Response to acute changes in salinity of two different muscle type creatine kinase isoforms, from euryhaline teleost (Oreochromis mossambicus) gills

Two CKM isoforms (CKM1 and CKM2) from the gills of tilapia (Oreochromis mossambicus) were obtained after transfer from freshwater (FW) to seawater (SW, 25 ppt). Based on the 5' and 3' RACE, the identity of CKM1 and CKM2 was determined to be 59% in the 5'-untranslated region (5'-UTR) and 41.9% in the 3'-UTR. Using Northern blot hybridization with the CKM1 and CKM2 3'-UTR probes, CKM1 and CKM2 were found to be expressed in muscle, heart and gill. The levels of these two different CK isoforms (CKM1 and CKM2) were shown to be different in FW than after acute SW transfer, showing that CKM isoforms respond to changes in salinity.     

Cellular distributions of creatine kinase in branchia of euryhaline tilapia (Oreochromis mossambicus)

Although euryhalineteleosts can adapt to environmental fluctuation of salinity, theirenergy source for responding to changes in salinity and osmolarityremains unclear. This study examines the cellular localization ofcreatine kinase (CK) expression in branchia of tilapia(Oreochromis mossambicus). Western blot analysis ofmuscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted toseawater did not change. With the use of immunocytochemistry, three CKisoforms (MM, MB, and BB) were localized in mitochondria-rich (MR)cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To ourknowledge, this work presents the first evidence of CK expression in MRcells of tilapia gills, highlighting the potential role of CK inproviding energy for ion transport.

     

Regulation of drinking rate in euryhaline tilapia larvae (Oreochromis mossambicus) during salinity challenges

Euryhaline tilapia larvae are capable of adapting to environmental salinity changes even when transferred from freshwater (FW) to seawater (SW) or vice versa. In this study, the water balance of developing tilapia larvae (Oreochromis mossambicus) adapted to FW or SW was compared, and the short-term regulation of drinking rate of the larvae during salinity adaptation was also examined. Following development, wet weight and water content of both SW- and FW-adapted larvae increased gradually, while the dry weight of both group larvae showed a slow but significant decline. On the other hand, the drinking rate of SW-adapted larvae was four- to ninefold higher than that of FW-adapted larvae from day 2 to day 5 after hatching. During acute salinity challenges, tilapia larvae reacted profoundly in drinking rate, that is, increased or decreased drinking rate within several hours while facing hypertonic or hypotonic challenges, to maintain their constancy of body fluid. This rapid regulation in water balance upon salinity challenges may be critical for the development and survival of developing larvae.     

Estradiol impairs hyposmoregulatory capacity in the euryhaline tilapia, Oreochromis mossambicus

Freshwater (FW)-adapted tilapia (Oreochromis mossambicus) were treated with estradiol (E(2)) for 4 days to stimulate protein synthesis and sampled at 0, 4, and 24 h after exposure to 50% seawater (SW). E(2) increased circulating vitellogenin (VTG) levels in large amounts, indicative of unusually high rates of hepatic protein synthesis. E(2) treatment prevented the recovery of plasma osmolality in 50% SW that was evident in the sham group. Plasma sodium concentration was significantly elevated with E(2) in FW, but the levels did not change in 50% SW. Gill Na(+)-K(+)-ATPase activity was significantly lower in the E(2) group compared with sham-injected tilapia in 50% SW. No significant differences were noted in plasma cortisol, thyroxine, triiodothyronine, or glucose concentration with E(2) in 50% SW. E(2) significantly lowered several key liver enzyme activities and also decreased gill lactate dehydrogenase and malate dehydrogenase activities over a 24-h period. Together, our results suggest that E(2) impairs ion regulation in tilapia, partially mediated by a decreased metabolic capacity in liver and gill. The decreased tissue metabolic capacity is likely due to E(2)-induced energy repartitioning processes that are geared toward VTG synthesis at the expense of other energy-demanding pathways.     

Time-course changes in the expression of Na, K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia (Oreochromis mossambicus) during freshwater acclimation

Changes in expression of Na, K-ATPase (NKA) and morphometry of mitochondrion-rich (MR) cells in gills of tilapia were investigated on a 96-hr time course following transfer from seawater (SW) to fresh water (FW). A transient decline in plasma osmolality and Na+, Cl- concentrations occurred from 3 hrs onward. Gills responded to FW transfer by decreasing NKA activity as early as 3 hrs from transfer. This response was followed by a significant decrease in the NKA isoform alpha1-mRNA abundance, which was detected by real-time PCR at 6 hrs post transfer. Next, a decrease of alpha1-protein amounts were observed from 6 hrs until 24 hrs post transfer. Additionally, during the time course of FW transfer, modifications in number and size of subtypes of gill MR cells were observed although no significant difference was found in densities of all subtypes of MR cells. These modifications were found as early as 3 hrs, evident at 6 hrs (exhibition of 3 subtypes of MR cells), and mostly completed by 24 hrs post transfer. Such rapid responses (in 3 hrs) as concurrent changes in branchial NKA expression and modifications of MR cell subtypes are thought to improve the osmoregulatory capacity of tilapia in acclimation from hypertonic SW to hypotonic FW.     

Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells

We have developed a technique for immunocytochemistry of fish gill cells that we used to quantify tilapia (Oreochromis mossambicus) mitochondria-rich cells (MRC) and other gill cells (non-MRC) within different cell cycle phases by laser scanning cytometry. Gill cells fixed on coverslips were triple stained with propidium iodide to distinguish G1 vs. G2 phases, Ser10-phosphorylated histone H3 antibody to label mitotic cells, and Na(+)/K(+) ATPase antibody to label MRC. These parameters were measured at 0 (control), 4, 8, 16, 24, 48, 72, and 168 hr (1 week) following exposure of freshwater (FW) acclimated fish to 2/3 seawater (SW). MRC increased mitotic activity very rapidly peaking at 8 hr following SW exposure. This change in mitotic MRC is indicative of epithelial reorganization during SW acclimation. In contrast to MRC, the proportion of non-MRC (likely pavement cells (PVC)) in mitosis did not change significantly in response to SW exposure. Moreover, twice as many MRC were in mitosis compared with non-MRC, suggesting that MRC turn over faster than other cell types during SW acclimation. Following the mitosis peak, MRC accumulated in G2 phase over a period of 16-72 hr post-SW exposure. We also observed G2 arrest with similar kinetics following SW exposure in tilapia non-MRC (likely PVC). We interpret the G2 arrest that occurs after an initial wave of transient increase in MRC mitosis as a means for conserving energy for dealing with the osmotic stress imposed during the exposure of FW fish to SW.     

Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus)

The purpose of the present article is to examine the relationships between ion uptakes and morphologies of gill mitochondria-rich (MR) cells in freshwater tilapia. Tilapia were acclimated to three different artificial freshwaters (high Na [10 mM], high Cl [7.5 mM]; high Na, low Cl [0.02-0.07 mM], and low Na [0.5 mM], low Cl) for 1 wk, and then morphological measurements of gill MR cells were made and ion influxes were determined. The number and the apical size of wavy-convex MR cells positively associated with the level of Cl(-) influx. Conversely, Na(+) influx showed no positive correlation with the morphologies of MR cells. The dominant MR cell type in tilapia gills changed from deep-hole to wavy-convex within 6 h after acute transfer from a high-Cl(-) to a low-Cl(-) environment. Deep-hole MR cells became dominant 24-96 h after acute transfer from a low-Cl(-) to a high-Cl(-) environment. We conclude that wavy-convex MR cells associate with Cl(-) uptake but not Na(+) uptake, and the rapid formation of wavy-convex MR cells reflects the timely stimulation of Cl(-) uptake to recover the homeostasis of internal Cl(-) levels on acute challenge with low environmental Cl(-).     

Surfactant-induced lipid peroxidation in a tropical euryhaline teleost Oreochromis mossambicus (Tilapia) adapted to fresh water

Exposure to anionic (sodium dodecyl sulfate, SDS), cationic (cetyl trimethyl ammonium bromide CTAB) and non ionic (Triton X-100) surfactants at a sub lethal concentration of 1 ppm resulted in severe oxidative stress in the hepatic, renal and cardiac tissues of fresh water adapted Oreochromis mossambicus. Hepatic catalase showed significant increase (P<0.001) in all the surfactant exposed fish, but the renal enzyme was significantly increased only in CTAB dosed fish (P<0.001) and the cardiac enzyme showed significant increase in Triton (P<0.05) and CTAB dosed fish (P<0.001). SOD levels were significantly increased (P<0.001) in hepatic, renal and cardiac tissues of all the surfactant-treated fish. Glutathione reductase also was significantly increased (P<0.001) in the hepatic and renal tissues of surfactant dosed fish except cardiac tissues of CTAB exposed animals. Glutathione levels in the tissues studied were significantly higher in the surfactant treated animals (P<0.001) whereas malondialdehyde levels were significantly elevated only in the hepatic tissues of animals exposed to Triton (P<0.001). The surfactants based on their charge, antioxidant profile and in vivo metabolism may be arranged in the order of decreasing toxicity as CTAB > Triton > SDS. Thus it may be inferred from the present study that the antioxidant defenses and the in vivo metabolism of the surfactants are key factors in deciding the surfactant toxicity.     

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus): effect of seawater exposure and cortisol treatment

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.     

Diurnal changes in the territorial behaviour of the tilapia Oreochromis mossambicus (Peters)

The numbers of black, territorial, male tilapia in large tanks undergoes a clear diurnal cyclicity, with peak numbers seen in the afternoon. It is suggested that this cyclicity is functionally related to an observed spawning cyclicity.     

Salinity effects on cytometrical parameters of the kidney in the euryhaline teleost Oreochromis mossambicus Peters

Study of the effects of environmental salinity on the glomerular areas and the nuclear areas of the renal tubules in tilapia showed significant differences ( P < 0.001 or 0.05) between seawater- and freshwater-adapted fish. Upon seawater adaptation, there were decreases in the glomerular areas and the nuclear areas in the main segments of the nephron: glomerular, −24.1%; 1st proximal, −17.1%; 2nd proximal, −21.5%; distal, −9.1%; collecting, −26.5%. These cytometrical changes are discussed in relation to the different osmoregulatory functions of euryhaline teleosts adapted to sea water and to fresh water.     

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.     

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60 S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Effects of cortisol and salinity challenge on water balance in developing larvae of tilapia (Oreochromis mossambicus)

Effects of exogenous cortisol on drinking rate and water content in developing larvae of tilapia (Oreochromis mossambicus) were examined. Both freshwater- and seawater-adapted larvae showed increases in drinking rates with development. Drinking rates of seawater-adapted larvae were about four- to ninefold higher than those of freshwater-adapted larvae from day 2 to day 5 after hatching. Seawater-adapted larvae showed declines in drinking rate and water content at 4 and 14 h, respectively, after immersion in 10 mg L(-1) cortisol. In the case of freshwater-adapted larvae, the drinking rate decreased after 8 h of cortisol immersion, while the water content did not show a significant change even after 32 h of cortisol immersion. In a subsequent experiment of transfer from freshwater to 20 ppt (parts per thousand, salinity) seawater, immersion in 10 mg L(-1) cortisol for 8-24 h enhanced the drinking rate in larvae at 4 h after transfer, but no significant difference was found in water contents between cortisol-treated and control groups following transfer. These results suggest that cortisol is involved in the regulation of drinking activity in developing tilapia larvae.