Prevalence of Francisella noatunensis subsp. orientalis in cultured tilapia on the island of Oahu, Hawaii.

Francisellosis is an emergent disease in cultured and wild aquatic animals. The causative agent, Francisella noatunensis subsp. orientalis (Fno), is a gram-negative bacterium recognized as one of the most virulent pathogens of warmwater fish. The main objective of this project was to investigate the prevalence of Fno in cultured tilapia (specifically, Mozambique Tilapia Oreochromis mossambicus, Koilapia [also known as Wami Tilapia] O. hornorum, Blue Tilapia O. aureus, and Nile Tilapia O. niloticus hybrids) on the island of Oahu, Hawaii, using conventional and real-time PCR assays followed by statistical modeling to compare the different diagnostic methods and identify potential risk factors. During 2010 and 2012, 827 fish were collected from different geographical locations throughout the island of Oahu. Upon collection of fish, the water temperature in the rearing system and the length of individual fish were measured. Extraction of DNA from different tissues collected aseptically during necropsy served as a template for molecular diagnosis. High correlation between both molecular methods was observed. Moreover, the bacterium was isolated from infected tilapia on selective media and confirmed to be Fno utilizing a species-specific Taqman-based real-time PCR assay. Although a direct comparison of the prevalence of Fno between the different geographical areas was not possible, the results indicate a high prevalence of Fno DNA in cultured tilapia throughout the farm sites located on Oahu. Of the different tilapia species and hybrids currently cultured in Hawaii, Mozambique Tilapia were more susceptible to infection than Koilapia. Water temperature in the rearing systems and fish size also had a strong effect on the predicted level of infection, with fish held at lower temperatures and smaller fish being more susceptible to piscine francisellosis.

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.

Gene expression of growth hormone family and glucocorticoid receptors, osmosensors, and ion transporters in the gill during seawater acclimation of Mozambique tilapia, Oreochromis mossambicus.

This study characterized endocrine and ionoregulatory responses accompanying seawater (SW) acclimation in Mozambique tilapia (Oreochromis mossambicus). Changes in plasma hormones and gene expression of hormone receptors, putative osmosensors, and ion transporters in the gill were measured. Transfer of freshwater (FW)-acclimated tilapia to SW resulted in a marked elevation in plasma osmolality and a significant rise in plasma growth hormone (GH) levels at 12 hr and 14 days after transfer. Significant reductions in plasma prolactin (PRL(177) and PRL(188)) levels also occurred in SW-transferred fish; no effect of transfer upon plasma cortisol or insulin-like growth factor I was observed. Gene expression of GH receptor increased strongly 6 hr after transfer, whereas PRL receptor was lower than controls at 12 hr. By contrast, mRNA levels of somatolactin and glucocorticoid receptors were unaffected by SW transfer. Osmotic stress transcription factor 1 mRNA levels rose significantly between 3 and 12 hr, whereas the calcium-sensing receptor was unaffected. Aquaporin-3 gene expression was strongly down-regulated during SW acclimation from 12 hr until the conclusion of the experiment. Na(+)/K(+)/2Cl(-) cotransporter gene expression increased significantly 3 hr after transfer, whereas expression of Na(+)/Cl(-) cotransporter, specific to FW-type chloride cells, declined by 6 hr into SW acclimation. The response of Na(+)/H(+) exchanger was less pronounced, but showed a similar pattern to that of the Na(+)/Cl(-) cotransporter. These results suggest that acquisition of hyposmoregulatory mechanisms in Mozambique tilapia entails the coordinated interaction of systemic hormones with local factors in the gill, including hormone receptors, ion transporters, and osmosensors.

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia.

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.

Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets.

Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERbeta, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.

Effects of short- and long-term fasting on plasma and stomach ghrelin, and the growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus.

Ghrelin is a highly conserved peptide hormone secreted by the stomach, which is involved in the regulation of food intake and energy expenditure. Ghrelin stimulates growth hormone (GH) release, and increases appetite in a variety of mammalian and non-mammalian vertebrates, including several fish species. Studies were conducted to investigate the effect of feeding and fasting on plasma and stomach ghrelin, and the growth hormone/insulin-like growth factor I (IGF-I) axis in the Mozambique tilapia, a euryhaline teleost. No postprandial changes in plasma and stomach ghrelin levels or stomach ghrelin mRNA levels were observed. Plasma levels of GH, IGF-I and glucose all increased postprandially which agrees with the anabolic roles of these factors. Fasting for 4 and 8d did not affect ghrelin levels in plasma or stomach. Plasma GH was elevated significantly after 4 and 8d of fasting, while plasma IGF-I levels were reduced. Plasma ghrelin levels were elevated significantly after 2 and 4 wk of fasting, but no change was detected in stomach ghrelin mRNA levels. Four weeks of fasting did not affect plasma GH levels, although plasma IGF-I and glucose were reduced significantly, indicating that GH resistance exists during a prolonged nutrient deficit (catabolic state). These results indicate that ghrelin may not be acting as a meal-initiated signal in tilapia, although it may be acting as a long-term indicator of negative energy balance.

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.

Absence of effects of short-term fasting on plasma ghrelin and brain expression of ghrelin receptors in the tilapia, Oreochromis mossambicus.

Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin's orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.

In vitro effects of homologous natriuretic peptides on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus.

C-type natriuretic peptide (CNP) cDNA was cloned from the tilapia brain and its inferred mature sequence was chemically synthesized together with previously cloned tilapia A-type and B-type natriuretic peptides (ANP and BNP). The cloned CNP belongs to the CNP-1 type of teleosts. Reverse-transcription polymerase chain reaction showed that the ANP and BNP genes were hardly expressed in the tilapia brain and pituitary, whereas the CNP gene was expressed strongly in the brain and slightly in the pituitary. Effects of homologous natriuretic peptides (100 nM each) on growth hormone (GH) and prolactin (PRL) release were examined using dispersed tilapia pituitary cells. Tilapia ANP and BNP stimulated GH and PRL release during 4-8, and 8-24 h of incubation. BNP appeared to be more potent than ANP, also stimulating GH and PRL release during 0-4 h of incubation. CNP stimulated GH release only during 4-8 h of incubation; CNP was without effect on PRL release. All three NPs stimulated GH and PRL mRNA expression in dispersed pituitary cells following 24 h of incubation. ANP and BNP significantly elevated intracellular cGMP accumulation in dispersed pituitary cells after 15 min of exposure, whereas no effect of CNP was observed. These results indicate a long-lasting stimulation of GH and PRL release by ANP and BNP that is mediated, at least in part, by the guanylyl cyclase-linked NP receptor.

Ionotropic glutamate receptor activation increases intracellular calcium in prolactin-releasing cells of the adenohypophysis.

Endocrine cells of the anterior pituitary are controlled by the central nervous system through hormonal interactions and are not believed to receive direct synaptic connections from the brain. Studies suggest that some pituitary cells may be modulated by the neurotransmitter glutamate. We investigated prolactin (PRL)-releasing cells of the anterior pituitary of a euryhaline fish, the tilapia (Oreochromis mossambicus), for the presence of possible glutamate receptors (GluRs). Fura-2 imaging addressed the ability of glutamate to increase intracellular calcium. We observed a dose-dependent increase in intracellular calcium with transient perfusion (1-2 min) of glutamate (10 nM to 1 mM) in two-thirds of imaged cells. This increase was attenuated by the ionotropic GluR antagonist kynurenic acid (0.5-1.0 mM). The increase was also blocked or attenuated by antagonists of L-type voltage-gated calcium channels. The GluR agonist alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA; 100 microM) produced intracellular calcium increases that were reversibly blocked by the selective AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, the selective agonist N-methyl-D-aspartate (NMDA; 100 microM to 1 mM in magnesium-free solution with 10 microM glycine) had no effect on intracellular calcium. Radioimmunoassays demonstrated that glutamate stimulated PRL release. CNQX but not the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid blocked this release. Antibodies for mammalian AMPA- and NMDA-type GluR produced a similar punctate immunoreactivity in the periphery of PRL cells. However, the NMDA antibody recognized a protein of a different molecular mass in PRL cells compared with brain cells. These results clearly indicate the presence of GluRs on tilapia PRL cells that can stimulate PRL release.

Long-term treatment of ghrelin stimulates feeding, fat deposition, and alters the GH/IGF-I axis in the tilapia, Oreochromis mossambicus.

Recently, a new peptide, ghrelin, which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue receptor. We have identified two ghrelins (ghrelin-C8 and -C10) in the stomach of tilapia, a euryhaline fish. The current study was aimed at investigating the in vivo effect of the two tilapia ghrelins on feeding, fat deposition, and on the GH/IGF-I axis. Tilapia were implanted with micro-osmotic pumps containing either ghrelin-C8, ghrelin-C10 or saline (control). Ghrelin was delivered at a continuous rate of 10 ng/h for 21 days. Food consumption increased significantly in those animals that received ghrelin-C10 but not ghrelin-C8. Treatment with ghrelin-C10 increased body weight significantly without altering body length. Hence, the condition factor was significantly higher in the ghrelin-C10 group compared with the control. Liver weight and total fat content in the liver were also elevated significantly in the fish treated with ghrelin-C10. There was no effect of either ghrelin on plasma GH levels, whereas plasma IGF-I levels were reduced significantly in the ghrelin-C10 group. These findings demonstrate that ghrelin plays a role in feeding and fat metabolism in the tilapia, and suggest that the two forms of ghrelin may be acting through different receptors.