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.

Regulation of gill claudin paralogs by salinity, cortisol and prolactin in Mozambique tilapia (Oreochromis mossambicus).

In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin.

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.

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.

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.

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.

Effects of salinity on metabolic rate and branchial expression of genes involved in ion transport and metabolism in Mozambique tilapia (Oreochromis mossambicus).

This study investigated the effects of two rearing salinities, and acute salinity transfer, on the energetic costs of osmoregulation and the expression of metabolic and osmoregulatory genes in the gill of Mozambique tilapia. Using automated, intermittent-flow respirometry, measured standard metabolic rates (SMRs) of tilapia reared in seawater (SW, 130 mg O2 kg⁻¹ h⁻¹) were greater than those reared in fresh water (FW, 103 mg O2 kg⁻¹ h⁻¹), when normalized to a common mass of 0.05 kg and at 25±1°C. Transfer from FW to 75% SW increased SMR within 18h, to levels similar to SW-reared fish, while transfer from SW to FW decreased SMR to levels similar to FW-reared fish. Branchial gene expression of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), an indicator of SW-type mitochondria-rich (MR) cells, was positively correlated with SMR, while Na⁺-Cl⁻ cotransporter (NCC), an indicator of FW-type MR cells, was negatively correlated. Principal Components Analysis also revealed that branchial expression of cytochrome c oxidase subunit IV (COX-IV), glycogen phosphorylase (GP), and a putative mitochondrial biogenesis regulator in fish, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), were correlated with a higher SMR, plasma osmolality, and environmental salinity, while expression of glycogen synthase (GS), PGC-1ß, and nuclear respiratory factor 1 (NRF-1) had negative correlations. These results suggest that the energetic costs of osmoregulation are higher in SW than in FW, which may be related to the salinity-dependent differences in osmoregulatory mechanisms found in the gills of Mozambique tilapia.

Estrogenic compounds decrease growth hormone receptor abundance and alter osmoregulation in Atlantic salmon.

Exposure of Atlantic salmon smolts to estrogenic compounds is shown to compromise several aspects of smolt development. We sought to determine the underlying endocrine mechanisms of estrogen impacts on the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Smolts in freshwater (FW) were either injected 3 times over 10 days with 2 µgg(-1) 17ß-estradiol (E2) or 150µgg(-1) 4-nonylphenol (NP). Seawater (SW)-acclimated fish received intraperitoneal implants of 30 µgg(-1) E2 over two weeks. Treatment with these estrogenic compounds increased hepatosomatic index and total plasma calcium. E2 and NP reduced maximum growth hormone binding by 30-60% in hepatic and branchial membranes in FW and SW, but did not alter the dissociation constant. E2 and NP treatment decreased plasma levels of IGF-I levels in both FW and SW. In FW E2 and NP decreased plasma GH whereas in SW plasma GH increased after E2 treatment. Compared to controls, plasma chloride concentrations of E2-treated fish were decreased 5.5mM in FW and increased 10.5mM in SW. There was no effect of NP or E2 on gill sodium-potassium adenosine triphosphatase (Na(+)/K(+)-ATPase) activity in FW smolts, whereas E2 treatment in SW reduced gill Na(+)/K(+)-ATPase activity and altered the number and size of ionocytes. Our data indicate that E2 downregulates the GH/IGF-I-axis and SW tolerance which may be part of its normal function for reproduction and movement into FW. We conclude that the mechanism of endocrine disruption of smolt development by NP is in part through alteration of the GH/IGF-I axis via reduced GH receptor abundance.

Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia.

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca(2+) through a stretch-activated Ca(2+) channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca(2+) ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.

Experimental Approaches for Characterizing the Endocrine-Disrupting Effects of Environmental Chemicals in Fish.

Increasing industrial and agricultural activities have led to a disturbing increase of pollutant discharges into the environment. Most of these pollutants can induce short-term, sustained or delayed impacts on developmental, physiological, and behavioral processes that are often regulated by the endocrine system in vertebrates, including fish, thus they are termed endocrine-disrupting chemicals (EDCs). Physiological impacts resulting from the exposure of these vertebrates to EDCs include abnormalities in growth and reproductive development, as many of the prevalent chemicals are capable of binding the receptors to sex steroid hormones. The approaches employed to investigate the action and impact of EDCs is largely dependent on the specific life history and habitat of each species, and the type of chemical that organisms are exposed to. Aquatic vertebrates, such as fish, are among the first organisms to be affected by waterborne EDCs, an attribute that has justified their wide-spread use as sentinel species. Many fish species are exposed to these chemicals in the wild, for either short or prolonged periods as larvae, adults, or both, thus, studies are typically designed to focus on either acute or chronic exposure at distinct developmental stages. The aim of this review is to provide an overview of the approaches and experimental methods commonly used to characterize the effects of some of the environmentally prevalent and emerging EDCs, including 17 α-ethinylestradiol, nonylphenol, BPA, phthalates, and arsenic; and the pervasive and potential carriers of EDCs, microplastics, on reproduction and growth. In vivo and in vitro studies are designed and employed to elucidate the direct effects of EDCs at the organismal and cellular levels, respectively. In silico approaches, on the other hand, comprise computational methods that have been more recently applied with the potential to replace extensive in vitro screening of EDCs. These approaches are discussed in light of model species, age and duration of EDC exposure.

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.