Adaptive branchial mechanisms in the sturgeon Acipenser naccarii during acclimation to saltwater

Variations of Na(+)/K(+)-ATPase activity and fatty-acid composition in the gills of the sturgeon Acipenser naccarii subjected to progressive acclimation to full seawater (35 ppt) were determined in relation to the hypo-osmoregulatory capacity of this species in the hyperosmotic medium. Blood samples were taken and gills arches were removed at intermediate salinity levels between 0 and 35 ppt and after 20 days at constant salinity (35 ppt). Plasma osmolality and Na(+)/K(+)-ATPase activity increased significantly with growing environmental salinity. Total saturated fatty acids (SFAs) decreased, while total polyunsaturated fatty acids (PUFAs) increased significantly with increasing salinity due mainly to changes in n-3 PUFAs (20:5n-3 and 22:6n-3). The n-3/n-6 ratio increased significantly during the acclimation process. The results show a direct relationship between salinity, increased gill Na(+)/K(+)-ATPase activity and ultrastructural changes of the gill chloride cells. Changes in the fatty-acid composition in gills of A. naccarii during progressive acclimation to full seawater suggest that variations of gill fatty acids may also have a role in osmoregulatory mechanisms.     

Effects of long-term exposure to different salinities on the location and activity of Na+-K+-ATPase in the gills of juvenile mitten crab, Eriocheir sinensis

The euryhalinity of mitten crab, Eriocheir sinensis, is based on osmoregulation, and thus on the activity of Na(+)-K(+)-ATPase. We studied location and activity of this enzyme in gills of juvenile crabs exposed to 5 per thousand, 25 per thousand, and 40 per thousand salinity. The posterior gills showed always a high number of immunopositive cells (IPC), staining with fluorescent antibody against Na(+)-K(+)-ATPase, covering at 5 per thousand the entire lamellae. At 25 per thousand, they showed fewer IPC which occurred only at the bases of the lamellae. Enzyme activity was consistently higher in posterior than in anterior gills. Low salinity stimulated the activity only in posterior gills. Both histochemical and enzymatic results are consistent with previous ultrastructural observations showing that the epithelial cells of the posterior, but not the anterior gills exhibit typical traits of ionocytes. While an increase in Na(+)-K(+)-ATPase activity at a reduced salinity is consistent with a strong hyper-osmoregulatory capacity in juvenile crabs, a low activity at an enhanced salinity suggests a physiological response, directed towards a reduction of Na(+) uptake. The activity increase of ion-transporting enzymes is directly related to spatial changes in their distribution along the osmoregulatory tissue, i.e. an enhanced number of IPC scattered along the entire lamellae. In juveniles, this allows for successful development and growth at reduced salinities.     

Influence of highly unsaturated fatty acids on the responses of white shrimp (Litopenaeus vannamei) postlarvae to low salinity

Salinity stress tests are commonly applied in shrimp hatcheries to estimate the quality of postlarvae (PL) to be used during growout. Higher larval survival during culture and to a salinity stress test in both fish and crustaceans have been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids (HUFA). However, it is not clear if increased survival is a result of better overall physiological condition resulting from the diet or a specific effect of HUFA on osmoregulatory mechanisms. This study analyzed if HUFA-rich diets could modify the fatty acid composition of membranes in gills, and if this change in composition could affect the activity of the Na⁺/K⁺ ATPase pump and carbonic anhydrase in relation to changes in salinity. One-day-old postlarvae (PL1) pooled from different spawns were fed for 20 days with Artemia sp. nauplii enriched with three levels of HUFA: low, medium and high. At PL20, survivals during culture and to salinity stress test (tap water for 30 min) were evaluated. Also at this stage, Na⁺/K⁺-ATPase and carbonic anhydrase activity, morphometric variables, and fatty acid composition in the hepatopancreas and gills were measured after they were submitted to a salinity challenge in dilute seawater (10 ppt) for 3 h. No significant differences were observed in survival rates during culture, but survival to a salinity stress test was higher and gill area was larger in PL20 fed the Artemia sp. nauplii enriched with medium HUFA levels, probably as a result of an increased 22:6n-3 content and higher 22:6n-3/20:5n-3 ratio in this diet and in the tissues of the organisms fed this diet. Na⁺/K⁺-ATPase specific activity was significantly higher in posterior gills, while the specific activity of the carbonic anhydrase was higher in anterior gills. Enzymatic activities increased significantly in PL20 submitted to a salinity challenge, and HUFA levels in the diet affected both. The proportion of fatty acids in hepatopancreas and gills were significantly affected not only by diet, but also by exposure to dilute media. This effect is discussed in relation to an increase in gill surface and changes in fatty acid composition in the phospholipids present in gill membranes, which can modify the permeability and the activity of the Na⁺/K⁺-ATPase pump. The beneficial effect of HUFA supplementation in the diet on survival to salinity stress test is partially related to modification of fatty acid composition of gills and to a larger gill area, which in turn enhances osmoregulatory mechanisms, namely Na⁺/K⁺-ATPase and carbonic anhydrase activities.     

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.     

Gill Na(+),K(+)-ATPase and osmoregulation in the estuarine crab, Chasmagnathus granulata Dana, 1851 (Decapoda, Grapsidae)

Some kinetic properties of gill Na(+),K(+)-ATPase of the estuarine crab, Chasmagnathus granulata, and its involvement in osmotic adaptation were analyzed. Results suggest the presence of different Na(+),K(+)-ATPase isoforms in anterior and posterior gills. They have different affinities for Na(+), but similar affinity values for K(+), Mg(2+), ATP and similar enzymatic profiles as a function of temperature of the incubation medium. Ouabain concentrations which inhibit 50% of enzyme activity were also similar in the two types of gills. Enzyme activity and affinity for Na(+) are higher in posterior gills than in anterior ones. Furthermore, affinities of Na(+),K(+)-ATPase of posterior gills for Na(+) and K(+) were similar to or higher than those of gills or other structures involved in the osmoregulation in several euryaline decapod crustaceans. Acclimation to low salinity was related to a significant increase in the maximum Na(+), K(+)-ATPase activity, mainly in posterior gills. On the other hand, crab acclimation to high salinity induced a significant decrease in maximum enzyme activity, both in anterior and posterior gills. These results are in accordance to the osmoregulatory performance showed by C. granulata in diluted media, and point out the major role of posterior gills in the osmoregulation of this species.     

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.     

Plasma thyroxine and cortisol profiles and gill and kidney Na+/K+-ATPase and SDH activities during acclimation of the catfish Heteropneustes fossilis (bloch) to higher salinity, with special reference to the effects of exogenous cortisol on hypo-osmoregulatory ability of the catfish

When the stenohaline catfish Heteropneustes fossilis was transferred from fresh water (FW) to 30% seawater (SW), the Na(+)/K(+)-ATPase activity significantly increased in the kidney, while in gills it remained more or less constant. A reverse pattern was observed for succinic dehydrogenase (SDH) activity inasmuch as it significantly increased in gills and remained unchanged in the kidney. Plasma osmolality significantly increased within 3 days of transfer to 30% SW and remained significantly higher throughout the duration of experiment. These results suggest that catfish gills may not be able to reverse their function from salt uptake in FW to salt excretion at higher salinity, and that the elimination of monovalent as well as divalent ions is performed by the kidney but not the gills. The significant decline in plasma cortisol (F) levels following transfer to higher salinity may not be due to reduced production but rather to an enhanced utilization and clearance rate, a conclusion supported by the fact that exogenous administration of cortisol acetate (FA) resulted in significant increases in branchial and renal Na(+)/K(+)-ATPase in FW and 30% SW. FA also improved the plasma osmotic regulatory ability of the catfish, possibly due to a change in branchial function from salt-absorption to salt excretion, as was evident from a significant increase in branchial Na(+)/K(+)-ATPase activity in the fish in 30% SW pretreated with FA for 5 days. Consistently higher levels of plasma thyroxine (T4) following transfer to higher salinity suggest the involvement of this hormone at higher salinity.     

Ultracytochemical location of Na(+)/K(+)-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N 相似文献   

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Effects of various salinities on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 expression profiles in juvenile mitten crabs, Eriocheir sinensis

Eriocheir sinensis is a euryhaline crab migrating from sea to freshwater habitats during the juvenile stage. We used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the gene expression profile of Na(+)-K(+)-ATPase, Hsp70 (heat shock protein 70) and Hsp90 in megalopa exposed to salinities of 0, 2, 5, 10, and 15 parts per thousand. Both low and high salinities markedly stimulated expression of Na(+)-K(+)-ATPase, Hsp70 and Hsp90 genes of Chinese mitten crab megalopa; salinity had different effects on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 levels depending on the duration of salinity stress, implying that Na(+)-K(+)-ATPase, Hsp70 and Hsp90 may play an important role in salinity tolerance in this crab species.     

Rapid modulation of gill Na+ + K+-dependent ATPase activity during acclimation of the killifish Fundulus heteroclitus to salinity change.

The enzymatic properties of membrane-bound Na+ + K+-ATPase from gills of killifish acclimated to fresh water, to 16% sea water, or to 30% sea water appear to be identical, indicating that the same enzyme may function to absorb Na+ in low salinities and excrete Na+ at the gills in high salinities. Ammonium ion is an effective substitute for K+: in the ATPase reaction itself, in blocking phosphorylation of the ATPase protein, and in inhibiting the binding of ouabain to the enzyme. The specific activities of the Na+ + K+-ATPase in the three different salinities are consistent with the expected Na+ pumping rates: higher in fresh water and 30% sea water than in 16% sea water. Within one-half hour after transfer of killifish from one salinity to another, gill Na+ + K+-ATPase activities reach equilibrium levels. The rapid increase in Na+ + K+-ATPase activity in gill microsomes of fish acclimating from fresh water to 30% sea water is accompanied by a slow decrease in the number of binding sites for ouabain, supporting the idea that acclimation to short-term salinity changes may involve modifications in the catalytic rate rather than the number of Na+ + K+-ATPase molecules.     

Hyper-osmoregulatory capacity of the Chinese mitten crab (Eriocheir sinensis) exposed to cadmium; acclimation during chronic exposure

The aim of this study was to evaluate the effects of waterborne cadmium on hyper-osmoregulatory capacity of the Chinese mitten crab Eriocheir sinensis acclimated to freshwater. For this purpose, crabs were submitted to acute (0.5 mg Cd L(-1) for 1, 2 or 3 days), chronic (10 or 50 microg Cd L(-1) for 30 days) or chronic, immediately followed by acute, exposure. While no effect was observed after 1 or 2 days, hemolymph osmolality, Na(+) and Cl(-) concentrations were significantly reduced after 3 days of acute exposure. Under this latter condition, the respiratory anterior gill ultrastructure, Na(+)/K(+)-ATPase and cytochrome c oxidase activities were significantly impaired. In contrast, the osmoregulatory posterior gill was unaffected for all treatments. As a consequence, we suggest that the observed hyper-osmoregulatory capacity impairment is the result of increased dissipative flow of ions and/or water through anterior gills. In contrast to acute exposure, chronic exposure did not induce any observable effect. However, crabs submitted to a known deleterious acute condition (0.5 mg Cd L(-1) for 3 days) directly after chronic exposure to 50 microg Cd L(-1) for 30 days showed normal hyper-osmoregulatory capacity with no change in gill Na(+)/K(+)-ATPase activity, and only little disturbance of anterior gill ultrastructure. These results demonstrate that a chronic cadmium exposure can induce acclimation mechanisms related to osmoregulation in this euryhaline decapod crustacean.     

Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister

In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H(+)-ATPase (subunit B), Na(+)/K(+)-ATPase (α-subunit) and Na(+)/H(+)-exchanger (NHE) were investigated. Under control conditions, the crabs' hemolymph exhibited a total ammonia concentration of 179.3±14.5μmol L(-1), while urea accounted for 467.2±33.5μmol L(-1), respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmol L(-1) NH(4)Cl, hemolymph ammonia increased within the first 12h to ca. 500μmol L(-1) and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H(+)-ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H(+)-ATPase, Na(+)/K(+)-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far unknown form.     

Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia, Oreochromis mossambicus, is capable of surviving a wide range of salinities and temperatures. The present study was undertaken to investigate the influence of environmental salinity and temperature on osmoregulatory ability, organic osmolytes and plasma hormone profiles in the tilapia. Fish were acclimated to fresh water (FW), seawater (SW) or double-strength seawater (200% SW) at 20, 28 or 35 degrees C for 7 days. Plasma osmolality increased significantly as environmental salinity and temperature increased. Marked increases in gill Na(+), K(+)-ATPase activity were observed at all temperatures in the fish acclimated to 200% SW. By contrast, Na(+), K(+)-ATPase activity was not affected by temperature at any salinity. Plasma glucose levels increased significantly with the increase in salinity and temperature. Significant correlations were observed between plasma glucose and osmolality. In brain and kidney, content of myo-inositol increased in parallel with plasma osmolality. In muscle and liver, there were similar increases in glycine and taurine, respectively. Glucose content in liver decreased significantly in the fish in 200% SW. Plasma prolactin levels decreased significantly after acclimation to SW or 200% SW. Plasma levels of cortisol and growth hormone were highly variable, and no consistent effect of salinity or temperature was observed. Although there was no significant difference among fish acclimated to different salinity at 20 degrees C, plasma IGF-I levels at 28 degrees C increased significantly with the increase in salinity. Highest levels of IGF-I were observed in SW fish at 35 degrees C. These results indicate that alterations in gill Na(+), K(+)-ATPase activity and glucose metabolism, the accumulation of organic osmolytes in some organs as well as plasma profiles of osmoregulatory hormones are sensitive to salinity and temperature acclimation in tilapia.     

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.     

Effects of gradual salinity increase on osmoregulation in Caspian roach Rutilus caspicus

This study was carried out to determine the effects of gradual salinity increase on osmoregulatory ability of the Caspian roach Rutilus caspicus, under conditions which mimic stocking conditions of hatchery-raised fish. Initially, 30 juvenile fish (mean ± S.D. 3.20 ± 0.34 g) were transferred to 20 l circular tanks, in which salinities were changed in a stepwise fashion, from 0 to 5, 10 or 15 at 48 h intervals. The fish at salinity 15 were held for an additional 48 h at this salinity. Forty-eight hours after salinity transfer, survival rate, haematocrit, plasma Cl(-) , Na(+) and K(+) concentrations, osmolality and gill Na(+) /K(+) -ATPase (NKA) activity were measured. The only effect of exposure to 5 was a significant reduction in haematocrit compared to the freshwater control group. Exposure to salinity 10 raised haematocrit, Cl(-) and Na(+) concentrations and osmolality. At 48 h exposure to salinity 15, haematocrit, Cl(-) and Na(+) concentrations and osmolality were significantly higher than freshwater controls, and gill NKA activity was significantly lower, but the effect on NKA was no longer evident at 96 h exposure. There were no effects on survival. These results indicate that R. caspicus juveniles experience an initial non-lethal iono-osmotic perturbation following salinity increase but can adapt to brackish water at salinity 15.