Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia.

In estuarine environments, euryhaline fish maintain a narrow range of internal osmolality despite daily changes in environmental salinity that can range from fresh water (FW) to seawater (SW). The capacity of euryhaline fish to maintain homeostasis in a range of environmental salinities is primarily facilitated by the neuroendocrine system. One such system, the hypothalamic-pituitary-interrenal (HPI) axis, culminates in the release of corticosteroids such as cortisol into circulation. Cortisol functions as both a mineralocorticoid and glucocorticoid in fish because of its roles in osmoregulation and metabolism, respectively. The gill, a key site for osmoregulation, and the liver, the primary storage site for glucose, are known targets of cortisol's actions during salinity stress. While cortisol facilitates acclimation to SW environments, less is known on its role during FW adaptation. In this study, we characterized the responses of plasma cortisol, mRNA expression of pituitary pro-opiomelanocortin (pomc), and mRNA expression of liver and gill corticosteroid receptors (gr1, gr2, and mr) in the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity challenges. Specifically, tilapia were subjected to salinity transfer regimes from steady-state FW to SW, SW to FW (experiment 1) or steady state FW or SW to tidal regimen (TR, experiment 2). In experiment 1, fish were sampled at 0 h, 6 h, 1, 2, and 7 d post transfer; while in experiment 2, fish were sampled at day 0 and day 15. We found a rise in pituitary pomc expression and plasma cortisol following transfer to SW while branchial corticosteroid receptors were immediately downregulated after transfer to FW. Moreover, branchial expression of corticosteroid receptors changed with each salinity phase of the TR, suggesting rapid environmental modulation of corticosteorid action. Together, these results support the role of the HPI-axis in promoting salinity acclimation, including in dynamically-changing environments.

Osmosensitive transcription factors in the prolactin cell of a euryhaline teleost.

In euryhaline fish, prolactin (Prl) plays a key role in freshwater acclimation. Prl release in the rostral pars distalis (RPD) of the pituitary is directly stimulated by a fall in extracellular osmolality. Recently, we identified several putative transcription factor modules (TFM) predicted to bind to the promoter regions of the two prl isoforms in Mozambique tilapia, Oreochromis mossambicus. We characterized the effects of extracellular osmolality on the activation of these TFMs from RPDs, in vivo and in vitro. OCT1_PIT1 01, CEBP_CEBP 01 and BRNF_RXRF 01 were significantly activated in freshwater (FW)- acclimated tilapia RPDs while SORY_PAX3 02 and SP1F_SP1F 06, SP1F_SP1F 09 were significantly activated in seawater (SW)- counterparts. Short-term incubation of SW- acclimated tilapia RPDs in hyposmotic media (280 mOsm/kg) resulted in activation of CAAT_AP1F 01, OCT1_CEBP 01, AP1F_SMAD 01, GATA_SP1F 01, SORY_PAX6 01 and CREB_EBOX 02, EBOX_AP2F 01, EBOX_MITF 01 while hyperosmotic media (420 mOsm/kg) activated SORY_PAX3 02 and AP1F_SMAD 01 in FW- tilapia. Short-term incubation of dispersed Prl cells from FW- acclimated fish exposed to hyperosmotic conditions decreased pou1f1, pou2f1b, stat3, stat1a and ap1b1 expression, while pou1f1, pou2f1b, and stat3 were inversely related to osmolality in their SW- counterparts. Further, in Prl cells of SW- tilapia, creb3l1 was suppressed in hyposmotic media. Collectively, our results indicate that multiple TFMs are involved in regulating prl transcription at different acclimation salinities and, together, they modulate responses of Prl cells to changes in extracellular osmolality. These responses reflect the complexity of osmosensitive molecular regulation of the osmoreceptive Prl cell of a euryhaline teleost.

Tilapia prolactin cells are thermosensitive osmoreceptors.

Prolactin (PRL) cells within the rostral pars distalis (RPD) of euryhaline and eurythermal Mozambique tilapia, Oreochromis mossambicus, rapidly respond to a hyposmotic stimulus by releasing two distinct PRL isoforms, PRL188 and PRL177. Here, we describe how environmentally relevant temperature changes affected mRNA levels of prl188 and prl177 and the release of immunoreactive prolactins from RPDs and dispersed PRL cells. When applied under isosmotic conditions (330 mosmol/kgH2O), a 6°C rise in temperature stimulated the release of PRL188 and PRL177 from both RPDs and dispersed PRL cells under perifusion. When exposed to this same change in temperature, ∼50% of dispersed PRL cells gradually increased in volume by ∼8%, a response partially inhibited by the water channel blocker, mercuric chloride. Following their response to increased temperature, PRL cells remained responsive to a hyposmotic stimulus (280 mosmol/kgH2O). The mRNA expression of transient potential vanilloid 4, a Ca2+-channel involved in hyposmotically induced PRL release, was elevated in response to a rise in temperature in dispersed PRL cells and RPDs at 6 and 24 h, respectively; prl188 and prl177 mRNAs were unaffected. Our findings indicate that thermosensitive PRL release is mediated, at least partially, through a cell-volume-dependent pathway similar to how osmoreceptive PRL release is achieved.

Endocrine and osmoregulatory responses to tidally-changing salinities in fishes.

Salinity is one of the main physical properties that govern the distribution of fishes across aquatic habitats. In order to maintain their body fluids near osmotic set points in the face of salinity changes, euryhaline fishes rely upon tissue-level osmotically-induced responses and systemic endocrine signaling to direct adaptive ion-transport processes in the gill and other critical osmoregulatory organs. Some euryhaline teleosts inhabit tidally influenced waters such as estuaries where salinity can vary between fresh water (FW) and seawater (SW). The physiological adaptations that underlie euryhalinity in teleosts have been traditionally identified in fish held under steady-state conditions or following unidirectional transfers between FW and SW. Far fewer studies have employed salinity regimes that simulate the tidal cycles that some euryhaline fishes may experience in their native habitats. With an emphasis on prolactin (Prl) signaling and branchial ionocytes, this mini-review contrasts the physiological responses between euryhaline fish responding to tidal versus unidirectional changes in salinity. Three patterns that emerged from studying Mozambique tilapia (Oreochromis mossambicus) subjected to tidally-changing salinities include, 1) fish can compensate for continuous and marked changes in external salinity to maintain osmoregulatory parameters within narrow ranges, 2) tilapia maintain branchial ionocyte populations in a fashion similar to SW-acclimated fish, and 3) there is a shift from systemic to local modulation of Prl signaling.

Sex-specific responses to growth hormone and luteinizing hormone in a model teleost, the Mozambique tilapia.

Across the vertebrate lineage, sexual dimorphism in body size is a common phenomenon that results from trade-offs between growth and reproduction. To address how key hormones that regulate growth and reproduction interact in teleost fishes, we studied Mozambique tilapia (Oreochromis mossambicus) to determine whether the activities of luteinizing hormone (Lh) are modulated by growth hormone (Gh), and conversely, whether targets of Gh are affected by the presence of Lh. In particular, we examined how gonadal morphology and specific gene transcripts responded to ovine GH (oGH) and/or LH (oLH) in hypophysectomized male and female tilapia. Hypophysectomized females exhibited a diminished gonadosomatic index (GSI) concomitant with ovarian follicular atresia. The combination of oGH and oLH restored GSI and ovarian morphology to conditions observed in sham-operated controls. A similar pattern was observed for GSI in males. In control fish, gonadal gh receptor (ghr2) and estrogen receptor ß (erß) expression was higher in females versus males. A combination of oGH and oLH restored erß and arß in females. In males, testicular insulin-like growth factor 3 (igf3) expression was reduced following hypophysectomy and subsequently restored to control levels by either oGH or oLH. By contrast, the combination of both hormones was required to recover ovarian igf3 expression in females. In muscle, ghr2 expression was more responsive to oGH in males versus females. In the liver of hypophysectomized males, igf2 expression was diminished by both oGH and oLH; there was no effect of hypophysectomy, oGH, or oLH on igf2 expression in females. Collectively, our results indicate that gene transcripts associated with growth and reproduction exhibit sex-specific responses to oGH and oLH. These responses reflect, at least in part, how hormones mediate trade-offs between growth and reproduction, and thus sexual dimorphism, in teleost fishes.

Enhanced expression of ncc1 and clc2c in the kidney and urinary bladder accompanies freshwater acclimation in Mozambique tilapia.

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities via the activities of multiple osmoregulatory organs, namely the gill, gastrointestinal tract, skin, kidney, and urinary bladder. Teleosts residing in freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water, and, therefore, the kidney and urinary bladder reabsorb Na+ and Cl- to support the production of dilute urine. Nonetheless, the regulated pathways for Na+ and Cl- transport by euryhaline fishes, especially in the urinary bladder, have not been fully resolved. Here, we first investigated the ultrastructure of epithelial cells within the urinary bladder of FW-acclimated Mozambique tilapia (Oreochromis mossambicus) by electron microscopy. We then investigated whether tilapia employ Na+/Cl- cotransporter 1 (Ncc1) and Clc family Cl- channel 2c (Clc2c) for the reabsorption of Na+ and Cl- by the kidney and urinary bladder. We hypothesized that levels of their associated gene transcripts vary inversely with environmental salinity. In whole kidney and urinary bladder homogenates, ncc1 and clc2c mRNA levels were markedly higher in steady-state FW- versus SW (seawater)-acclimated tilapia. Following transfer from SW to FW, ncc1 and clc2c in both the kidney and urinary bladder were elevated within 48 h. A concomitant increase in branchial ncc2, and decreases in Na+/K+/2Cl-cotransporter 1a (nkcc1a) and cystic fibrosis transmembrane regulator 1 (cftr1) levels indicated a transition from Na+ and Cl- secretion to absorption by the gills in parallel with the identified renal and urinary bladder responses to FW transfer. Our findings suggest that Ncc1 and Clc2c contribute to the functional plasticity of the kidney and urinary bladder in tilapia.

Growth hormone regulates intestinal gene expression of nutrient transporters in tilapia (Oreochromis mossambicus).

Among the various ways that growth hormone (GH) underlies the growth physiology of teleost fishes, GH stimulates transport pathways that facilitate the absorption of nutrients across intestinal epithelia. The current study investigated the effects of GH on the gene expression of nutrient transporters in an omnivorous teleost, the Mozambique tilapia (Oreochromis mossambicus). We employed pituitary gland removal (hypophysectomy) and hormone replacement to assess whether GH directs the gene expression of the GH receptor (ghr2), the peptide transporters, pept1a, pept1b and pept2, the amino acid transporter, slc7a9, the Na+/glucose cotransporter, sglt1, the glucose transporter, glut2, and the myo-inositol transporter, smit2, in anterior, middle, and posterior intestine. ghr2 was predominantly expressed in posterior intestine, while pept1a, pept1b, slc7a9, sglt1, glut2, and smit2 exhibited the highest mRNA levels in anterior and/or middle intestine. While hypophysectomized tilapia exhibited diminished expression of ghr2, pept1a, pept1b, slc7a9, and glut2 compared with intact and sham-operated controls, only ghr2, pept1a, pept1b and glut2 levels were restored by GH replacement. Our findings indicate that GH supports growth, at least in part, by stimulating the gene expression of its cognate receptor and key nutrient transporters in the intestine.

Sex, salinity and sampling period dependent patterns of growth hormone mRNA expression in Mozambique tilapia.

Tilapias comprise the second most aquacultured finfish group in the world. Such popularity stems in part from their tolerance to a wide range of environmental conditions and their sexually dimorphic nature, where males grow larger than females. As in other vertebrates, growth in tilapia is regulated by the growth hormone/ insulin like growth factor (GH/IGF) system. Moreover, environmental salinity has previously been shown to directly modulate growth in tilapia. Less is known, however, regarding how salinity may modulate sexually dimorphic growth. Utilizing a species of tilapia of high salinity tolerance, the Mozambique tilapia, Oreochromis mossambicus, we compared gh expression from the pituitary of male and female adults reared in fresh water (FW), seawater (SW), and a tidal regime (TR) characterized by dynamically changing salinities between FW and SW every six hours, over a 24 h period. We found significant effects of sex, salinity regime and whether fish were sampled during daylight or dark hours. In both sexes, gh expression was greater in fish reared in SW and TR compared with those in FW, and greater in fish sampled during dark hours, compared with those sampled in daylight hours. Pituitary gh expression was greater in males than in females reared in SW and TR, but not in FW. These results provide insight on the sex-specific modulation of gh expression by environmental factors in Mozambique tilapia.

The effects of transfer from steady-state to tidally-changing salinities on plasma and branchial osmoregulatory variables in adult Mozambique tilapia.

The Mozambique tilapia, Oreochromis mossambicus, is a teleost fish native to estuarine waters that vary in salinity between fresh water (FW) and seawater (SW). The neuroendocrine system plays a key role in salinity acclimation by directing ion uptake and extrusion in osmoregulatory tissues such as gill. While most studies with O. mossambicus have focused on acclimation to steady-state salinities, less is known about the ability of adult fish to acclimate to dynamically-changing salinities. Plasma osmolality, prolactin (PRL) levels, and branchial gene expression of PRL receptors (PRLR1 and PRLR2), Na+/Cl- and Na+/K+/2Cl- co-transporters (NCC and NKCC), Na+/K+-ATPase (NKAα1a and NKAα1b), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 3 (AQP3) were measured in fish reared in FW and SW steady-state salinities, in a tidal regimen (TR) where salinities changed between FW and SW every six hours, and in fish transferred from FW or SW to TR. Regardless of rearing regimen, plasma osmolality was higher in fish in SW than in FW fish, while plasma PRL was lower in fish in SW. Furthermore, branchial gene expression of effectors of ion transport in TR fish showed greater similarity to those in steady-state SW fish than in FW fish. By seven days of transfer from steady-state FW or SW to TR, plasma osmolality, plasma PRL and branchial expression of effectors of ion transport were similar to those of fish reared in TR since larval stages. These findings demonstrate the ability of adult tilapia reared in steady-state salinities to successfully acclimate to dynamically-changing salinities. Moreover, the present findings suggest that early exposure to salinity changes does not significantly improve survivability in future challenge with dynamically-changing salinities.

Acute salinity tolerance and the control of two prolactins and their receptors in the Nile tilapia (Oreochromis niloticus) and Mozambique tilapia (O. mossambicus): A comparative study.

Osmoregulation in vertebrates is largely controlled by the neuroendocrine system. Prolactin (PRL) is critical for the survival of euryhaline teleosts in fresh water by promoting ion retention. In the euryhaline Mozambique tilapia (Oreochromis mossambicus), pituitary PRL cells release two PRL isoforms, PRL188 and PRL177, in response to a fall in extracellular osmolality. Both PRLs function via two PRL receptors (PRLRs) denoted PRLR1 and PRLR2. We conducted a comparative study using the Nile tilapia (O. niloticus), a close relative of Mozambique tilapia that is less tolerant to increases in environmental salinity, to investigate the regulation of PRLs and PRLRs upon acute hyperosmotic challenges in vivo and in vitro. We hypothesized that differences in the regulation of PRLs and PRLRs underlie the variation in salinity tolerance of tilapias within the genus Oreochromis. When transferred from fresh water to brackish water (20‰), Nile tilapia increased plasma osmolality and decreased circulating PRLs, especially PRL177, to a greater extent than Mozambique tilapia. In dispersed PRL cell incubations, the release of both PRLs was less sensitive to variations in medium osmolality in Nile tilapia than in Mozambique tilapia. By contrast, increases in pituitary and branchial prlr2 gene expression in response to a rise in extracellular osmolality were more pronounced in Nile tilapia relative to its congener, both in vitro and in vivo. Together, these results support the conclusion that inter-specific differences in salinity tolerance between the two tilapia congeners are tied, at least in part, to the distinct responses of both PRLs and their receptors to osmotic stimuli.

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.

Rearing Mozambique tilapia in tidally-changing salinities: Effects on growth and the growth hormone/insulin-like growth factor I axis.

The growth hormone (GH)/insulin-like growth factor (IGF) axis plays a central role in the regulation of growth in teleosts and has been shown to be affected by acclimation salinity. This study was aimed at characterizing the effects of rearing tilapia, Oreochromis mossambicus, in a tidally-changing salinity on the GH/IGF axis and growth. Tilapia were raised in fresh water (FW), seawater (SW), or in a tidally-changing environment, in which salinity is switched between FW (TF) and SW (TS) every 6h, for 4months. Growth was measured over all time points recorded and fish reared in a tidally-changing environment grew significantly faster than other groups. The levels of circulating growth hormone (GH), insulin-like growth factor I (IGF-I), pituitary GH mRNA, gene expression of IGF-I, IGF-II, and growth hormone receptor 2 (GHR) in the muscle and liver were also determined. Plasma IGF-I was higher in FW and TS than in SW and TF tilapia. Pituitary GH mRNA was higher in TF and TS than in FW and SW tilapia. Gene expression of IGF-I in the liver and of GHR in both the muscle and liver changed between TF and TS fish. Fish growth was positively correlated with GH mRNA expression in the pituitary, and GHR mRNA expression in muscle and liver tissues. Our study indicates that rearing fish under tidally-changing salinities elicits a distinct pattern of endocrine regulation from that observed in fish reared in steady-state conditions, and may provide a new approach to increase tilapia growth rate and study the regulation of growth in euryhaline fish.

Prolactin 177, prolactin 188, and extracellular osmolality independently regulate the gene expression of ion transport effectors in gill of Mozambique tilapia.

This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380, and 450 mosmol/kg H2O. The mRNA expression of Na(+)/K(+)-ATPase α1a (NKA α1a) and Na(+)/Cl(-) cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na(+)/K(+)/2Cl(-) cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL177 and tPRL188). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKAα1a, and PRLR1 expression; Na(+)/H(+) exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL177 and tPRL188 differentially induce expression of these ion transporters, according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors, as well as directly respond to PRL to modulate branchial ionoregulatory functions.

The effects of acute salinity challenges on osmoregulation in Mozambique tilapia reared in a tidally changing salinity.

This study characterizes the differences in osmoregulatory capacity among Mozambique tilapia, Oreochromis mossambicus, reared in freshwater (FW), in seawater (SW) or under tidally driven changes in salinity. This was addressed through the use of an abrupt exposure to a change in salinity. We measured changes in: (1) plasma osmolality and prolactin (PRL) levels; (2) pituitary expression of prolactin (PRL) and its receptors, PRLR1 and PRLR2; (3) branchial expression of PRLR1, PRLR2, Na(+)/Cl(-) co-transporter (NCC), Na(+)/K(+)/2Cl(-) co-transporter (NKCC), α1a and α1b isoforms of Na(+)/K(+)-ATPase (NKA), cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 3 (AQP3) and Na(+)/H(+) exchanger 3 (NHE3). Mozambique tilapia reared in a tidal environment successfully adapted to SW while fish reared in FW did not survive a transfer to SW beyond the 6 h sampling. With the exception of CFTR, the change in the expression of ion pumps, transporters and channels was more gradual in fish transferred from tidally changing salinities to SW than in fish transferred from FW to SW. Upon transfer to SW, the increase in CFTR expression was more robust in tidal fish than in FW fish. Tidal and SW fish successfully adapted when transferred to FW. These results suggest that Mozambique tilapia reared in a tidally changing salinity, a condition that more closely represents their natural history, gain an adaptive advantage compared with fish reared in FW when facing a hyperosmotic challenge.

Discovery of conventional prolactin from the holocephalan elephant fish, Callorhinchus milii.

The conventional prolactin (PRL), also known as PRL1, is an adenohypophysial hormone that critically regulates various physiological events in reproduction, metabolism, growth, osmoregulation, among others. PRL1 shares its evolutionary origin with PRL2, growth hormone (GH), somatolactin and placental lactogen, which together form the GH/PRL hormone family. Previously, several bioassays implied the existence of PRL1 in elasmobranch pituitaries. However, to date, all attempts to isolate PRL1 from chondrichthyans have been unsuccessful. Here, we cloned PRL1 from the pituitary of the holocephalan elephant fish, Callorhinchus milii, as the first report of chondrichthyan PRL1. The putative mature protein of elephant fish PRL1 (cmPRL1) consists of 198 amino acids, containing two conserved disulfide bonds. The orthologous relationship of cmPRL1 to known vertebrate PRL1s was confirmed by the analyses of molecular phylogeny and gene synteny. The cmPRL1 gene was similar to teleost PRL1 genes in gene synteny, but was distinct from amniote PRL1 genes, which most likely arose in an early amphibian by duplication of the ancestral PRL1 gene. The mRNA of cmPRL1 was predominantly expressed in the pituitary, but was considerably less abundant than has been previously reported for bony fish and tetrapod PRL1s; the copy number of cmPRL1 mRNA in the pituitary was less than 1% and 0.1% of that of GH and pro-opiomelanocortin mRNAs, respectively. The cells expressing cmPRL1 mRNA were sparsely distributed in the rostral pars distalis. Our findings provide a new insight into the studies on molecular and functional evolution of PRL1 in vertebrates.

In vivo and in vitro effects of high-K(+) stress on branchial expression of ROMKa in seawater-acclimated Mozambique tilapia.

Recently, a teleost ortholog of renal outer medullary K(+) channel (ROMK) expressed in gill ionocytes (ROMKa) has emerged as a primary K(+)-excreting pathway in fish. However, the mechanisms by which ROMKa expression is regulated in response to perturbations of plasma K(+) levels are unknown. In this study, we aimed to identify potential links between the endocrine system and K(+) regulation in a euryhaline fish. We assessed time-course changes in multiple endocrine parameters, including plasma cortisol and gene expression of branchial glucocorticoid and mineralocorticoid receptors (GR1, GR2, and MR) and pituitary hormones, in seawater (SW)-acclimated Mozambique tilapia (Oreochromis mossambicus) exposed to high-K(+) (H-K) SW. Exposure to H-K SW elicited little effects on plasma cortisol or mRNA levels of GRs and pituitary hormones. Since plasma K(+) and branchial ROMKa expression was increased within 6h after H-K treatment in vivo, the effect of high K(+) was subsequently tested in a gill filament incubation experiment using media with differing K(+) concentrations. ROMKa mRNA levels were induced following incubation of filaments in H-K medium for 6h. The present study is the first to demonstrate that the expression of ROMKa in teleost ionocytes can respond to high K(+) conditions independent from systemic signaling.

The osmoregulatory effects of rearing Mozambique tilapia in a tidally changing salinity.

The native distribution of Mozambique tilapia, Oreochromis mossambicus, is characterized by estuarine areas subject to salinity variations between fresh water (FW) and seawater (SW) with tidal frequency. Osmoregulation in the face of changing environmental salinity is largely mediated through the neuroendocrine system and involves the activation of ion uptake and extrusion mechanisms in osmoregulatory tissues. We compared plasma osmolality, plasma prolactin (PRL), pituitary PRL mRNA, and mRNA of branchial ion pumps, transporters, channels, and PRL receptors in tilapia reared in FW, SW, brackish water (BW) and in tidally-changing salinity, which varied between FW (TF) and SW (TS) every 6h. Plasma PRL was higher in FW tilapia than in SW, BW, TF, and TS tilapia. Unlike tilapia reared in FW or SW, fish in salinities that varied tidally showed no correlation between plasma osmolality and PRL. In FW fish, gene expression of PRL receptor 1 (PRLR1), Na(+)/Cl(-) cotransporter (NCC), aquaporin 3 (AQP3) and two isoforms of Na(+)/K(+)-ATPase (NKA α1a and NKA α1b) was higher than that of SW, BW or tidally-changing salinity fish. Gene expression of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC1a), and the cystic fibrosis transmembrane conductance regulator (CFTR) were higher in fish in SW, BW or a tidally-changing salinity than in FW fish. Immunocytochemistry revealed that ionocytes of fish in tidally-changing salinities resemble ionocytes of SW fish. This study indicated that tilapia reared in a tidally-changing salinity can compensate for large changes in external osmolality while maintaining osmoregulatory parameters within a narrow range closer to that observed in SW-acclimated fish.

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.

Nutritional status and growth hormone regulate insulin-like growth factor binding protein (igfbp) transcripts in Mozambique tilapia.

Growth in teleosts is controlled in large part by the activities of the growth hormone (Gh)/insulin-like growth factor (Igf) system. In this study, we initially identified igf-binding protein (bp)1b, -2b, -4, -5a and -6b transcripts in a tilapia EST library. In Mozambique tilapia (Oreochromis mossambicus), tissue expression profiling of igfbps revealed that igfbp1b and -2b had the highest levels of expression in liver while igfbp4, -5a and -6b were expressed at comparable levels in most other tissues. We compared changes in hepatic igfbp1b, -2b and -5a expression during catabolic conditions (28days of fasting) along with key components of the Gh/Igf system, including plasma Gh and Igf1 and hepatic gh receptor (ghr2), igf1 and igf2 expression. In parallel with elevated plasma Gh and decreased Igf1 levels, we found that hepatic igfbp1b increased substantially in fasted animals. We then tested whether systemic Gh could direct the expression of igfbps in liver. A single intraperitoneal injection of ovine Gh into hypophysectomized tilapia specifically stimulated liver igfbp2b expression along with plasma Igf1 and hepatic ghr2 levels. Our collective data suggest that hepatic endocrine signaling during fasting may involve post-translational regulation of plasma Igf1 via a shift towards the expression of igfbp1b. Thus, Igfbp1b may operate as a molecular switch to restrict Igf1 signaling in tilapia; furthermore, we provide new details regarding isoform-specific regulation of igfbp expression by Gh.

Effects of salinity and prolactin on gene transcript levels of ion transporters, ion pumps and prolactin receptors in Mozambique tilapia intestine.

Euryhaline teleosts are faced with significant challenges during changes in salinity. Osmoregulatory responses to salinity changes are mediated through the neuroendocrine system which directs osmoregulatory tissues to modulate ion transport. Prolactin (PRL) plays a major role in freshwater (FW) osmoregulation by promoting ion uptake in osmoregulatory tissues, including intestine. We measured mRNA expression of ion pumps, Na(+)/K(+)-ATPase α3-subunit (NKAα3) and vacuolar type H(+)-ATPase A-subunit (V-ATPase A-subunit); ion transporters/channels, Na(+)/K(+)/2Cl(-) co-transporter (NKCC2) and cystic fibrosis transmembrane conductance regulator (CFTR); and the two PRL receptors, PRLR1 and PRLR2 in eleven intestinal segments of Mozambique tilapia (Oreochromis mossambicus) acclimated to FW or seawater (SW). Gene expression levels of NKAα3, V-ATPase A-subunit, and NKCC2 were generally lower in middle segments of the intestine, whereas CFTR mRNA was most highly expressed in anterior intestine of FW-fish. In both FW- and SW-acclimated fish, PRLR1 was most highly expressed in the terminal segment of the intestine, whereas PRLR2 was generally most highly expressed in anterior intestinal segments. While NKCC2, NKAα3 and PRLR2 mRNA expression was higher in the intestinal segments of SW-acclimated fish, CFTR mRNA expression was higher in FW-fish; PRLR1 and V-ATPase A-subunit mRNA expression was similar between FW- and SW-acclimated fish. Next, we characterized the effects of hypophysectomy (Hx) and PRL replacement on the expression of intestinal transcripts. Hypophysectomy reduced both NKCC2 and CFTR expression in particular intestinal segments; however, only NKCC2 expression was restored by PRL replacement. Together, these findings describe how both acclimation salinity and PRL impact transcript levels of effectors of ion transport in tilapia intestine.