiod in Atlantic salmon. The results demonstrated that the expression of the Atlantic salmon kisspeptin-receptor (skissr) was not always consistent with the expression pattern of Atlantic salmon GnRH3 (sGnRH3) during all developmental processes. Kisspeptin may exert its influence primarily in the early and later stages of gonad development by promoting the secretion of sGnRH3. Meanwhile, the expression levels of kissr were higher in fish with gonads at stage II and stage V under the long-day photoperiod regime than under the short-day regime. In addition, both skissr and sGnRH3 were also expressed in the saccus vasculosus (SV), an organ only found in fish. The SV might be a seasonal sensor regulating reproduction in addition to the hypothalamus (Hyp).

Cloning and localization of immediate early response 2 (ier2) gene in the brain of medaka.

Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.

In vitro Assessment of Hg Toxicity in Hepatocytes from Heat-Stressed Atlantic Salmon.

Global warming may alter the bioavailability of contaminants in aquatic environments. In this work, mercury (Hg2+) toxicity was studied in cells obtained from Atlantic salmon smolt kept at 15 °C (optimal growth temperature) for 3 months or at a stepwise increase to 20 °C (temperature-stress) during 3 months prior to cell harvest to evaluate whether acclimation temperature affects Hg toxicity. To examine possible altered dietary requirements in warmer seas, one group of fish following the stepwise temperature regimes was fed a diet spiked with antioxidants. Atlantic salmon hepatocytes were exposed in vitro to 0, 1.0, or 100 µM Hg2+ for 48 h. Cytotoxicity, determined as electrical impedance changes with the xCELLigence system, and transcriptional responses, determined with RT-qPCR, were assessed as measures of toxicity. The results showed that inorganic Hg at a concentration up to 100 µM is not cytotoxic to Atlantic salmon hepatocytes. Significance and directional responses of the 18 evaluated target genes suggest that both Hg and temperature stress affected the transcription of genes encoding proteins involved in the protection against ROS-generated oxidative stress. Both stressors also affected the transcription of genes linked to lipid metabolism. Spiking the diet with antioxidants resulted in higher concentrations of Se and vitamin C and reduced concentration of Hg in the liver in vivo, but no interactions were seen between the dietary supplementation of antioxidants and Hg toxicity in vitro. In conclusion, no evidence was found suggesting that inorganic Hg is more toxic in cells harvested from temperature-stressed fish.

Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

The Atlantic salmon protein tyrosine kinase Tyk2: molecular cloning, modulation of expression and function.

Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/ß binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway.

Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for ß-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and ß-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

High levels of dietary phytosterols affect lipid metabolism and increase liver and plasma TAG in Atlantic salmon (Salmo salar L.).

Replacing dietary fishmeal (FM) and fish oil (FO) with plant ingredients in Atlantic salmon (Salmo salar L.) diets decreases dietary cholesterol and introduces phytosterols. The aim of the present study was to assess the effect of dietary sterol composition on cholesterol metabolism in Atlantic salmon. For this purpose, two dietary trials were performed, in which Atlantic salmon were fed either 100 % FM and FO (FM-FO) diet or one of the three diets with either high (80 %) or medium (40 %) plant protein (PP) and a high (70 %) or medium (35 %) vegetable oil (VO) blend (trial 1); or 70 % PP with either 100 % FO or 80 % of the FO replaced with olive, rapeseed or soyabean oil (trial 2). Replacing ≥ 70 % of FM with PP and ≥ 70 % of FO with either a VO blend or rapeseed oil increased plasma and liver TAG concentrations. These diets contained high levels of phytosterols and low levels of cholesterol. Fish fed low-cholesterol diets, but with less phytosterols, exhibited an increased expression of genes encoding proteins involved in cholesterol uptake and synthesis. The expression of these genes was, however, partially inhibited in rapeseed oil-fed fish possibly due to the high dietary and tissue phytosterol:cholesterol ratio. Atlantic salmon tissue and plasma cholesterol concentrations were maintained stable independent of the dietary sterol content.

Cloning and expression of Na+/K+ -ATPase and osmotic stress transcription factor 1 mRNA in black porgy, Acanthopagrus schlegeli during osmotic stress.

We cloned complementary DNA (cDNA) encoding the Na(+)/K(+)-ATPase (NKA) and the osmotic stress transcription factor 1 (OSTF1) from the kidney and gill, respectively, of the black porgy, Acanthopagrus schlegeli. Black porgy NKA full-length cDNA consists of 3078 base pairs (bp) and encodes a protein of 1025 amino acids; OSTF1 partial cDNA consists of 201 bp. To investigate the osmoregulatory ability of black porgy when black porgy were transferred to freshwater (FW), we examined the expression of NKA and OSTF1 mRNA in osmoregulatory organs, i.e., gill, kidney and intestine, using quantitative polymerase chain reaction (QPCR). To determine the hypoosmotic stressor specificity of the induction of NKA and OSTF1, black porgy were exposed to 30 degrees C water temperature for 24 h. In the gill, NKA mRNA was 4.2 times higher in FW, its expression in the kidney was 5.7 times higher in 10 per thousand seawater (10 per thousand SW) than in SW. In contrast, OSTF1 mRNA in the gill was 3.7 times higher in FW than in SW. The expression of heat shock protein 90 (HSP90) mRNA occurred not only during transfer to FW, but also in high-temperature water in all tested tissues, although the mRNA levels were not significantly different. Plasma osmolality level was decreased and cortisol level was increased when the fish were transferred from SW to FW. These results suggest that NKA and OSTF1 genes play important roles in hormonal regulation in osmoregulatory organs and that these genes are specific to hypoosmotic stress, improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.

Cloning of the prepro C-RFa gene and brain localization of the active peptide in Salmo salar.

In all vertebrates, the synthesis and release of prolactin (Prl) from pituitary lactotroph cells is tightly controlled by hypothalamic factors. We have cloned and characterized a hypothalamic cDNA from Atlantic salmon (Salmo salar) encoding C-RFa, a peptide structurally related to mammalian Prl-releasing peptide (PrRP). The deduced preprohormone precursor is composed of 155 amino acid residues presenting a 87.1% similarity to chum salmon C-RFa and a 100% similarity to all fish C-RFa in the bioactive precursor motifs. C-RFa-immunoreactive perikarya and fibres were located in the brain of S. salar, especially in the hypothalamus, olfactory tract, optic tectum and cerebellum. In contrast, immunolabelled fibres were not observed in the pituitary stalk or in the hypophysis. However, interestingly, we detected immunolabelled cells in the rostral pars distalis of the pituitary in the basolateral region in which Prl is synthesized. These results were confirmed by obtaining a strong signal by using reverse transcription/polymerase chain reaction (RT-PCR) on mRNA from both hypothalamus and pituitary. These data show, for the first time, by immunohistochemistry and RT-PCR, that C-RFa is produced in pituitary cells. Finally, based on these results, a possible function for C-RFa as a locally produced PrRP in this teleost is discussed.