Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10 kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3 nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004 mmol L-1) and high (VM = 153.4 ± 7.7 nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006 mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5 nmol Pi min-1 mg-1 protein, K0.5 = 0.16 ± 0.01 mmol L-1), K+ (VM = 485.3 ± 24.3 nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05 mmol L-1), Na+ (VM = 425.0 ± 23.4 nmol Pi min-1 mg-1 protein, K0.5 = 5.1 ± 0.3 mmol L-1) and NH4+ (VM = 497.9 ± 24.9 nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5 mmol L-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8 µmol L-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Cortisol is an osmoregulatory and glucose-regulating hormone in Atlantic sturgeon, a basal ray-finned fish.

Our current understanding of the hormonal control of ion regulation in aquatic vertebrates comes primarily from studies on teleost fishes, with relatively little information on more basal fishes. We investigated the role of cortisol in regulating seawater tolerance and its underlying mechanisms in an anadromous chondrostean, the Atlantic sturgeon (Acipenser oxyrinchus). Exposure of freshwater-reared Atlantic sturgeon to seawater (25 ppt) resulted in transient (1-3 day) increases in plasma chloride, cortisol and glucose levels and long-term (6-14 day) increases in the abundance of gill Na+/K+/2Cl- cotransporter (NKCC), which plays a critical role in salt secretion in teleosts. The abundance of gill V-type H+-ATPase, which is thought to play a role in ion uptake in fishes, decreased after exposure to seawater. Gill Na+/K+-ATPase activity did not increase in 25 ppt seawater, but did increase in fish gradually acclimated to 30 ppt. Treatment of Atlantic sturgeon in freshwater with exogenous cortisol resulted in dose-dependent increases in cortisol, glucose and gill NKCC and H+-ATPase abundance. Our results indicate that cortisol has an important role in regulating mechanisms for ion secretion and uptake in sturgeon and provide support for the hypothesis that control of osmoregulation and glucose by corticosteroids is a basal trait of jawed vertebrates.

Short- and long-term salinity challenge, osmoregulatory ability, and (Na+, K+)-ATPase kinetics and α-subunit mRNA expression in the gills of the thinstripe hermit crab Clibanarius symmetricus (Anomura, Diogenidae).

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na+, K+)-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L-1) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na+, K+)-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na+, K+)-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na+, K+)-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes.

Effects of ocean acidification on salinity tolerance and seawater growth of Atlantic salmon Salmo salar smolts.

Human activity has resulted in increasing atmospheric carbon dioxide (CO2 ), which will result in reduced pH and higher levels of CO2 in the ocean, a process known as ocean acidification. Understanding the effects of ocean acidification (OA) on fishes will be important to predicting and mitigating its consequences. Anadromous species such as salmonids may be especially at risk because of their rapid movements between fresh water and seawater, which could minimize their ability to acclimate. In the present study, we examine the effect of future OA on the salinity tolerance and early seawater growth of Atlantic salmon Salmo salar smolts. Exposure to 610 and 1010 µatm CO2 did not alter salinity tolerance but did result in slightly lower plasma chloride levels in smolts exposed to seawater compared with controls (390 µatm). Gill Na+ -K+ -ATPase activity, plasma cortisol, glucose and haematocrit after seawater exposure were not altered by elevated CO2 . Growth rate in the first 2 weeks of seawater exposure was greater at 1010 µatm CO2 than under control conditions. This study of the effects of OA on S. salar during the transition from fresh water to seawater indicates that elevated CO2 is not likely to affect osmoregulation negatively and may improve early growth in seawater.

A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110 kDa and hydrolyzes ATP with V M = 146.8 ± 6.3 nmol Pi min-1 mg protein-1 and K M = 0.05 ± 0.003 mmol L-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V M = 139.4 ± 6.9 nmol Pi min-1 mg protein-1, K M = 4.50 ± 0.22 mmol L-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M = 136.8 ± 6.5 nmol Pi min-1 mg protein-1, K 0.5 = 0.27 ± 0.04 mmol L-1), K+ (V M = 140.2 ± 7.0 nmol Pi min-1 mg protein-1, K 0.5 = 0.17 ± 0.008 mmol L-1), and NH4+ (V M = 149.1 ± 7.4 nmol Pi min-1 mg protein-1, K 0.5 = 0.60 ± 0.03 mmol L-1) shows cooperative kinetics. Ouabain (K I = 52.0 ± 2.6 µmol L-1) and orthovanadate (K I = 1.0 ± 0.05 µmol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

Ecological risk assessment of silicon dioxide nanoparticles in a freshwater fish Labeo rohita: Hematology, ionoregulation and gill Na(+)/K(+) ATPase activity.

The fate and effect of nanomaterials in the environment has raised concern about their environmental risk to aquatic organisms. Silica nanoparticles (SiO2-NPs) find its uses in various fields and are inevitably released into the environment. However, the ecotoxicological effects of SiO2-NPs on the freshwater fish remain poorly understood. The aim of this study was to evaluate the effect of different concentrations (1, 5 and 25mgL(-1)) of SiO2-NPs on certain hematological, ionoregulatory and enzymological profiles of a freshwater teleost fish Labeo rohita. Hematological parameters such as hemoglobin (Hb), hematocrit (Hct), red blood cells (RBC), white blood cells (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values were altered in SiO2-NPs treated groups. Likewise, plasma electrolytes such as plasma sodium (Na(+)), potassium (K(+)) and chloride (Cl(-)) levels and Na(+)/K(+) ATPase activity in gill of SiO2-NPs treated groups were altered in all concentrations throughout the study period (96h). The alterations of these parameters were found to be dependent on dose and exposure period. The results of the present study indicate that the alterations of these parameters may relate to physiological stress system to SiO2-NPs toxicity and also demonstrate that manufactured metal oxide NPs in aquatic environment may affect the health condition of the aquatic organisms.

Smolting in coastal cutthroat trout Onchorhynchus clarkii clarkii.

Gill Na(+) , K(+) -ATPase activity, condition factor and seawater (SW) challenges were used to assess the development of smolt characteristics in a cohort of hatchery coastal cutthroat trout Oncorhynchus clarkii clarkii from the Cowlitz River in Washington State, U.S.A. Gill Na(+) , K(+) -ATPase activity increased slightly in the spring, coinciding with an increase in hypo-osmoregulatory ability. These changes were of lesser magnitude than are observed in other salmonine species. Even at the peak of tolerance, these fish exhibited notable osmotic perturbations in full strength SW. Condition factor in these hatchery fish declined steadily through the spring. Wild captured migrants from four tributaries of the Columbia River had moderately elevated gill Na(+) , K(+) -ATPase activity, consistent with smolt development and with greater enzyme activity than autumn captured juveniles from one of the tributaries, Abernathy Creek. Migrant fish also had reduced condition factor. General linear models of 7 years of data from Abernathy Creek suggest that yearly variation, advancing photoperiod (as ordinal date) and fish size (fork length) were significant factors for predicting gill Na(+) , K(+) -ATPase activity in these wild fish. Both yearly variation and temperature were significant factors for predicting condition factor. These results suggest that coastal O. c. clarkii exhibit weakly developed characteristics of smolting. These changes are influenced by environmental conditions with great individual variation. The data suggest great physiological plasticity consistent with the variable life-history tactics observed in this species.

Migratory patterns of hatchery and stream-reared Atlantic salmon Salmo salar smolts in the Connecticut River, U.S.A.

The timing of downstream migration and detection rates of hatchery-reared Atlantic salmon Salmo salar smolts and stream-reared smolts (stocked 2 years earlier as fry) were examined in the Connecticut River (U.S.A.) using passive integrated transponder (PIT) tags implanted into fish and then detected at a downstream fish bypass collection facility at Turners Falls, MA (river length 192 km). In two successive years, hatchery-reared smolts were released in mid-April and early May at two sites: the West River (river length 241 km) or the Passumpsic (river length 450 km). Hatchery-reared smolts released higher in the catchment arrived 7 to 14 days later and had significantly lower detection rates than smolts stocked lower in the catchment. Hatchery-reared smolts released 3 weeks apart at the same location were detected downstream at similar times, indicating that early-release smolts had a lower average speed after release and longer residence time. The size and gill Na(+) /K(+) -ATPase (NKA) activity of smolts at the time of release were significantly greater for detected fish (those that survived and migrated) than for those that were not detected. Stream-reared pre-smolts (>11·5 cm) from four tributaries (length 261-551 km) were tagged in autumn and detected during smolt migration the following spring. Stream-reared smolts higher in the catchment arrived later and had significantly lower detection rates. The results indicate that both hatchery and stream-reared smolts from the upper catchment will arrive at the mouth of the river later and experience higher overall mortality than fish from lower reaches, and that both size and gill NKA activity are related to survival during downstream migration.

Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the Blue crab Callinectes danae (Decapoda, Brachyura).

We used the gill (Na+, K+)-ATPase as a molecular marker to provide a comprehensive kinetic analysis of the effects of Co2+in vitro on the modulation of K+-phosphatase activity in the Blue crab Callinectes danae. Co2+ can stimulate or inhibit K+-phosphatase activity. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Co2+ stimulates K+-phosphatase activity similarly to Mg2+ although with a ≈4.5-fold greater affinity. At saturating Mg2+ concentrations, Mg2+ displaces bound Co2+ from the Mg2+-binding site in a concentration dependent manner, but Co2+ cannot displace Mg2+ from its binding site even at millimolar concentrations. Saturation by Co2+ of the Mg2+ binding site does not affect pNPP recognition by the enzyme. Substitution of Mg2+ by Co2+ slightly increases enzyme affinity for K+ and NH4+. Independently of Mg2+, inhibition by ouabain or sodium ions is unaffected by Co2+. Investigation of gill (Na+, K+)-ATPase K+-phosphatase activity provides a reliable tool to examine the kinetic effects of Co2+ with and without Na+ and ATP. Given that the toxic effects of Co2+ at the molecular level are poorly understood, these findings advance our knowledge of the mechanism of action of Co2+ on the crustacean gill (Na+, K+)-ATPase.

Effects of ammonia on gill (Na+, K+)-ATPase kinetics in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum.

Water quality is essential for successful aquaculture. For freshwater shrimp farming, ammonia concentrations can increase considerably, even when culture water is renewed frequently, consequently increasing the risk of ammonia intoxication. We investigated ammonia lethality (LC50-96 h) in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum from the Paraná/Paraguay River basin, including the effects of exposure to 4.93 mg L-1 total ammonia concentration on gill (Na+, K+)-ATPase activity. The mean LC50-96 h was 49.27 mg L-1 total ammonia, corresponding to 1.8 mg L-1 un-ionized ammonia. Except for NH4+ affinity that increased 2.5-fold, that of the gill (Na+, K+)-ATPase for ATP, Mg2+, Na+, K+ and ouabain was unchanged after ammonia exposure. Western blotting of gill microsomal preparations from fresh caught shrimps showed a single immunoreactive band of ≈110 kDa, corresponding to the gill (Na+, K+)-ATPase α-subunit. Ammonia exposure increased (Na+, K+)-ATPase activity by ≈25%, coincident with an additional 130 kDa α-subunit immunoreactive band, and increased K+-stimulated and V(H+)-ATPase activities by ≈2.5-fold. Macrobrachium amazonicum from the Paraná/Paraguay River basin is as tolerant to ammonia as are other Amazon River basins populations, showing toxicity comparable to that of marine crustaceans.

Environmental Salinity Modifies Mucus Exudation and Energy Use in European Sea Bass Juveniles.

The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p < 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p < 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

Response of branchial Na(+)/K(+) ATPase to changes in ambient temperature in Atlantic cod (Gadus morhua) and whiting (Merlangius merlangus).

The maintenance of ion and pH homeostasis despite changes in ambient temperature is crucial for ectothermic organisms. Thermal sensitivity of Na(+)/K(+) ATPase mRNA expression, protein expression and activity was determined in gills of North Sea cod (NC) and Northeastern Arctic cod (NEAC), acclimated for 6 weeks at 4 and 10 °C and compared to field samples of North Sea cod (sNC), acclimatized to early spring (4 °C) and summer (18 °C) conditions. The same analyses were conducted in gills of the confamiliar whiting, acclimated at 4 and 10 °C. Branchial Na(+)/K(+) ATPase capacities remained uncompensated at functional and protein levels in NC and NEAC at both acclimation temperatures. Na(+)/K(+) ATPase mRNA expression in NEAC acclimated at 10 °C was about twofold higher compared to NC, indicating some population-specific differentiation at this level. Lower Na(+)/K(+) ATPase capacities in gills of warm-acclimatized sNC at common assay temperatures indicate thermal compensation between seasonal extremes, and post-translational modifications contributed to this mitigation at high assay temperature. Together, cod compensates Na(+)/K(+) ATPase capacities on the warm edge of the thermal window and below 4 °C, respectively. In contrast, whiting Na(+)/K(+) ATPase capacities were cold compensated at 4 °C, supported by 1.5-fold higher mRNA and protein expression. Besides, capacities were lower in whiting compared to NC and NEAC at optimum temperature, which may be advantageous in terms of reduced maintenance cost, but at temperatures ≤4 °C, compensation may represent an energy trade-off to maintain homeostasis. The species-specific response of gadid Na(+)/K(+) ATPase indicates certain threshold temperatures beyond which compensation of the pump is elicited, possibly related to the different biogeography of these species.

Effects of metal contamination in situ on osmoregulation and oxygen consumption in the mudflat fiddler crab Uca rapax (Ocypodidae, Brachyura).

The contamination of estuaries by metals can impose additional stresses on estuarine species, which may exhibit a limited capability to adjust their regulatory processes and maintain physiological homeostasis. The mudflat fiddler crab Uca rapax is a typical estuarine crab, abundant in both pristine and contaminated areas along the Atlantic coast of Brazil. This study evaluates osmotic and ionic regulatory ability and gill Na(+)/K(+)-ATPase activity in different salinities (<0.5, 25 and 60‰ S) and oxygen consumption rates at different temperatures (15, 25 and 35°C) in U. rapax collected from localities along the coast of São Paulo State showing different histories of metal contamination (most contaminated Ilha Diana, Santos>Rio Itapanhaú, Bertioga>Picinguaba, Ubatuba [pristine reference site]). Our findings show that the contamination of U. rapax by metals in situ leads to bioaccumulation and induces biochemical and physiological changes compared to crabs from the pristine locality. U. rapax from the contaminated sites exhibit stronger hyper- and hypo-osmotic regulatory abilities and show greater gill Na(+)/K(+)-ATPase activities than crabs from the pristine site, revealing that the underlying biochemical machinery can maintain systemic physiological processes functioning well. However, oxygen consumption, particularly at elevated temperatures, decreases in crabs showing high bioaccumulation titers but increases in crabs with low/moderate bioaccumulation levels. These data show that U. rapax chronically contaminated in situ exhibits compensatory biochemical and physiological adjustments, and reveal the importance of studies on organisms exposed to metals in situ, particularly estuarine invertebrates subject to frequent changes in natural environmental parameters like salinity and temperature.