Impact of environmental DDT concentrations on gill adaptation to increased salinity in the tilapia Sarotherodon melanotheron

Estuaries of tropical developing countries suffering from severe droughts induced by climate change are habitats to fish, which face drastic salinity variations and the contact with pollutants. The Western Africa tilapia Sarotherodon melanotheron is highly resistant to hypersalinity, but the effect of human-released xenobiotics on its adaptation is barely known. Controlled experiments were conducted to observe S. melanotheron gill adaptation to abrupt salinity variations in the presence of waterborne DDT, at concentrations detected in their natural habitat. The gills appeared as an important site of DDT conversion to DDD and/or depuration. A 12-days DDT exposure resulted in decreased gill epithelium thickness at all salinities (from fresh- to hypersaline-water), and the structure of gills from freshwater fish was particularly altered, relative to controls. No unbalance in tilapia blood osmolality was observed following DDT exposure, which however caused a decrease in branchial Na(+)-K(+)-ATPase (NKA) activity. Gill cellular NKA expression was reduced in salt-water, together with the expression of the CFTR chloride channel in hypersaline water. Although S. melanotheron seems very resistant (especially in seawater) to short-term waterborne DDT contamination, the resulting alterations of the gill tissue, cells and enzymes might affect longer term respiration, toxicant depuration and/or osmoregulation in highly fluctuating salinities.     

Short-term low-salinity tolerance by the longhorn sculpin, Myoxocephalus octodecimspinosus

The bottom-dwelling, longhorn sculpin, Myoxocephalus octodecimspinosus, is traditionally viewed as a stenohaline marine fish, but fishermen have described finding this sculpin in estuaries during high tide. Little is known about the salinity tolerance of the longhorn sculpin; thus, the purposes of these experiments were to explore the effects of low environmental salinity on ion transporter expression and distribution in the longhorn sculpin gill. Longhorn sculpin were acclimated to either 100% seawater (SW, sham), 20% SW, or 10% SW for 24 or 72 hr. Plasma osmolality, sodium, potassium, and chloride concentrations were not different between the 20 and 100% treatments; however, they were 20-25% lower with exposure to 10% SW at 24 and 72 hr. In the teleost gill, regulation of Na(+), K(+)-ATPase (NKA), Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR) are necessary for ion homeostasis. We immunolocalized these proteins to the mitochondrion-rich cell of the gill and determined that acclimation to low salinity does not affect their localization. Also, there was not a downregulation of gill NKA, NKCC1, and CFTR mRNA or protein during acclimation to low salinities. Collectively, these results suggest that down to 20% SW longhorn sculpin are capable of completely regulating ion levels over a 72-hr period, whereas 10% SW exposure results in a significant loss of ions and no change in ion transporter density or localization in the gill. We conclude that longhorn sculpin can tolerate low-salinity environments for days but, because they cannot regulate ion transporter density, they are unable to tolerate low salinity for longer periods or enter freshwater (FW). The genus Myoxocephalus has three FW species, making this group an excellent model to test evolutionary and physiological mechanisms that allow teleosts to invade new low salinities successfully.     

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.     

The effect of temperature on juvenile Mozambique tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) exposed to full-strength and hypersaline seawater

The effects of temperature on the salinity tolerance of Mozambique-Wami tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) were investigated by transferring 35 g/l, 25 degrees C-acclimated fish to 35, 43, 51 or 60 g/l salinity at 15, 25 or 35 degrees C for 24 h, and by assaying gill tissue for branchial Na(+), K(+)-ATPase activity at the three temperatures after acclimating the fish to 15, 25 or 35 degrees C for 2 weeks. Tilapia survived all salinities at 25 and 35 degrees C; however, at 15 degrees C, mortality was 85.7% and 100% in the 51 g/l and 60 g/l groups, respectively. There was a significant interaction between temperature and salinity, as plasma osmolality, [Na(+)] and [Cl(-)] were significantly increased at 51 and 60 g/l salinity in 35 degrees C water (P<0.001). Additionally, muscle water content was significantly reduced at 43 g/l, 15 degrees C relative to pre-transfer values (P<0.001). Branchial Na(+), K(+)-ATPase activity was reduced at 15 degrees C regardless of acclimation temperature, and 25 degrees C-acclimated gill tissue did not show an increase in activity when assayed at 35 degrees C. Results indicate that the effects of a combined temperature-salinity transfer on plasma osmolality and ion concentrations, as well as muscle water content, are greater than when either challenge is given alone. Additionally, branchial Na(+), K(+)-ATPase activity is altered when assayed at varying temperatures; in the case of 15 degrees C, regardless of acclimation temperature. Our enzyme activity data may indicate the presence of a high temperature isoform of branchial Na(+), K(+)-ATPase enzyme.     

Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge

Freshwater (FW) spotted green pufferfish (Tetraodon nigroviridis) were transferred directly from a local aquarium to fresh water (FW; 0 per thousand ), brackish water (BW; 15 per thousand ), and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. No mortality was found. To investigate the efficient mechanisms of osmoregulation in the euryhaline teleost, distribution and expression of Na,K-ATPase (NKA) in gill and kidney of the pufferfish were examined and the osmolality, [Na+] and [Cl-] of the blood were assayed. The lowest levels of both relative protein abundance and activity were found to be exhibited in the BW group, and higher levels in the SW group than FW group. In all salinities, branchial NKA immunoreactivity was found in epithelial cells of the interlamellar region of the filament and not on the lamellae. Relative abundance of kidney NKA alpha-subunit, as well as the NKA activity, was found to be higher in the FW pufferfish than fish in BW or SW. Renal NKA appeared in the epithelial cells of distal tubules, proximal tubules, and collecting tubules, but not in glomeruli, in fish groups of various salinities. Plasma osmolality and chloride levels were significantly lower in FW pufferfish than those in BW and SW, whereas plasma sodium did not differ among the groups. Although identical distributions of NKA were found in either gill or kidney of FW-, BW- or SW-acclimated spotted green pufferfish, differential NKA expression in fish of various salinity groups was associated with physiological homeostasis (stable blood osmolality), and illustrated the impressive osmoregulatory ability of this freshwater and estuarine species in response to salinity challenge.     

急性盐度胁迫对军曹鱼稚鱼渗透压调节的影响

研究了环境盐度急性胁迫对军曹鱼(Rachycentron canadum)稚鱼鳃Na⁺-K⁺ATPase(NKA)活性及血清渗透压、Na⁺、K⁺和Cl^-离子调节的影响.结果表明:将稚鱼从盐度37中直接转移至盐度0、5、15、25、37（对照）和45的水体中,12 h后仅盐度0处理出现死亡(死亡率100％).各处理鳃NKA活性和血清渗透压在最初3 h内出现一定波动,随后变化平稳.试验结束时(12 h), NKA活性与盐度梯度呈“U”型分布,盐度5处理酶活性显著高于其它处理(P<0.05),盐度15处理活性最低,而各处理的血清渗透压大小(293～399 mOsmol·kg^-1)与盐度呈正相关;在3～12 h内稚鱼血清Na⁺和Cl^-浓度随盐度升高而升高,但增幅较小,血清K⁺浓度则与盐度呈负相关;12 h稚鱼的等渗点为328.2 mOsm·kg^-1,相当于盐度11.48,而Na⁺、K⁺和Cl^-等离子点分别为155.2、6.16和137.1 mmol·L^-1,分别相当于盐度10.68、20.44及8.41.军曹鱼在生理上具有广盐性鱼类的“低渗环境高NKA活性”特征,有较强及迅速的渗透压和离子调节与平衡能力.     

Modulation of ion transporter expression in gill mitochondrion-rich cells of eels acclimated to low-Na(+) or-Cl(-) freshwater

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Osmoregulatory effects of hypophysectomy and homologous prolactin replacement in hybrid striped bass

The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na(+),K(+)-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilisxMorone chrysops). They were kept in dilute (isoosmotic) seawater for about 10 days after surgery. Seven days after transfer to FW, Hx fish had lower plasma osmolality and lower levels of Na(+), Cl(-), and Ca(2+) than sham-operated and intact fish. Fish were injected four times with oPRL (1, 5, or 20 microg/g body mass), sbPRL (10 or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na(+), Cl(-), and Ca(2+) above values seen in saline-injected controls. Hypophysectomy did not affect branchial Na(+),K(+)-ATPase activity, but enzyme activity was significantly reduced in Hx fish receiving oPRL (20 mug/g) or sbPRL (10 or 100 ng/g). These results indicate that PRL acts to maintain plasma osmotic and ionic balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na(+),K(+)-ATPase activity.     

Short-term effects of hyposmotic shock on Na+/K+ -ATPase expression in gills of the euryhaline milkfish, Chanos chanos

Changes in expression of gill Na+/K+ -ATPase (NKA) on a short-term (96 h) time-course following hyposmotic shock (direct transfer to fresh water) of the euryhaline, marine milkfish were studied on gene, protein, and cell levels in this paper. Plasma osmolality and [Na+] responded with rapid declines in 3 h post-transfer yet, thereafter, remained constant. Plasma [Cl-] gradually fell to a significantly lower level at 6 h post-transfer. Gills responded to hyposmotic shock by a dual phase enhancement of NKA activity and protein abundance; (a) Before 24 h: NKA activity increased as early as 3 h and reached a maximum level from 6 to 12 h post-transfer coincided with the sustained lower levels of plasma osmolality, [Na+], and [Cl-] since 3 h post-transfer. This was followed by a gradual rise in alpha-subunit protein levels that peaked at 12 h post-transfer. Meanwhile, alpha-mRNA of NKA did no show significant change. (b) After 24 h: NKA activity as well as the amounts of alpha-subunit mRNA and protein increased significantly. Direct freshwater transfer induced a prompt and significant decrease of NKA immunoreactive (NKIR) cell abundance in filaments before 24 h, followed by a significant increase after 24 h due to their development in filaments and lamellae. Increased number of NKIR cells after 24 h of hyposmotic shock may occur in conjunction with rise of NKA activity as well as alpha-subunit mRNA and protein abundance. In conclusion, milkfish is able to avoid an excessive drop in plasma ions immediately upon hyposmotic shock and maintain plasma ions on a marginal lower level in fresh water. Notably, the initial increase in NKA activity (adjustive phase; 3-12 h) and delayed increase in NKA mRNA and protein abundance (regulatory phase; 48-96 h) indicate the importance of a higher level of the gill enzyme in milkfish upon hyposmotic shock.     

Acid-base balance during social interactions in rainbow trout (Oncorhynchus mykiss)

Socially subordinate rainbow trout (Oncorhynchus mykiss) experience chronic stress that impacts upon a variety of physiological functions, including Na(+) regulation. Owing to the tight coupling between Na(+) and Cl(-) uptake and, respectively, H(+) and HCO(3)(-) loss at the gill, ionoregulatory changes associated with social status may affect acid-base regulation. The present study assessed the responses of dominant, subordinate and control trout to hypercapnia (1% CO(2)) to test this hypothesis. Social status appeared to impact net acid excretion (J(net)H(+)) as subordinate individuals failed to increase net acid flux in response to hypercapnia. However, blood acid-base status was found to be unaffected by social status before or during hypercapnic exposure, indicating that subordinate fish were as effective as dominant or control trout in achieving compensation for the acid-base disturbance induced by hypercapnic exposure. Compensation in all groups involved decreasing Cl(-) uptake in response to hypercapnia. The branchial activities of both Na(+),K(+)-ATPase (NKA) and V-type H(+)-ATPase were affected by social interactions and/or exposure to hypercapnia. Branchial NKA activity was higher but V-ATPase activity was lower in control fish than in dominant or subordinate trout. In addition, control and subordinate but not dominant trout exposed to 24h of hypercapnia exhibited significantly higher branchial V-ATPase activity than fish maintained in normocapnia. Collectively, the data suggest that subordinate trout are able to regulate blood pH during a respiratory acidosis.     

The osmorespiratory compromise in sculpins: impaired gas exchange is associated with freshwater tolerance

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater ( approximately 0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl-], or mortality. Gill Na+/K+-ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher P(crit) than fish acclimated to brackish water or seawater; P(crit) is the environmental P(O2) below which an animal can no longer maintain a routine (.-)M(O2), and an increase in P(crit) represents a compromise of respiratory gas exchange. The higher P(crit) observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by approximately 1 microm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%-20%, reductions in plasma osmolality and [Cl-], and these losses in plasma ions resulted in a 1.4-fold increase in gill Na+/K+-ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in P(crit), but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.     

Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.     

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

Physiological mechanisms involved in acclimation to variable salinity and oxygen levels and their interaction were studied in European flounder. The fish were acclimated for 2 weeks to freshwater (1 per thousand salinity), brackish water (11 per thousand) or full strength seawater (35 per thousand) under normoxic conditions (water Po(2) = 158 mmHg) and then subjected to 48 h of continued normoxia or hypoxia at a level (Po(2) = 54 mmHg) close to but above the critical Po(2). Plasma osmolality, [Na(+)] and [Cl(-)] increased with increasing salinity, but the rises were limited, reflecting an effective extracellular osmoregulation. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na(+)/K(+)-ATPase activity did not change with salinity, but hypoxia caused a 25% decrease in branchial Na(+)/K(+)-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant decrease in mRNA levels of the Na(+)/K(+)-ATPase alpha1-subunit, signifying a reduced expression of the transporter gene. The reduced ATPase activity did not influence extracellular ionic concentrations. Blood [Hb] was stable with salinity, and it was not increased by hypoxia. Instead, hypoxia decreased the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O(2) affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na(+)/K(+)-ATPase activity, which did not compromise extracellular osmoregulation.     

Przewalski's naked carp (Gymnocypris przewalskii): an endangered species taking a metabolic holiday in Lake Qinghai, China

The naked carp is an endangered cyprinid that migrates annually between freshwater rivers, where it spawns, and Lake Qinghai, where it feeds and grows. Lake Qinghai is a high-altitude lake (3,200 m) in western China that currently exhibits the following composition (in mmol L(-1): [Na(+)] 200, [Cl(-)] 173, [Mg(2+)] 36, [Ca(2+)] 0.23, [K(+)] 5.3, total CO(2) 21, titration alkalinity 29; osmolality 375 mOsm kg(-1); pH 9.3), but concentrations are increasing because of water diversion and climate change. We studied the physiology of river water to lake water transfer. When river fish are transferred to lake water, there is a transitory metabolic acidosis followed by a slight respiratory alkalosis, and hemoconcentration occurs. All plasma electrolytes rise over the initial 48 h, and final levels in lake water-acclimated fish are very close to lake water concentrations for [Na(+)], [Cl(-)], [K(+)], and osmolality, whereas [Ca(2+)] continues to be regulated well above ambient levels. However, [Mg(2+)] rises to a much greater extent (fourfold in 48 h); final plasma levels in lake fish may reach 12 mmol L(-1) but are still much lower than in lake water (36 mmol L(-1)). At the same time, urine flow rate decreases drastically to <5% of river water values; only the renal excretion of Mg(2+) is maintained. Both gill and kidney Na(+),K(+)-ATPase rapidly decline, with final levels in lake water fish only 30% and 70%, respectively, of those in river water fish. Metabolic rate also quickly decreases on exposure to lake water, with O(2) consumption and ammonia-N excretion rates eventually falling to only 60% and 30%, respectively, of those in river fish, while plasma ammonia rises fivefold. The fish appear to be benefiting from a metabolic holiday at present because of decreases in iono- and osmoregulatory costs while in lake water; elevated plasma [Mg(2+)] and ammonia may be additional factors depressing metabolic rate. If the lake continues to dehydrate, these benefits may change to pathology.     

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss)

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.