Recruitment and degeneration of mitochondrion-rich cells in the gills of Mozambique tilapia Oreochromis mossambicus during adaptation to a hyperosmotic environment

Cellular recruitment and degeneration of branchial mitochondrion-rich (MR) cells were examined in Mozambique tilapia transferred from hypoosmotic to hyperosmotic water. To examine apoptosis in the gills associated with salinity change, tilapia were directly transferred from freshwater to 70% seawater. The TUNEL assay showed that apoptotic cells in the gills were significantly increased at 1 day after transfer, which was supported by an electron-microscopic observation that gill MR cells underwent morphological changes characteristic of apoptosis such as an irregularly shaped electron-dense nucleus and distension of the tubular system. To further examine MR-cell recruitment, freshwater-acclimated tilapia were transferred either to freshwater or to 70% seawater after BrdU injection. Immunohistochemical detection of BrdU-labeled nuclei and Na(+)/K(+)-ATPase-rich MR cells allowed us to classify BrdU-labeled MR cells into two subtypes: a single MR cell and an MR-cell complex. Although newly generated single MR cells were observed similarly in both freshwater and 70% seawater-transferred fish, the density of MR-cell complexes was much higher in 70% seawater than in freshwater. Our findings indicated that transfer from hypoosmotic to hyperosmotic water enhanced apoptosis of freshwater-type MR cells, resulting in reduction in hyperosmoregulatory ability for freshwater adaptation, and stimulated the recruitment of MR-cell complexes to develop hypoosmoregulatory ability for seawater adaptation.     

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.     

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(-).     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

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.     

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.     

Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout (Salmo trutta) during acclimation to seawater

Changes in protein and mRNA expression of Na(+),K(+)-ATPase in gills and pyloric caeca of brown trout were investigated on a detailed time course after transfer from freshwater to 25 ppt seawater (SW). A transient deflection in plasma osmolality and muscle water content lasting from 4 h until day 3 was followed by restoration of hydromineral balance from day 5 onward. Gills and pyloric caeca responded to SW transfer by increasing Na(+),K(+)-ATPase activity from days 5 and 3, respectively, onward. In both tissues, this response was preceded by an increase in alpha-subunit Na(+), K(+)-ATPase mRNA as early as 12 h posttransfer. The similarity of the response in these two organs suggests that they both play significant physiological roles in restoring hydromineral balance after abrupt increase in salinity. Further, SW transfer induced a slight, though significant, increase in primary gill filament Na(+), K(+)-ATPase immunoreactive (NKIR) cell abundance. This was paralleled by a marked (50%) decrease in secondary lamellar NKIR cell abundance after less than 1 d in SW. Thus, SW acclimation in brown trout is characterised by a lasting decrease in overall NKIR cell abundance in the gill. We propose that SW transfer stimulates Na(+),K(+)-ATPase enzymatic activity within individual chloride cells long before (<1 d) it becomes apparent in measurements of whole-gill homogenate enzymatic activity. This is supported by the early stabilisation (12 h) of hydromineral balance.     

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.     

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.     

Phenotypic changes in mitochondrion-rich cells and responses of Na+/K+-ATPase in gills of tilapia exposed to deionized water

The aim of this study was to illustrate the phenotypic modification of mitochondrion-rich (MR) cells and Na(+)/K(+)-ATPase (NKA) responses, including relative protein abundance, specific activity, and immunolocalization in gills of euryhaline tilapia exposed to deionized water (DW) for one week. The plasma osmolality was not significantly different between tilapia of the local fresh water (LFW) group and DW group. Remodeling of MR cells occurred in DW-exposed fish. After transfer to DW for one week, the relative percentage of subtype-I (wavy-convex) MR cells with apical size ranging from 3 to 9 microm increased and eventually became the dominant MR cell subtype. In DW tilapia gills, relative percentages of lamellar NKA immunoreactive (NKIR) cells among total NKIR cells increased to 29% and led to significant increases in the number of NKIR cells. In addition, the relative protein abundance and specific activity of NKA were significantly higher in gills of the DW-exposed fish. Our study concluded that tilapia require the development of subtype-I MR cells, the presence of lamellar NKIR cells, and enhancement of NKA protein abundance and activity in gills to deal with the challenge of an ion-deficient environment.     

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.     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

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.     

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.     

Incorporation and metabolism of cortisol in oocytes of tilapia (Oreochromis mossambicus)

The entry and metabolism of 3H-cortisol in oocytes were investigated using isolated follicles of the tilapia (Oreochromis mossambicus) in order to examine the mechanisms of incorporation of maternal hormones into oocytes. The composition of 3H-labeled steroids in the oocyte was analyzed by high-performance liquid chromatography. A significant amount of cortisol was converted to cortisone and an unidentified molecule by the follicular layer. The contents of 3H-cortisol and 3H-cortisone in the oocyte reached an equilibrium level within 12 hr, whereas the content of the unidentified metabolite continued to increase for 36 hr. The total content of the incorporated cortisol and its metabolites was proportional to cortisol in the medium over the concentration range of 5 ng/ml to 5 microg/ml. The amounts of cortisone and the unidentified molecule increased proportionally when the concentration of cortisol in the medium was lower than 500 ng/ml, whereas they reached a plateau when the concentration of cortisol exceeded 500 ng/ml. Cortisol entry was reversible, because 90% of cortisol and cortisone in the oocyte was lost within 18 hr when the medium was changed to that without 3H-cortisol. On the other hand, 50% of the unidentified molecule was preserved at the end of the incubation. In conclusion, the entry of cortisol into the oocyte was considered to be nonspecific and due probably to simple diffusion. However, a considerable amount of cortisol (50-70%) was specifically converted to cortisone and another unidentified molecule during passage through the follicular layer.     

Metallothionein induction in early larval stages of tilapia (Oreochromis mossambicus)

Amounts of whole-body metallothionein (MT) in tilapia (Oreochromis mossambicus) larvae increased to a peak (1,500 ng mg(-1) protein) 1 d after hatching (H1), decreased rapidly thereafter, and was maintained at a constant level (700 ng mg(-1)) 3 d after hatching (H3). Waterborne Cd(2+) could stimulate MT expression in newly hatched (H0) larvae in dose-dependent and time-dependent patterns. H0 larvae, which were treated with 35 microg L(-1) Cd(2+) for 24 h, showed a 1.7-fold increase in the MT amount (174.0+/-64.7) and a 6. 5-fold increase in accumulated Cd(2+) but no significant change in Ca(2+) content, compared with the H0 control (MT, 102.6+/-48.1). H3 larvae with the same treatment revealed about a 10-fold increase in accumulated Cd(2+), a 10% decrease in Ca(2+) content, but no change in MT (261.2+/-120.0), compared with the H3 control (MT, 330+/-74.0). H0 larvae could synthesize more MT to bind Cd(2+) for detoxification in 35 microg L(-1) Cd(2+), a dose that would not affect normal physiology or survival of H0 larvae. On the other hand, 35 microg L(-1) Cd(2+) caused H3 larvae to experience hypocalcemia, an abnormal physiological condition, in which H3 larvae could not synthesize sufficient MT, thus causing greater than 25% mortality. These results indicate for the first time that the inducibility of MT by waterborne Cd(2+) is development dependent, being correlated with inconsistent sensitivities to Cd(2+) during larval development.     

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.     

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.