Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide 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 (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

Adult neurogenesis in the brain of the Mozambique tilapia, Oreochromis mossambicus

Although the generation of new neurons in the adult nervous system ('adult neurogenesis') has been studied intensively in recent years, little is known about this phenomenon in non-mammalian vertebrates. Here, we examined the generation, migration, and differentiation of new neurons and glial cells in the Mozambique tilapia (Oreochromis mossambicus), a representative of one of the largest vertebrate taxonomic orders, the perciform fish. The vast majority of new cells in the brain are born in specific proliferation zones of the olfactory bulb; the dorsal and ventral telencephalon; the periventricular nucleus of the posterior tuberculum, optic tectum, and nucleus recessi lateralis of the diencephalon; and the valvula cerebelli, corpus cerebelli, and lobus caudalis of the cerebellum. As shown in the olfactory bulb and the lateral part of the valvula cerebelli, some of the young cells migrate from their site of origin to specific target areas. Labeling of mitotic cells with the thymidine analog 5-bromo-2'-deoxyuridine, combined with immunostaining against the neuron-specific marker protein Hu or against the astroglial marker glial fibrillary acidic protein demonstrated differentiation of the adult-born cells into both neurons and glia. Taken together, the present investigation supports the hypothesis that adult neurogenesis is an evolutionarily conserved vertebrate trait.     

Influence of steroid hormones on plasma proteins in freshwater tilapia Oreochromis mossambicus

The effect of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen, diethylstilbestrol on plasma proteins of tilapia (Oreochromis mossambicus) was investigated. SDS-PAGE clearly revealed the appearance of several new bands of protein, which were not present in the control plasma and were comparable to the known bands of the molecular markers. Of the different bands appeared in the steroids treated plasma, the most important ones were the presumed vitellogenin and corticotrophin binding globulin with a molecular weight of 180 and 17 kDa, respectively. Increase in protein bands in the steroid treated plasma of O. mossambicus confirmed the anabolic role of steroids in teleost.     

Effects of acclimation salinity on the expression of selenoproteins in the tilapia,Oreochromis mossambicus

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.     

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

Effects of fasting on the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis were examined in the tilapia (Oreochromis mossambicus) acclimated to fresh water. Fasting for 2 weeks resulted in significant reductions in body weight, specific growth rate and hepatosomatic index in both males and females. Significant reductions in specific growth rates were observed after 1 and 2 weeks in both sexes, although the decrease in body weight was not significant in the female. A significant reduction was also seen in the condition factor of females after 2 weeks. No change was seen in the gonadosomatic index in either sex. Two weeks of fasting also produced a significant reduction in plasma IGF-I but not in plasma GH, prolactin (PRL(188)) or cortisol. Significant reductions in the hepatic IGF-I mRNA were seen in both sexes. On the other hand, a significant increase was observed in cortisol receptor mRNA in the female liver. Plasma IGF-I levels were correlated significantly with specific growth rate, condition factor and hepatosomatic index, indicating that plasma IGF-I is a good indicator of growth in the tilapia. No change was seen in plasma glucose or osmolality after 2 weeks of fasting. During fasting, tilapia appears to convert metabolic energy from growth to basal metabolism including maintenance of ion and water balance.     

Metabolic effects of bovine growth hormone in the tilapia Oreochromis mossambicus.

1. Bovine growth hormone (bGH) was injected into tilapia intramuscularly at a dose of 50 micrograms/100 g/day for a total of five injections. Control fish received saline instead. 2. The serum concentrations of amino acid and glucose were significantly higher and hepatic glycogen concentration and glycogen synthetase activity significantly lower in the bGH-treated fish than those in the control fish. 3. The serum concentrations of protein, lipid and cholesterol, and the hepatic concentrations of protein and lipid, remained unaltered after bGH treatment. 4. The results suggest that bGH exerts anti-insulin effects in tilapia.     

Evidence of steroidogenesis in postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were examined with electron microscopy and enzyme histochemistry for evidence of steroid-hormone production. Light microscopy was also used to examine changes in the ovary with time after spawning. Electron microscopy detected the presence of smooth endoplasmic reticulum, lipid droplets, and mitochondria with tubular cristae in certain cells of the theca interna. These structures are suggestive of cells that synthesize steroid hormones. Granulosa cells also contained some smooth endoplasmic reticulum, along with an augmentation of Golgi complexes, vesicles, microvilli, and microfilaments within 5–7 days after spawning. Enzyme histochemistry demonstrated an intense reaction of 5, 3-hydroxysteroid dehydrogenase (3-HSD) in variably placed thecal cells up to 7 days after spawning. At this time, the thecal cells of vitellogenic oocyte follicles also began to show strong 3-HSD activity. During the first 7 days after spawning, there was an increase in young primary oocytes and recruitment of some of these to vitellogenic oocytes. By 10 days after spawning, certain thecal cells in the follicles of these vitellogenic oocytes showed an intense 3-HSD reaction, while the postovulatory follicular tissue demonstrated a weak reaction. This arrangement continued for the lifespan of the postovulatory follicular tissue. Postovulatory follicles had a lifespan of up to 25 days after spawning in females that continued to hold the developing fry inside their mouths, i.e., mouthbrooders. At 25 days after spawning, the postovulatory follicular tissue showed signs of degeneration with the presence of vacuoles and lysosomes. In females that ate the zygotes, therefore exhibiting no parental behavior, the postovulatory follicular tissue showed signs of degeneration at l0days after spawning. In these females, the next clutch of eggs also developed at a higher rate than in mouthbrooders.     

Effects of temperature on the deformity and sex differentiation of tilapia, Oreochromis mossambicus

The effects of temperature on the deformity and sex differentiation of tilapia, Oreochromis mossambicus, were investigated. Zero- (the hatching day), 5-, and 10-day-old tilapia were respectively divided into 4 groups that were reared at 20, 24, 28, and 32 degrees C for 5 days. Percentages of deformity were significantly increased when tilapia were kept in the elevated temperatures (28 and 32 degrees C) before 5 days old during this experiment, whereas the lower temperature (20 degrees C) had no effect on the development of morphology. On the other hand, exposure to the lower temperature before 10 days old induced a high proportion of females whereas a high proportion of males was induced by the elevated temperature after 10 days old during this experiment. These results indicate that morphological development is influenced by temperature, particularly by the elevated temperature during a restricted developmental period. Both lower and elevated temperatures induce the gonadal feminization and masculinization, respectively, during its restricted developmental period. J. Exp. Zool. 286:534-537, 2000.     

The effects of short-term acute cadmium exposure on blue tilapia,Oreochromis aureus

Synopsis Disease-free blue tilapia,Oreochromis aureus, exposed to cadmium (0.5 ppm and 10 ppm) showed very significant and dramatic decrease in food consumptions. Food consumptions returned to near normal 21 days after fish were returned to cadmium-free water. Along with anorexia there were decreases in body weights during the period when fish were exposed to cadmium. Fish in cadmium-free water that were pair-fed to cadmium-exposed fish (0.5 ppm) did not gain weight but there were no decreases in body weights. Cadmium was still in tissues 34 days after the fish were placed in cadmium-free water and the accumulation was highest in the kidney; this was followed by liver, brain, gill filaments and muscles. The accumulations of the heavy metal (in kidneys and gills) were significantly higher in fish exposed to high than low cadmium levels. There were no differences in complement levels (haemolytic acitivity) in cadmium-exposed and cadmium-free fish. However, cadmium-exposed fish did not produce detectable haemagglutinating antibodies against sheep red blood cells while cadmium-free fish responded well to the antigen. The anorexia in cadmium-exposed fish contributed to the depression in antibody production.     

Pattern formation of the 'Tilapia mark' in the tilapia fish, Oreochromis mossambicus

The dorsal fin of the larval and juvenile Oreochromis mossambicus exhibits a unique black spot known as the ' Tilapia mark'. We traced its development and found that it occupied a specific position in the dorsal fin bounded by rays 15 and 20. Ablation experiments carried out on the larval dorsal fin showed that this spot region constituted a developmental positional field. This positional field in the fin could regenerate and re-establish the spot pattern despite repeated perturbation. The re-establishment of spot was not simply due to fin injury since ablation of the non-spotted region of the dorsal and the tail fin did not result in aggregation of melanophores. We propose that that' Tilapia mark' is a result of positional information in operation.     

Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus

The effect of exposure to sublethal concentrations (0.017 mg L(-1), 1/10 of LC50) of the novel organophosphate (OP) insecticide, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II) on biochemical parameters in Oreochromis mossambicus was studied during exposure for 3, 7, 15, 30 and its recovery response after seven days. Acetylcholinesterase (AChE) activity of brain, gill and muscle was inhibited by 67%, 77% and 73% respectively on day-30. The plasma and kidney alanine aminotransferase (AlaAT), and aspartate aminotransferase (AspAT) activity increased, while decreases were observed in gill and liver. Increases in acid phosphatase (AcP), and alkaline phosphatase (AP) activities were observed in plasma, gill, and kidney, and reductions of 20% and 61% in liver AcP and AP, respectively. Depletion of glycogen was observed in all tissues, an indication of typical stress related response of the fish with pesticide. Lactate dehydrogenase (LDH) activity decreased in liver and muscle, indicating tissue damage but a significant increase in LDH activity in gill and brain was observed. Depletion of glutathione (GSH) was observed in all tissues, thereby enhancing lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in the above biochemical parameters, in all tissues of fish after a recovery period of seven days. These results revealed that the OP insecticide RPR-II is highly toxic and affects the intermediary metabolism of O. mossambicus.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.     

Effect of blood withdrawal and angiotensin II on prolactin release in the tilapia,Oreochromis mossambicus

Repeated blood withdrawal (5% of estimated blood volume at 0, 1, 4, 8, 24, 48 and 76 h) from tilapia acclimated to fresh water (FW) resulted in a marked increase in plasma levels of prolactin (PRL) during the first 8 h, reaching a peak above 300 ng/ml after 4 h. The increase in plasma PRL levels was significant except for the level after 72 h. A slight but significant decrease in plasma osmolality was observed at all time points after the blood withdrawal. Repeated blood withdrawal from fish acclimated to seawater (SW) resulted in a marked increase in plasma osmolality after 4 and 8 h. A significant increase was observed in plasma growth hormone (GH) in the fish in SW until the end of the experiment, but there was no change in plasma PRL. Plasma levels of cortisol were significantly higher in the fish in SW than in those in FW during the first 24 h. Blood withdrawal resulted in a significant reduction in hematocrit values in both FW- and SW-adapted fish, suggesting hemodilution. In a separate experiment, a single blood withdrawal (20% of total blood) stimulated drinking after 5 h, regardless of whether the fish were held in FW or SW. Plasma PRL level was also elevated following a single blood withdrawal in the fish acclimated to FW, but not in the fish in SW. Intraperitoneal injection of ANG II (1.0 microg/g) into the fish in FW significantly increased plasma PRL levels after 1 h. Activation of the renin-angiotensin system after blood withdrawal and the dipsogenic action of angiotensin II (ANG II) are well established in fish. The reduction in plasma osmolality after repeated blood withdrawal in FW and the increased osmolality in SW suggest that blood volume is restored, at least in part, by drinking environmental water. These results suggest that the marked increase in PRL concentration after blood withdrawal from the fish in FW is due, at least in part, to a facilitative effect between ANG II and reduced plasma osmolality.     

Insulin-like growth factor I-like immunoreactivity in serum and tissues of the tilapia, Oreochromis mossambicus

Tilapia serum was acidified with 0.5 M HCl and then chromatographed on an octadecasily-silica column. After washing with 4% acetic acid, the column was eluted with methanol. The eluate was evaporated to dryness. The sample cross-reacted in a human insulin-like growth factor I (IGF-I) radioimmunoassay, suggesting immunochemical similarity to human IGF-I. IGF-I-like immunoreactivity was present at high levels in tilapia liver. Other tissues containing IGF-I-like immunoreactivity included the gonad, kidney, heart, spleen, brain and muscle. The serum IGF-I-like immunoreactivity was attributed to substances with a molecular weight of 9,000 and 45,000 respectively, and it was elevated after treatment with bovine growth hormone and carp pituitary extract.     

Photic and circadian regulation of melatonin production in the Mozambique tilapia Oreochromis mossambicus

Diverse circadian systems related to phylogeny and ecological adaptive strategies are proposed in teleosts. Recently, retinal photoreception was reported to be important for the circadian pacemaking activities of the Nile tilapia Oreochromis niloticus. We aimed to confirm the photic and circadian responsiveness of its close relative-the Mozambique tilapia O. mossambicus. Melatonin production in cannulated or ophthalmectomized fish and its secretion from cultured pineal glands were examined under several light regimes. Melatonin production in the cannulated tilapias was measured at 3-h intervals; it fluctuated daily, with a nocturnal increase and a diurnal decrease. Exposing the cannulated fish to several light intensities (1500-0.1 lx) and to natural light (0.1 and 0.3 lx) suppressed melatonin levels within 30 min. Static pineal gland culture under light-dark and reverse light-dark cycles revealed that melatonin synthesis increased during the dark periods. Rhythmic melatonin synthesis disappeared on pineal gland culture under constant dark and light conditions. After ophthalmectomy, plasma melatonin levels did not vary with light-dark cycles. These results suggest that (1) Mozambique tilapias possess strong photic responsiveness, (2) their pineal glands are sensitive to light but lack circadian pacemaker activity, and (3) they require lateral eyes for rhythmic melatonin secretion from the pineal gland.     

Changes of glycogen metabolism in the gills and hepatic tissue of tilapia (Oreochromis mossambicus) during short-term Cd exposure

The aim of the study was to test the hypothesis that the mechanism of glycogen metabolism has taken place in gills rather than in liver during Cd exposure. Male tilapia were exposed to 44.45 μM ambient Cd for 12 h, and we found blood glucose significantly increased, however, lactate levels showed no significant changes. The glycogen phosphorylase (GP) activity increased immediately after 0.75 to 3 h of Cd exposure in the gills, and after 1 to 6 h in the liver, respectively. In addition, the glycogen level depleted faster in the gills than in the liver. Plasma cortisol level increased from 0.25 to 1 h and recovered after 3 h, while the glucagon did not significantly change during Cd exposure. Glucocorticoid receptor (GR) mRNA expression decreased after 0.75 h in the gills, while it significantly increased after 6 h in the liver. Ca²⁺, Na⁺, Cl⁻, and K⁺ significantly decreased upon Cd exposure within 6 h following Cd-induced toxic stress. We suggested that the cortisol is the spontaneous stimulation of glycogen metabolism in the gills, and it triggers a subsequent energy supply later in the liver. Taken together, the profile of glycogen metabolism between gills and liver during Cd-exposure stress provide good support to our hypothesis.     

Sodium and calcium balance in Mozambique tilapia, Oreochromis mossambicus, raised at different salinities

Mozambique tilapia, Oreochromis mossambicus, born and raised in five salinities, viz. (relatively soft) fresh water, 25, 50, 75% and full-strength sea-water, were analyzed for ionoregulatory performance (in particular sodium and calcium handling) and growth. This tilapia regulates its blood serum mineral composition rather effectively; however, in sea-water serum concentrations of sodium, chloride and calcium (in males only) were increased, as was the serum osmolarity. In sea-water, the total body sodium pool was significantly enlarged. With increasing salinity, sodium turnover increased. Serum calcium levels and the total body calcium pool were more strictly controlled than those of sodium. The lowest density of chloride cells in opercular epithelium and the lowest branchial Na+-K+-ATPase activity were observed in 50% sea-water; these values were higher in fish kept in waters of lower or higher salinities. Fish grew more rapidly in brackish water. Fish kept in brackish water appeared to depend on food-related calcium for growth as branchial calcium uptake provides no more than 20% of growth related Ca-accumulation.