Combinatorial metabolomic and transcriptomic analysis of muscle growth in hybrid striped bass (female white bass Morone chrysops x male striped bass M. saxatilis).

BACKGROUND: Understanding growth regulatory pathways is important in aquaculture, fisheries, and vertebrate physiology generally. Machine learning pattern recognition and sensitivity analysis were employed to examine metabolomic small molecule profiles and transcriptomic gene expression data generated from liver and white skeletal muscle of hybrid striped bass (white bass Morone chrysops x striped bass M. saxatilis) representative of the top and bottom 10 % by body size of a production cohort. RESULTS: Larger fish (good-growth) had significantly greater weight, total length, hepatosomatic index, and specific growth rate compared to smaller fish (poor-growth) and also had significantly more muscle fibers of smaller diameter (≤ 20 µm diameter), indicating active hyperplasia. Differences in metabolomic pathways included enhanced energetics (glycolysis, citric acid cycle) and amino acid metabolism in good-growth fish, and enhanced stress, muscle inflammation (cortisol, eicosanoids) and dysfunctional liver cholesterol metabolism in poor-growth fish. The majority of gene transcripts identified as differentially expressed between groups were down-regulated in good-growth fish. Several molecules associated with important growth-regulatory pathways were up-regulated in muscle of fish that grew poorly: growth factors including agt and agtr2 (angiotensins), nicotinic acid (which stimulates growth hormone production), gadd45b, rgl1, zfp36, cebpb, and hmgb1; insulin-like growth factor signaling (igfbp1 and igf1); cytokine signaling (socs3, cxcr4); cell signaling (rgs13, rundc3a), and differentiation (rhou, mmp17, cd22, msi1); mitochondrial uncoupling proteins (ucp3, ucp2); and regulators of lipid metabolism (apoa1, ldlr). Growth factors pttg1, egfr, myc, notch1, and sirt1 were notably up-regulated in muscle of good-growing fish. CONCLUSION: A combinatorial pathway analysis using metabolomic and transcriptomic data collectively suggested promotion of cell signaling, proliferation, and differentiation in muscle of good-growth fish, whereas muscle inflammation and apoptosis was observed in poor-growth fish, along with elevated cortisol (an anti-inflammatory hormone), perhaps related to muscle wasting, hypertrophy, and inferior growth. These findings provide important biomarkers and mechanisms by which growth is regulated in fishes and other vertebrates as well.

Epinephrine and glucose regulation of leptin synthesis and secretion in a teleost fish, the tilapia (Oreochromis mossambicus).

Acute stress is regulated through the sympathetic adrenergic axis where catecholamines mobilize energy stores including carbohydrates as a principal element of the endocrine stress response. Leptin is a cytokine critical for regulating energy expenditure in vertebrates and is stimulated by various stressors in fish such as fasting, hyperosmotic challenge, and hypoxia. However, little is known about the regulatory interactions between leptin and the endocrine stress axis in fishes and other ectothermic vertebrates. We evaluated the actions of epinephrine and glucose in regulating leptin A (LepA) in vivo and in vitro in tilapia. Using hepatocyte incubations and a homologous LepA ELISA, we show that LepA synthesis and secretion decline as ambient glucose levels increase (10-25 mM). By contrast, bolus glucose administration in tilapia increases lepa mRNA levels 14-fold at 6 h, suggesting systemic factors regulated by glucose may counteract the direct inhibitory effects of glucose on hepatic lepa mRNA observed in vitro. Epinephrine stimulated glucose and LepA secretion from hepatocytes in a dose-dependent fashion within 15 min but had little effect on lepa mRNA levels. An in vivo injection of epinephrine into tilapia stimulated a rapid rise in blood glucose which was followed by a 4-fold increase in hepatic lepa mRNA levels at 2.5 and 6 h. Plasma LepA was also elevated by 6 h relative to controls. Recombinant tilapia LepA administration in vivo did not have any significant effect on plasma epinephrine levels. The results of this study demonstrate LepA is negatively regulated by rises in extracellular glucose at the level of the hepatocyte but stimulated by hyperglycemia in vivo. Further, epinephrine increases LepA. This, along with previous work demonstrating a hyperglycemic and glycogenolytic effect of LepA in tilapia, suggests that epinephrine may stimulate leptin secretion to augment and fine tune glucose mobilization and homeostasis as part of the integrated, adaptive stress response.

Control of leptin by metabolic state and its regulatory interactions with pituitary growth hormone and hepatic growth hormone receptors and insulin like growth factors in the tilapia (Oreochromis mossambicus).

Leptin is an important cytokine for regulating energy homeostasis, however, relatively little is known about its function and control in teleost fishes or other ectotherms, particularly with regard to interactions with the growth hormone (GH)/insulin-like growth factors (IGFs) growth regulatory axis. Here we assessed the regulation of LepA, the dominant paralog in tilapia (Oreochromis mossambicus) and other teleosts under altered nutritional state, and evaluated how LepA might alter pituitary growth hormone (GH) and hepatic insulin-like growth factors (IGFs) that are known to be disparately regulated by metabolic state. Circulating LepA, and lepa and lepr gene expression increased after 3-weeks fasting and declined to control levels 10days following refeeding. This pattern of leptin regulation by metabolic state is similar to that previously observed for pituitary GH and opposite that of hepatic GHR and/or IGF dynamics in tilapia and other fishes. We therefore evaluated if LepA might differentially regulate pituitary GH, and hepatic GH receptors (GHRs) and IGFs. Recombinant tilapia LepA (rtLepA) increased hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 from isolated hepatocytes following 24h incubation. Intraperitoneal rtLepA injection, on the other hand, stimulated hepatic igf-1, but had little effect on hepatic igf-2, ghr1, or ghr2 mRNA abundance. LepA suppressed GH accumulation and gh mRNA in pituitaries in vitro, but had no effect on GH release. We next sought to test if abolition of pituitary GH via hypophysectomy (Hx) affects the expression of hepatic lepa and lepr. Hypophysectomy significantly increases hepatic lepa mRNA abundance, while GH replacement in Hx fish restores lepa mRNA levels to that of sham controls. Leptin receptor (lepr) mRNA was unchanged by Hx. In in vitro hepatocyte incubations, GH inhibits lepa and lepr mRNA expression at low concentrations, while higher concentration stimulates lepa expression. Taken together, these findings indicate LepA gene expression and secretion increases with fasting, consistent with the hormones function in promoting energy expenditure during catabolic stress. It would also appear that LepA might play an important role in stimulating GHR and IGFs to potentially spare declines in these factors during catabolism. Evidence also suggests for the first time in teleosts that GH may exert important regulatory effects on hepatic LepA production, insofar as physiological levels (0.05-1 nM) suppresse lepa mRNA accumulation. Leptin A, may in turn exert negative feedback effects on basal GH mRNA abundance, but not secretion.

Leptin stimulates hepatic growth hormone receptor and insulin-like growth factor gene expression in a teleost fish, the hybrid striped bass.

Leptin is an anorexigenic peptide hormone that circulates as an indicator of adiposity in mammals, and functions to maintain energy homeostasis by balancing feeding and energy expenditure. In fish, leptin tends to be predominantly expressed in the liver, another important energy storing tissue, rather than in fat depots as it is in mammals. The liver also produces the majority of circulating insulin-like growth factors (IGFs), which comprise the mitogenic component of the growth hormone (GH)-IGF endocrine growth axis. Based on similar regulatory patterns of leptin and IGFs that we have documented in previous studies on hybrid striped bass (HSB: Morone saxatilis×Morone chrysops), and considering the co-localization of these peptides in the liver, we hypothesized that leptin might regulate the endocrine growth axis in a manner that helps coordinate somatic growth with energy availability. Using a HSB hepatocyte culture system to simulate autocrine or paracrine exposure that might occur within the liver, this study examines the potential for leptin to modulate metabolism and growth through regulation of IGF gene expression directly, or indirectly through the regulation of GH receptors (GHR), which mediate GH-induced IGF expression. First, we verified that GH (50nM) has a classical stimulatory effect on IGF-1 and additionally show it stimulates IGF-2 transcription in hepatocytes. Leptin (5 and/or 50nM) directly stimulated in vitro GHR2 gene expression within 8h of exposure, and both GHR1 and GHR2 as well as IGF-1 and IGF-2 gene expression after 24h. Cells were then co-incubated with submaximal concentrations of leptin and GH (25nM each) to test if they had a synergistic effect on IGF gene expression, possibly through increased GH sensitivity following GHR upregulation by leptin. In combination, however, the treatments only had an additive effect on stimulating IGF-1 mRNA despite their capacity to increase GHR mRNA abundance. This suggests that leptin's stimulatory effect on GHRs may be limited to enhancing transcription or mRNA stability rather than inducing full translation of functional receptors, at least within a 24-h time frame. Finally, leptin was injected IP (100ng/g and 1µg/gBW) to test the in vivo regulation of hepatic IGF-1 and GHR1 gene expression. The 100ng/g BW leptin dose significantly upregulated in vivo IGF-1 mRNA levels relative to controls after 24h of fasting, but neither dosage was effective at regulating GHR1 gene expression. These studies suggest that stimulation of growth axis component transcripts by leptin may be an important mechanism for coordinating somatic growth with nutritional state in these and perhaps other fish or vertebrates, and represent the first evidence of leptin regulating GHRs in vertebrates.

Prolactin is a major inhibitor of hepatic Leptin A synthesis and secretion: studies utilizing a homologous Leptin A ELISA in the tilapia.

The present study identifies regulatory interactions between leptin A (LepA) and the pituitary hormone prolactin (PRL). In order to measure tilapia (Oreochromis mossambicus) LepA, an enzyme-linked immunosorbent assay (ELISA) utilizing a rabbit polyclonal antibody specific to tilapia LepA was first developed. The antibody shows strong cross reactivity to recombinant tilapia LepA (rtLepA), and a corresponding 16kDa protein in both tilapia and striped bass plasma, but not to recombinant human leptin (rhLep). The assay has a linear detection range of 0.25-1000nM, with intra- and interassay variability of 9% and 16%, respectively. Plasma LepA levels measured in tilapia ranged from 0.8 to 3.9nM, similar to that found for other vertebrates. Hypophysectomy (Hx) increased circulating LepA and lepa mRNA levels in the liver, the dominant source of hormone production. Adminstration of ovine PRL (oPRL, 5µg/g BW) to Hx fish restored circulating LepA and hepatic lepa mRNA levels to those of control fish. Additionally, oPRL reduced lepa mRNA levels in a dose-dependent fashion in cultured hepatocytes following an 18h incubation. Previous work in our lab indicates that rhLep stimulates PRL release in vitro from tilapia pituitaries. Here, both rtLepA and rhLep (0.5µg/g BW) increased mRNA expression of tilapia prolactin mRNAs (prl1, prl2) in the pituitary in vivo. These results demonstrate that LepA enhances pituitary prolactin synthesis and release, while PRL in turn inhibits hepatic leptin secretion and synthesis in teleosts. We postulate this regulatory interaction may be necessary for mobilizing energy reserves during acute hyperosmotic adaptation.

Temperature regulates sex determination and growth in the paralichthid flatfish California halibut.

California halibut (Paralichthys californicus) is a candidate species for aquaculture and stock enhancement. These applications rely on sex control, either to maximize the production of faster growing females or to match sex ratios in the wild. Other paralichthids exhibit temperature-dependent sex determination (TSD), but the presence and pattern of TSD is not well defined in California halibut. Juvenile California halibut were cultured at three distinct temperatures (15°C, 19°C, and 23°C) through the developmental period presumed to be thermosensitive based on findings from congeners. Sex ratios were quantified in each treatment using phenotypic sex identification techniques applied early (molecular biomarkers; 51-100 mm total length [TL]) and late (visual examination of the gonads; ≥100 mm TL) in the juvenile phase. Both techniques indicated similar sex determination trends at each temperature, with overall sex ratios assessed as 49.9% male at 15°C, 74.5% male at 19°C, and 98.2% male at 23°C. Growth rates were highest at 23°C and lowest at 15°C, with intrinsically fast- and slow-growing individuals at all temperatures. At 15°C and 19°C, females comprised a higher proportion among the fast growers than they did among the slow growers. These data show that California halibut exhibit TSD, with temperatures of 19°C and 23°C masculinizing fish while 15°C appears to produce a 1:1 sex ratio. This study will help optimize sex ratios and growth in hatcheries through thermal manipulation. Furthermore, the developed biomolecular tools and identified temperature thresholds will be important in future work to understand the influence of global warming on wild population demographics.

Characterization of membrane receptor binding activity for cortisol in the liver and kidney of the euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus).

Glucocorticoids (GCs) regulate an array of physiological responses in vertebrates. Genomic GC actions mediated by nuclear steroid receptors require a lag time on the order of hours to days to generate an appreciable physiological response. Experimental evidence has accumulated that GCs, can also act rapidly through a nongenomic mechanism to modulate cellular physiology in vertebrates. Causal evidence in the Mozambique tilapia (Oreochromis mossambicus) suggests that the GC cortisol exerts rapid, nongenomic actions in the gills, liver, and pituitary of this euryhaline teleost, but the membrane receptor mediating these actions has not been characterized. Radioreceptor binding assays were conducted to identify a putative GC membrane receptor site in O. mossambicus. The tissue distribution, binding kinetics, and pharmacological signature of the GC membrane-binding activity were characterized. High affinity (Kd=9.527±0.001 nM), low-capacity (Bmax=1.008±0.116 fmol/mg protein) [(3)H] cortisol binding was identified on plasma membranes prepared from the livers and a lower affinity (Kd=30.08±2.373 nM), low capacity (Bmax=4.690±2.373 fmol/mg protein) binding was found in kidney membrane preparations. Competitors with high binding affinity for nuclear GC receptors, mifepristone (RU486), dexamethasone, and 11-deoxycorticosterone, displayed no affinity for the membrane GC receptor. The association and dissociation kinetics of [(3)H] cortisol binding to membranes were orders of magnitude faster (t1/2=1.7-2.6 min) than those for the intracellular (nuclear) GC receptor (t1/2=10.2h). Specific [(3)H] cortisol membrane binding was also detected in the gill and pituitary but not in brain tissue. This study represents the first characterization of a membrane GC receptor in fishes and one of only a few characterized in vertebrates.

Circulating levels of plasma IGF-I during recovery from size-selective harvesting in Menidia menidia.

Selection for growth-related traits in domesticated fishes often results in predictable changes within the growth hormone-insulin-like growth factor (GH-IGF-1) axis. Little is known about the mechanisms controlling changes in growth capacity resulting from fishery-induced evolution. We took advantage of a long-term study where Menidia menidia were selected for size at age over multiple generations to mimic fisheries-induced selection. This selection regime produced three populations with significant differences in intrinsic growth rate. These growth differences partially rebounded, but persisted even after selection was relaxed, resulting in fast, intermediate, and slow-growing lines. Plasma IGF-1 was measured in these populations as a potential target of selection on growth. IGF-1 was significantly correlated with current length and mass, and was positively correlated with growth rate (g d(-1)) in two lines, indicating it may be an appropriate indicator of growth capacity. The slow-growing line exhibited higher overall IGF-1 levels relative to the depressed IGF-1 seen in the fast-growing line, contrary to our prediction. We offer possible explanations for this unusual pattern and argue that somatic growth is likely to be under control of mechanism(s) downstream to IGF-1. IGF-1 provides an interesting basis for understanding endocrine control of growth in response to artificial selection and recovery.

Nutritional programming in Nile tilapia (Oreochromis niloticus): Effect of low dietary protein on growth and the intestinal microbiome and transcriptome.

Nutritional programming is the idea that early nutrient contributions can influence organismal structure or function and is documented in a variety of vertebrates, yet studies in fish are largely lacking. Tilapia are an important foodfish, with global production having increased rapidly since the 1990s. They exhibit high disease-resistance and grow well on formulated feeds which makes them an ideal aquaculture species, however incorporating high quality proteins into feeds can be costly. As feed constitutes 50-70% of total production costs in aquaculture, reducing protein content could curb these costs and increase revenue. Thus, we examined the effects of feeding Nile tilapia (O. niloticus) fry a restricted protein diet for the first 7-21 days on growth, gut microbial flora, and the intestinal transcriptome. Fish were fed either a 25% restricted or 48% control crude protein starter (ST) diet for up to 21 days and then switched to a 25% or 38% control crude protein growout (GO) diet. Fish fed a 25% ST diet for 14 days followed by a 38% GO diet had significantly higher lengths and weights and better feed efficiency than fish fed the control 48% ST and 38% GO diet after 56 days of culture. Growth of fry on the 25% ST, 7-day/38% GO and the 25% ST,7-day/25% GO diets did not differ from the those fed the control protein diets, while fish fed the 25% ST diet for 21 days had significantly lower growth and survival rates. We observed no significant differences in either alpha or beta diversity of the gut microbial flora between diets, however species richness (Shannon Index) was higher in fry fed the 25% protein ST diet regardless of the GO diet. Similarly, fish fed the 25% ST diet for 14 days followed by the 38% GO diet had minimal changes to the intestinal transcriptome relative to fish fed the control 48% ST and 38% GO diet. However, those fed 25% ST and GO diets for the entire 56 days exhibited substantial differences in the gut transcriptome from other groups showing gene expression profiles characteristic of detrimental changes to gut physiology, protein metabolism and immune function. Results suggest protein restriction for up to 14 days early in development leads to enhanced growth and feed efficiency with minimal effects on gut microbes or intestinal function. Protein restriction beyond this period appears detrimental to fish growth and health as underscored by expression of disease related genes and higher mortality rates.

Cloning and characterization of leptin in a Perciform fish, the striped bass (Morone saxatilis): control of feeding and regulation by nutritional state.

In mammals, leptin is an anorexigenic peptide hormone that regulates energy homeostasis. It is produced predominantly by white adipose tissue and circulates as an endocrine indicator of energy reserves. Teleost leptin has been characterized in a few fish species, but its regulation is not well understood, particularly in response to nutritional status. In this study, we cloned a putative leptin in striped bass (Morone saxatilis) and report the first characterization of leptin in a Perciforme, the largest and most diverse order of fish. The striped bass leptin coding sequence was 65% homologous with pufferfish, 52% with Atlantic salmon, and 46% with human. PCR showed that leptin mRNA was exclusively expressed in the liver, and not adipose or other tissues. The leptin coding sequence of striped bass and the more widely cultured hybrid striped bass variety (HSB; Morone chrysops, white bass×M. saxatilis) were identical. We then evaluated whether the metabolic status of HSB might alter leptin gene expression. Juvenile HSB were subjected to 3weeks feed deprivation followed by 3weeks of refeeding. Quantitative PCR showed that fasting for 3weeks reduced hepatic leptin mRNA levels relative to fed controls. Leptin mRNA levels then increased upon refeeding, albeit levels were not completely restored to those seen in control fish fed throughout the experiment. Intraperitoneal injection of human leptin suppressed appetite in HSB. In as much as hepatic HSB leptin mRNA is regulated by nutritional state and has a corresponding anorexigenic effect, our results suggest that leptin may play a role in energy homeostasis in these advanced Perciformes.

Evidence for a leptin-insulin axis in a fish, the tilapia (Oreochromis mossambicus).

Leptin, insulin, and glucagon are involved in regulating glycaemia in vertebrates and play a role in the progression of obesity and type 2 diabetes. While mammals possess an 'adipoinsular axis' whereby insulin stimulates leptin release from adipocytes and leptin in turn feeds back on the pancreas to inhibit further insulin secretion, evidence of such an axis in non-mammalian vertebrates is unknown. We investigated the interactions between these glycaemic hormones and provide evidence for a leptin-insulin axis in a teleost fish, the tilapia. In the first study, we exposed hepatocytes to various concentrations of either insulin or glucagon to determine effects on leptin a (lepa) and then examined this in vivo with i.p. injections of both hormones. We also exposed isolated Brockmann bodies (pancreatic islets) to recombinant tilapia leptin A (rtLepA) and again followed this up with an i.p. injection to examine changes in insulin a and glucagon b. We found that glucagon increases lepa in vitroand in vivo, with the latter being 18-fold higher than saline-injected controls; however, the effects of rtLepA on glub were more variable. Insulin increased lepa by 2.5-fold in vitro and 70-fold in vivo, while rtLepA decreased insa at basal and increased it at high glucose concentrations. These data indicate that a leptin-insulin axis may be conserved among vertebrates and is thus essential for regulating nutrient balance but that the relationship is likely much more dynamic in teleosts as glycaemia is not as tightly regulated as it is in mammals.

FXYD-11 associates with Na+-K+-ATPase in the gill of Atlantic salmon: regulation and localization in relation to changed ion-regulatory status.

The Na(+)-K(+)-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na(+)-K(+)-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na(+)-K(+)-ATPase α-subunit. Transfer of freshwater (FW)-acclimated salmon to SW induced a parallel increase in total α-subunit and FXYD-11 protein expression. A similar concurrent increase was seen during smoltification in FW. In FW fish, cortisol induced an increase in both α-subunit and FXYD-11 abundance, and growth hormone further stimulated FXYD-11 levels. In SW fish, prolactin induced a decrease in FXYD-11 and α-subunit protein levels. In vitro cortisol (18 h, 10 µg/ml) stimulated FXYD-11, but not FXYD-9, mRNA levels in gills from FW and SW salmon. The data show that Na(+)-K(+)-ATPase expressed in branchial mitochondrion-rich cells is accompanied by FXYD-11, and that regulation of the two proteins is highly coordinated. The demonstrated association of FXYD-11 and α-subunit strengthens our hypothesis that FXYD-11 has a role in modulating the pump's kinetic properties. The presence of putative phosphorylation sites on the intracellular domain of FXYD-11 suggests the possibility that this protein also may transmit external signals that regulate Na(+)-K(+)-ATPase activity.

Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia under reduced frequency feeding strategies.

Feed constitutes 50-70% of total production costs of tilapia, one of the most widely cultured finfishes in the world. We evaluated reduced-feeding strategies for improving production efficiency of Nile tilapia (Oreochromis niloticus). In a 12-week pond trial, fish were fed daily, every other day, every third day, or not at all. Ponds were fertilized to enhance natural foods. In a fifth group fish were fed daily without pond fertilization. Fish fed daily with or without pond fertilization and fish fed every other day had higher specific growth rates, survivability, and net production than the other two treatments. Fish feed efficiency and benefit to cost ratio was highest for treatments fed in a pulsatile manner (i.e. fed every other day or every third day) with fish fed on alternate days providing the best net return among all groups. Fish fed on alternate days had more moderate gene expression levels of intestinal nutrient transporters which may allow for a more balanced and efficient nutrient uptake. Fecal microbe analyses identified 145 families of prokaryotic and 132 genera of eukaryotic organisms in tilapia. The highest diversity of prokaryotes was found in fish fed either every other day or daily in fertilized ponds and the highest diversity of eukaryotes was found in fish fed every other day. These studies indicate feeding Nile tilapia on alternate days along with weekly pond fertilization has no deleterious effects on growth, survivability, or production versus daily feeding regimes, but enhances feed efficiency by 76% and provides the greatest net return on investments. Our studies also suggest for the first time that combining alternate-day feeding with pond fertilization produces the greatest microbial biodiversity in the intestine that could contribute to enhanced feed efficiency and overall health of tilapia.

Transcriptional regulation of prolactin in a euryhaline teleost: Characterisation of gene promoters through in silico and transcriptome analyses.

The sensitivity of prolactin (Prl) cells of the Mozambique tilapia (Oreochromis mossambicus) pituitary to variations in extracellular osmolality enables investigations into how osmoreception underlies patterns of hormone secretion. Through the actions of their main secretory products, Prl cells play a key role in supporting hydromineral balance of fishes by controlling the major osmoregulatory organs (ie, gill, intestine and kidney). The release of Prl from isolated cells of the rostral pars distalis (RPD) occurs in direct response to physiologically relevant reductions in extracellular osmolality. Although the particular signal transduction pathways that link osmotic conditions to Prl secretion have been identified, the processes that underlie hyposmotic induction of prl gene expression remain unknown. In this short review, we describe two distinct tilapia gene loci that encode Prl177 and Prl188 . From our in silico analyses of prl177 and prl188 promoter regions (approximately 1000 bp) and a transcriptome analysis of RPDs from fresh water (FW)- and seawater (SW)-acclimated tilapia, we propose a working model for how multiple transcription factors link osmoreceptive processes with adaptive patterns of prl177 and prl188 gene expression. We confirmed via RNA-sequencing and a quantitative polymerase chain reaction that multiple transcription factors emerging as predicted regulators of prl gene expression are expressed in the RPD of tilapia. In particular, gene transcripts encoding pou1f1, stat3, creb3l1, pbxip1a and stat1a were highly expressed; creb3l1, pbxip1a and stat1a were elevated in fish acclimated to SW vs FW. Combined, our in silico and transcriptome analyses set a path for resolving how adaptive patterns of Prl secretion are achieved via the integration of osmoreceptive processes with the control of prl gene transcription.

cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression.

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca(2+) and acid-base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Plasma ghrelin and growth hormone regulation in response to metabolic state in hybrid striped bass: effects of feeding, ghrelin and insulin-like growth factor-I on in vivo and in vitro GH secretion.

The regulation of growth hormone (GH) secretion by ghrelin during variable metabolic states is poorly understood. We examined plasma GH and ghrelin in hybrid striped bass (HSB) undergoing seasonally-based feeding and temperature manipulations. Fasting for 21 days (d) at 24 degrees C resulted in catabolism and up-regulation of plasma GH and ghrelin relative to fed controls. Continued fasting during cold-banking (14 degrees C, 90 d) resulted in a further 43-fold increase in ghrelin while GH remained elevated. A subsequent 19 day refeeding period at 24 degrees C elicited hyperphagic and compensatory growth responses, accompanied by declines in ghrelin and GH. We then tested the role of ghrelin in stimulating GH release in vivo and in vitro. Intraperitoneal injections of ghrelin resulted in dose-dependent increases in plasma GH after 6 hours (h). Ghrelin also increased GH release from HSB pituitaries during 6h incubations. Lastly, we assessed how metabolic state, ghrelin and insulin-like growth factor-I (IGF-I) affect in vitro pituitary GH release. Spontaneous GH release was 5.2-fold higher from pituitaries of fasted compared with fed animals. Ghrelin was equally effective in stimulating GH release from pituitaries of fed and starved animals, while it was ineffective in enhancing GH release from pituitaries of starved (21 d) then refed (4d) HSB. Incubation with IGF-I inhibited GH release regardless of metabolic state. These studies are the first to show that seasonally-based periods of feed deprivation and low temperature yield sustained increases in GH secretion that are likely mediated, at least partially, through elevated ghrelin, reduced IGF-I negative feedback and fasting-induced spontaneous GH release.

Nongenomic glucocorticoid effects and their mechanisms of action in vertebrates.

Glucocorticoids (GC) act on multiple organ systems to regulate a variety of physiological processes in vertebrates. Due to their immunosuppressive and anti-inflammatory actions, glucocorticoids are an attractive target for pharmaceutical development. Accordingly, they are one of the most widely prescribed classes of therapeutics. Through the classical mechanism of steroid action, glucocorticoids are thought to mainly affect gene transcription, both in a stimulatory and suppressive fashion, regulating de novo protein synthesis that subsequently leads to the physiological response. However, over the past three decades multiple lines of evidence demonstrate that glucocorticoids may work through rapid, nonclassical mechanisms that do not require alterations in gene transcription or translation. This review assimilates evidence across the vertebrate taxa on the diversity of nongenomic actions of glucocorticoids and the membrane-associated cellular mechanisms that may underlie rapid glucocorticoid responses to include potential binding sites characterized to date.

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma).

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.

Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia.

We assessed if leptin, a cytokine hormone known to enhance energy expenditure by promoting lipid and carbohydrate catabolism in response to physiologic stress, might directly regulate cellular glycolysis. A transcriptomic analysis of prolactin cells in the tilapia (Oreochromis mossambicus) pituitary rostral pars distalis (RPD) revealed that recombinant leptin (rtLep) differentially regulates 1,995 genes, in vitro. Machine learning algorithms and clustering analyses show leptin influences numerous cellular gene networks including metabolism; protein processing, transport, and metabolism; cell cycle and the hypoxia response. Leptin stimulates transcript abundance of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh) in a covariate manner to the hypoxic stress gene network. Orthogonal tests confirm that rtLepA dose-dependently increases gapdh gene expression in the RPD along with transcript abundance of 6-phosphofructo-1-kinase (pfk1), the rate limiting glycolytic enzyme. Functional testing demonstrated that leptin stimulates PFK activity and glycolytic output, while Stattic (a STAT3 blocker) was sufficient to suppress these responses, indicating leptin stimulates glycolysis through a STAT3-dependent mechanism. Leptin also stimulated pfk1 gene expression and lactate production in primary hepatocyte incubations in a similar manner to those shown for the pituitary RPD. This work characterizes a critical metabolic action of leptin to directly stimulate glycolysis across tissue types in a teleost model system, and suggest that leptin may promote energy expenditure, in part, by stimulating glycolysis. These data in a teleost fish, suggest that one of leptin's ancient, highly-conserved functions among vertebrates may be stimulation of glycolysis to facilitate the energetic needs associated with various stressors.

Ecology of sleeping: the microbial and arthropod associates of chimpanzee beds.

The indoor environment created by the construction of homes and other buildings is often considered to be uniquely different from other environments. It is composed of organisms that are less diverse than those of the outdoors and strongly sourced by, or dependent upon, human bodies. Yet, no one has ever compared the composition of species found in contemporary human homes to that of other structures built by mammals, including those of non-human primates. Here we consider the microbes and arthropods found in chimpanzee beds, relative to the surrounding environment (n = 41 and 15 beds, respectively). Based on the study of human homes, we hypothesized that the microbes found in chimpanzee beds would be less diverse than those on nearby branches and leaves and that their beds would be primarily composed of body-associated organisms. However, we found that differences between wet and dry seasons and elevation above sea level explained nearly all of the observed variation in microbial diversity and community structure. While we can identify the presence of a chimpanzee based on the assemblage of bacteria, the dominant signal is that of environmental microbes. We found just four ectoparasitic arthropod specimens, none of which appears to be specialized on chimpanzees or their structures. These results suggest that the life to which chimpanzees are exposed while in their beds is predominately the same as that of the surrounding environment.