Primary amino acid sequences of decapod (Na+, K+)-ATPase provide evolutionary insights into osmoregulatory mechanisms.

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 ß-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-ß 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.

(Na+, K+)- ATPase kinetics in Macrobrachium pantanalense: highlighting intra- and interspecific variation within the Macrobrachium amazonicum complex.

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 (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-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 3-fold 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.

Minuca panema (Coelho, 1972): Resurrection of a Fiddler Crab Species from Brazil Closely Related to Minuca burgersi (Holthuis, 1967) (Crustacea, Decapoda, Brachyura, Ocypodidae).

We redescribe a species of fiddler crab, Minuca panema (Coelho, 1972), from the Atlantic coast of South America. It is closely related to M. mordax (Smith, 1870), and until now, the taxon has been considered to be synonymous with another closely related species Minuca burgersi (Holthuis, 1967). However, we found that two clades of M. burgersi sensu lato were restricted to the Caribbean Basin. This distribution differs from than that of M. panema, which occurs primarily along the eastern coast of South America, ranging from the island of Trinidad to Praia da Armação, Santa Catarina, Brazil. Based on our field studies, the geographical boundary between M. burgersi sensu stricto and M. panema is the Tobago Basin, north of Trinidad. Since the two species diverged only 3 to 4 million years ago, as dated from the phylogeny of the genus Minuca Bott 1954, there are few reliable morphological features that can be used to distinguish them clearly. In live crabs, there is a striking difference in coloration between the cherryred South American M. panema and the rusty-red Caribbean M. burgersi sensu lato. In males, the pattern of tubercles on the inner surface of the major cheliped varies between the two species. In females, the vulva is slightly larger in M. burgersi sensu stricto. Ocean tides and currents together with siltation owing to freshwater outflow from the Amazon and Orinoco rivers most likely have driven the divergence of these species. In the Caribbean, small tidal amplitudes have minimized long-distance gene flow in M. burgersi sensu stricto from isolated insular lagoons. In contrast, large tidal amplitudes and exposed habitats on riverbanks along the eastern Atlantic coast of South America have enabled long-distance dispersal in M. panema. DNA analysis reveals that haplotypes of cytochrome c oxidase subunit 1 are not shared between the species. Since natural selection and/or genetic drift have yet to produce extensive morphological divergences between M. panema and M. burgersi sensu stricto, we speculate that changes in the genes regulating mitochondrial DNA functions have led to speciation at the molecular level. According to the mitonuclear compatibility concept, we propose that mitochondrial DNA may be at the forefront of speciation events and that co-evolved mitonuclear interactions are responsible for some of the earliest genetic incompatibilities arising among isolated populations.

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.

A kinetic characterization of the gill V(H+)-ATPase from two hololimnetic populations of the Amazon River shrimp Macrobrachium amazonicum.

This investigation examines the kinetic characteristics and effect of acclimation to a brackish medium (21 ‰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 ‰S for 10 days. V(H+)-ATPase activity from the Tietê River population is ≈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 Tietê and the Grande River shrimps is abolished after 21 ‰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.

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

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 ‰ salinity) or acclimated to 21 ‰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 ≈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 Vmax of 54.9 ± 1.8 nmol min-1 mg-1 protein, corresponding 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 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-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 α-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.

Can tolerances of multiple stressors and calculated safety margins in fiddler crabs predict responses to extreme environmental conditions resulting from climate change?

To comprehend mangrove crab responses to predicted global climate changes, we assessed submersion and desiccation survival durations and salinity tolerances and upper thermal limits in fiddler crabs from Isla del Carmen, Yucatán Peninsula. Based on their tolerances of extreme ambient conditions, we also calculated safety margins using abiotic monitoring data. The two most terrestrial species, Minuca rapax and Leptuca panacea, exhibited submersion tolerances of from 22 to 40 h, and desiccation tolerances of from 30 to 55 h; LC50's were ≈45‰S and UT50's were ≈40 °C. The two least terrestrial species, M. vocator and L. speciosa, were less tolerant of all experimental challenges, showing submersion and desiccation tolerances of <6 h, and LC50's of 36‰S and UT50's of 38 °C. While these fiddler crabs inhabit niches closer to their salinity and desiccation/submersion tolerances than to their temperature limits, all are clearly vulnerable to the multiple stressors that accompany anticipated global climate change.

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.

Osmotic and ionic regulation, and modulation by protein kinases, FXYD2 peptide and ATP of gill (Na+, K+)-ATPase activity, in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae).

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.

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.

Dopamine binding directly up-regulates (Na+, K+)-ATPase activity in the gills of the freshwater shrimp Macrobrachium amazonicum.

We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100 mmol L-1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VM = 190.5 ±â€¯15.7 nmol Pi min-1 mg-1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

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.

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.

Macroevolution of thermal tolerance in intertidal crabs from Neotropical provinces: A phylogenetic comparative evaluation of critical limits.

Thermal tolerance underpins most biogeographical patterns in ectothermic animals. Macroevolutionary patterns of thermal limits have been historically evaluated, but a role for the phylogenetic component in physiological variation has been neglected. Three marine zoogeographical provinces are recognized throughout the Neotropical region based on mean seawater temperature (Tm): the Brazilian (Tm = 26 °C), Argentinian (Tm = 15 °C), and Magellanic (Tm = 9 °C) provinces. Microhabitat temperature (MHT) was measured, and the upper (UL 50) and lower (LL 50) critical thermal limits were established for 12 eubrachyuran crab species from intertidal zones within these three provinces. A molecular phylogenetic analysis was performed by maximum likelihood using the 16S mitochondrial gene, also considering other representative species to enable comparative evaluations. We tested for: (1) phylogenetic pattern of MHT, UL 50, and LL 50; (2) effect of zoogeographical province on the evolution of both limits; and (3) evolutionary correlation between MHT and thermal limits. MHT and UL 50 showed strong phylogenetic signal at the species level while LL 50 was unrelated to phylogeny, suggesting a more plastic evolution. Province seems to have affected the evolution of thermal tolerance, and only UL 50 was dependent on MHT. UL 50 was similar between the two northern provinces compared to the southernmost while LL 50 differed markedly among provinces. Apparently, critical limits are subject to different environmental pressures and thus manifest unique evolutionary histories. An asymmetrical macroevolutionary scenario for eubrachyuran thermal tolerance seems likely, as the critical thermal limits are differentially inherited and environmentally driven.

Effects of waterborne copper delivered under two different exposure and salinity regimes on osmotic and ionic regulation in the mudflat fiddler crab, Minuca rapax (Ocypodidae, Brachyura).

The effects of exposure to copper (Cu) on tissue Cu accumulation, on hemolymph osmotic, Na+ and Cl- regulation, and on gill Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) activities were evaluated in the fiddler crab Minuca rapax. Waterborne copper was delivered to the crabs at one of three salinities (seawater at 25‰ salinity [S] = isosmotic control; distilled water [<0.1‰ S] = hypo-osmotic medium; or 60‰ S = hyper-osmotic seawater) either for 5 days in a 0.5-cm water film containing 0, 50, 150, 250 or 500µg Cu/L with free access to a dry surface, or in crabs fully submerged for 5h at 0, 250 or 500µg Cu/L. In the crabs with free access to a dry surface, the highest Cu concentrations were found in the hemolymph and hepatopancreas with some accumulation in the gills; accumulation in the hemolymph and gills was enhanced in low salinity but was salinity independent in the hepatopancreas. Osmotic regulation was unaffected by Cu exposure; however Na+ and Cl- hypo- regulation was impaired by Cu in 25 and 60‰ S. Gill NKA activity was stimulated 2-fold at 50µg Cu/L and markedly inhibited at 150µg Cu/L and above in 0 and 25‰ S. Gill CA was inhibited in <0.1‰ S but stimulated in 25 and 60‰ S; an inverse concentration-CA activity response was seen above 150µg Cu/L for all salinities. In the submerged crabs, Cu accumulated in all tissues in 60‰ S; however, there was no clear-cut Cu concentration-accumulation relationship evident in any tissue for either exposure regime, likely owing to the crabs' ability to regulate Cu. Copper exposure diminished osmotic, [Na+] and [Cl-] hypo-regulatory ability, especially in higher salinities. Gill NKA activity was markedly inhibited by Cu overall, and particularly above 250µg Cu/L in <0.1‰ S. Gill CA activity was inhibited in 25‰ S but inconsistently affected in 0 and 60‰ S. These findings show that Minuca rapax is affected both physiologically and biochemically by Cu contamination, although to different degrees, depending on the delivery regime, salinity, copper concentration and target tissue.

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.

Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We evaluate the effects of total ammonia nitrogen-N (TAN) exposure for 72h on (Na(+),K(+))- and V(H(+))-ATPase activities and on their subunit expressions in gills of the diadromous freshwater shrimp Macrobrachium amazonicum. Specific (Na(+),K(+))- and V(H(+))-ATPase activities increased roughly 1.5- to 2-fold, respectively, after exposure to 2.0mmolL(-1) TAN. Quantitative RT-PCR analyses revealed a 2.5-fold increase in V(H(+))-ATPase B subunit mRNA expression while (Na(+),K(+))-ATPase α-subunit expression was unchanged. Immunohistochemical analyses of the gill lamellae located the (Na(+),K(+))-ATPase throughout the intralamellar septal cells, independently of TAN concentration, while the V(H(+))-ATPase was located in both the apical pillar cell flanges and pillar cell bodies. Systemic stress parameters like total hemocyte count decreased by 30% after exposure to 2.0mmolL(-1) TAN, accompanied by increased activities of the oxidative stress enzymes superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase in the gills. The stress responses of M. amazonicum to elevated TAN include increases in gill (Na(+),K(+))- and V(H(+))-ATPase activities that are accompanied by changes in oxidative stress enzyme activities, immune system effects and an increase in gill V(H(+))-ATPase gene expression. These findings likely underpin physiological effects in a crustacean like M. amazonicum that exploits multiple ecosystems during its life cycle, as well as under culture conditions that may significantly impact shrimp production by the aquaculture industry.

Gill-specific (Na(+), K(+))-ATPase activity and α-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura).

We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.

A kinetic characterization of the gill V(H(+))-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp.

Novel kinetic properties of a microsomal gill V(H(+))-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H(+))-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H(+))-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H(+))-ATPase B-subunit is identical. Juvenile (36.6±3.3 nmol Pi min(-1) mg(-1)) and adult (41.6±1.3 nmol Pi min(-1) mg(-1)) V(H(+))-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5=0.21±0.02 mmol L(-1)) being 3-fold greater than for juveniles (K0.5=0.61±0.01 mmol L(-1)). The K0.5 for Mg(2+) interaction with the juvenile V(H(+))-ATPase (1.40 ± 0.07 mmol L(-1)) is ≈6-fold greater than for adults (0.26±0.02 mmol L(-1)) while the bafilomycin A1 inhibition constant (KI) is 45.0±2.3 nmol L(-1) and 24.2±1.2 nmol L(-1), for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min(-1) mg(-1), suggesting the presence of ATPases other than the V(H(+))-ATPase. These differences may reflect a long-term regulatory mechanism of V(H(+))-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H(+))-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.