The remarkable diversity of decapod crustaceans inhabiting a wide range of osmotic environments reflects their evolutionary success, likely driven by the variety of mechanisms for body fluid regulation involving both molecular and systemic adaptations. In recent decades, considerable research has focused on the osmoregulatory strategies of decapod crustaceans occupying distinct osmotic niches. However, limited information is available on the effects of salinity on the activity and expression of gill (Na+, K+)-ATPase in terrestrial and semi-terrestrial species. This study investigates key aspects of osmoregulation in the semiterrestrial crab Goniopsis cruentata during acclimation to varying salinity levels. After exposure to 10, 20, 30, and 40 ‰S for 5 days, the crabs exhibited strong hyper- and hypo-osmoregulatory capabilities, reaching isosmotic conditions at approximately 30 ‰S. The Total Regulatory Capacity, expressed as a dimensionless ratio of hemolymph to external osmolality variation, was 0.08, with hyper- and hypo-osmoregulatory values of 0.06 and 0.12, respectively. Goniopsis cruentata demonstrated increased mRNA expression of (Na+, K+)-ATPase under hypo-osmotic conditions, despite a concurrent reduction in enzyme activity. These findings suggest that both hyper- and hypo-osmoregulatory mechanisms in G. cruentata are dependent only minimally on branchial (Na+, K+)-ATPase activity and instead rely more strongly on other ion transporters to maintain body fluid homeostasis. These results contrast with observations in aquatic crabs, in which branchial (Na+, K+)-ATPase plays a central role in osmoregulation.

Mangrove ecosystems are tidal wetlands with anaerobic, salt-saturated soils that support diverse fauna. Despite intensive studies on the osmoregulatory mechanisms of decapods inhabiting distinct osmotic niches, there is little information on the kinetic characteristics of the gill (Na+, K+)-ATPase of decapod crustaceans inhabiting mangroves. Here, we provide a comprehensive kinetic characterization of the K-phosphatase activity of the gill (Na+, K+)-ATPase in two semi-terrestrial brachyuran crabs, Goniopsis cruentata and Cardisoma guanhumi, which respectively inhabit intermittently flooded lower zones and drier upper zones along the vertical gradient of mangrove environments. This study revealed distinct kinetic profiles between the two species. Goniopsis cruentata shows twice as much K-phosphatase activity and a 3-fold greater affinity for p-nitrophenyl phosphate than Cardisoma guanhumi. Substrate hydrolysis by the Goniopsis cruentata enzyme follows cooperative kinetics, whereas Cardisoma guanhumi displays typical Michaelis-Menten behavior. In both species, stimulation by K+ ions shows cooperative kinetics. Notably, Cardisoma guanhumi also exhibits 25% higher K-phosphatase activity when both K+ and NH₄+ are present. However, whether these differences arise from phylogenetic divergence or reflect physiological adaptation to different mangrove microhabitats remains unresolved. Given the paucity of kinetic studies on (Na+, K+)-ATPase in semi-/terrestrial crabs, our data contribute novel insights into the enzymatic basis of ion transport in decapods inhabiting variable salinity environments.

The whiteleg marine shrimp Penaeus vannamei, originally from the Eastern Pacific Ocean, now inhabits tropical waters across Asia and Central and Southern America. This benthic species exhibits rapid growth, wide salinity and temperature tolerance, and disease resistance. These physiological traits have led to extensive research on its osmoregulatory mechanisms, including next-generation sequencing, transcriptomic analyses, and lipidomic responses. In crustaceans, osmotic and ionic homeostasis is primarily maintained by the membrane-bound metalloenzyme (Na+, K+)-ATPase. However, little is known about how various ligands modulate this enzyme in P. vannamei. Here, we examined the kinetic characteristics of the gill (Na+, K+)-ATPase to get biochemical insights into its modulation. A prominent immunoreactive band of similar to 120 kDa, corresponding to the (Na+, K+)-ATPase alpha-subunit, was identified. The enzyme exhibited two ATP hydrolyzing sites with K-0.5 = 0.0003 +/- 0.00002 and 0.05 +/- 0.003 mmol L-1 and was stimulated by low sodium ion concentrations. Potassium and ammonium ions also stimulated enzyme activity with similar K-0.5 values of 0.08 +/- 0.004 and 0.06 +/- 0.003 mmol L-1, respectively. Ouabain inhibition profile suggested a single enzyme isoform with a K-I value of 2.10 +/- 0.16 mmol L-1. Our findings showed significant kinetic differences in the (Na+, K+)-ATPase in Penaeus vannamei compared to marine and freshwater crustaceans. We expect our results to enhance understanding of the modulation of gill (Na+, K+)-ATPase in Penaeus vannamei and to provide a valuable tool for studying the shrimp's biochemical acclimation to varying salinity conditions.

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlandia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Parana-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 +/- 3.4 and 93.3 +/- 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.

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Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na+, K+)-ATPase and the apical V(H+)-ATPase. The kinetic characteristic of gill (Na+, K+)-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (∼94% identity) compared to the β-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-β interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na+, K+)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.

This investigation examines the kinetic characteristics and effect of acclimation to a brackish medium (21 parts per thousand S) on gill V(H+)-ATPase activity in two hololimnetic populations of M. amazonicum. We also investigate the cellular immunolocalization of the enzyme. Immunofluorescence findings demonstrate that the V(H+)-ATPase c-subunit is distributed in the apical pillar cells of shrimps in fresh water but is absent after acclimation to 21 parts per thousand S for 10 days. V(H+)-ATPase activity from the Tiete River population is approximate to 50% greater than the Grande River population, comparable to a wild population from the Santa Elisa Reservoir, but is 2-fold less than in cultivated shrimps. V (H+)-ATPase activity in the Tiete and the Grande River shrimps is abolished after 21 parts per thousand S acclimation. The apparent affinities of the V(H+)-ATPase for ATP (0.27 +/- 0.04 and 0.16 +/- 0.03 mmol L-1, respectively) and Mg2+ (0.28 +/- 0.05 and 0.14 +/- 0.02 mmol L-1, respectively) are similar in both populations. The absence of V(H+)-ATPase activity in salinity-acclimated shrimps and its apical distribution in shrimps in fresh water underpins the importance of the crustacean V(H+)-ATPase for ion uptake in fresh water.

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.

AbstractTo evaluate the crustacean gill (Na+, K+)-ATPase as a molecular marker for toxic contamination by heavy metals of estuarine and coastal environments, we provide a comprehensive analysis of the effects of Co2+in vitroon modulation of the K+-phosphatase activity of a gill (Na+, K+)-ATPase from the blue crabCallinectes danae. Usingp-nitrophenyl phosphate as a substrate, Co2+can act as both stimulator and inhibitor of K+-phosphatase activity. Without Mg2+, Co2+stimulates K+-phosphatase activity similarly but with a ≈4.5-fold greater affinity than with Mg2+. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Substitution of Mg2+by Co2+slightly increases enzyme affinity for K+and NH4+. Independently of Mg2+, ouabain inhibition is unaffected by Co2+. Mg2+displaces bound Co2+from the Mg2+-binding site in a concentration dependent mechanism. However, at saturating Mg2+concentrations, Co2+does not displace Mg2+from its binding site even at elevated concentrations. Saturation by Co2+of the Mg2+binding site does not affectpNPP recognition by the enzyme. Given that the interactions between heavy metal ions and enzymes are particularly complex, their toxic effects at the molecular level are poorly understood. Our findings elucidate partly the mechanism of action of Co2+on a crustacean gill (Na+, K+)-ATPase.HighlightsWithout Mg2+, cobalt ions stimulate the gill (Na+, K+)-ATPaseCo2+has a 4.5-fold greater affinity for the gill (Na+, K+)-ATPase than does Mg2+Mg2+displaces Co2+from the Mg2+-binding site in a concentration dependent mannerOuabain inhibition with Co2+or Mg2+is identicalSaturation by Co2+of Mg2+-binding sites does not affect substrate recognitionGraphical AbstractGraphical abstract (synopsis)Using a crab gill (Na+, K+)-ATPase, we demonstrate that Co2+inhibits K+-phosphatase activity with Mg2+, which is stimulated without Mg2+. Mg2+displaces Co2+from the Mg2+-binding site but Co2+cannot displace Mg2+. Ouabain inhibition is unaffected by Co2+, independently of Mg2+. The molecular mechanism of Co2+toxicity is partly elucidated.

•Mean LC50-96 h total ammonia concentration was 49.27 mg L−1•Ammonia exposure increased (Na+, K+)-, V(H+)- and (K+)-ATPase activities•Ammonia exposure increased (Na+, K+)-ATPase affinity for NH4+ by 2.5-fold•Ouabain inhibition of (Na+, K+)-ATPase was unaffected by ammonia exposure

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na+, K+)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 parts per thousand salinity) or acclimated to 21 parts per thousand S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by approximate to 50 %. PKA-mediated phosphorylation inhibited gill (Na+, K+)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na+, K+)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a V-max of 54.9 +/- 1.8 nmol min(-1) mg(-1) protein, corresponding to approximate to 60 % that of freshwater shrimps. Mg2+ affinity increased with salinity acclimation while K+ affinity decreased. (Ca2+, Mg2+)-ATPase activity increased while V(H+)- and Na+- or K+-stimulated activities decreased on salinity acclimation. The 120kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific alpha-subunit phosphorylation by PKA and/or PKC. These alterations in (Na+, K+)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different alpha-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.

Salt tolerance reflects ecophysiological adaptation, and the wide-ranging distribution of the Brachyura mirrors their ability to adjust body fluid concentrations. The gill (Na+, K+)-ATPase underpins such hyper/hypo-regulatory mechanisms. We evaluate osmotic and chloride regulation in Callinectes danae after 10 days acclimation to a wide salinity range (5–50 ‰S), accompanying alterations in hemolymph osmolality and [Cl−] during hypo- (15 ‰S) or hyper- (45 ‰S) osmotic challenge. Further, we investigate posterior gill (Na+, K+)-ATPase kinetics, α-subunit immunolocalization and its mRNA and protein expression (15, 30 and 40 or 45 ‰S). The crab is a moderate, asymmetrical hyper/hypo-osmoregulator but is a strong, asymmetrical hyper/hypo-chloride regulator. Hyper-regulation at low salinity is sustained by a threefold increase in (Na+, K+)-ATPase activity, a 3.5-fold increase in α-subunit mRNA expression and 1.6-fold increase in protein expression. α-Subunit signal is highest in 15 ‰S-acclimated crabs, and is uniformly distributed throughout the ionocytes and pillar cells. Activity in 30- and 40% S-acclimated crabs is similar. Affinity for ATP and Na+ increases on high salinity acclimation but decreases for ouabain. K+ apparent affinity is independent of salinity, while that for Mg2+ decreases and for NH4+ increases with increasing salinity. A high-affinity ATP-binding site disappears on acclimation at any salinity. FOF1- and Na+- or K+-ATPase activities decrease with increased salinity. Hemolymph chloride hypo-regulation depends little on gill (Na+, K+)-ATPase activity. Hyper-, and hypo-osmotic and ionic regulatory capabilities in C. danae are intricate physiological processes underpinned by multifarious gill (Na+, K+)-ATPase kinetics and altered mRNA and protein expressions.

We analyzed the modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+-calmodulin-dependent kinase, of gill (Na+, K+)-ATPase activity in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. Osmotic and ionic regulatory ability and gill (Na+, K+)-ATPase activity also were evaluated. (Na+, K+)-ATPase activity is stimulated by exogenous pig kidney FXYD2 peptide, while phosphorylation by endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase inhibits activity. Stimulation by FXYD2 and inhibition by protein kinase C and Ca2+/calmodulin-dependent kinase are salinity-dependent. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. At low salinities, the (Na+, K+)-ATPase exhibited a single, low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18‰S, a second, cooperative, high affinity ATP-binding site appeared, corresponding to 10–20% of total (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper−/hypo-regulated in crabs acclimated at 2 to 35‰S. Cl− was well hyper−/hypo-regulated although Na+ much less so, becoming isonatremic at elevated salinity. (Na+, K+)-ATPase activity was greatest in isosmotic crabs (26‰S), decreasing notably at 35‰S and also diminishing progressively from 18to 2‰S. Hyper-osmoregulation in U. cordatus showed little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of enzymatic and osmoregulatory adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.

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.