Na++K+-ATPase in the osmoregulating clam Rangia cuneata.

Of six tissues sampled from the osmoregulating clam Rangia cuneata, mantle contained the highest concentration of Na++K+-ATPase activity and ouabain binding sites. Acclimation to low salinities was accompanied by adaptive increases in Na++K+-ATPase activity in mantle but not in gill. Since the number of ouabain binding sites did not show parallel increases, the mechanism of acclimation to reduced salinity in Rangia appears to involve activation of pre-existing pump sites in the mantle epithelium.     

Teleost chloride cell. II. Autoradiographic localization of gill Na,K- ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments

The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.     

A carrier enzyme basis for ammonium excretion in teleost gill. NH+4-stimulated Na-dependent ATPase activity in Opsanus beta

1. Branchial Na+K+-ATPase specific activity is some 20% greater in hyposaline adapted Opsanus beta than in SW specimens. 2. Ouabain insensitive ATPase (Mg2+-ATPase) specific activities were similar, while whole body activity differences in low salinity and SW adapted fish could be accounted for by the 30% difference in extractable gill protein. 3. NH+4 ion was 15% more effective at dephosphorylation of the microsomal Na-dependent phosphoenzyme than either Rb+ or K+, and revealed a maximal ATPase affinity (Km = 0.2 mM) within the physiological range of blood [K+]. 4. Similar properties as pH optima, ATP and Mg2+ Km's, ouabain sensitivity, percent recoveries and subcell distribution indicated that the NH+4-stimulation acts through the Na+ K+-ATPase carrier enzyme and may be responsible for the Na+/NH+4 exchange in Opsanus beta.     

Na+-ATPase in the gills of bass (Dicentrarchus labrax)

The authors evidence a Mg2+ dependent ATPase activity stimulated by Na+ in absence of K+ in bass gill microsomes. As this stimulated ATPase shows different features from "baseline" activity measured in the absence of both Na+ and K+ ions (Mg2+-ATPase) and from 1mM ouabain sensitive (Na+ + K+)-ATPase, it has been ascribed to a distinct Na+-ATPase. In the present paper the optimal conditions for bass gill Na+-ATPase assay and the temperature dependence of the enzyme are reported. Moreover the Na+-ATPase appears to be insensitive to 1mM ouabain and 100% inhibited by 2,5mM ethacrynic acid. It is suggested a parallel diffusion of Na+- and (Na+ + K+)-ATPase and a possible physiological role of Na+ATPase in osmoregulation.     

Gill area, permeability and Na+ ,K+ -ATPase activity as a function of size and salinity in the blue crab, Callinectes sapidus

Juvenile blue crabs, Callinectes sapidus, extensively utilize oligohaline and freshwater regions of the estuary. With a presumptively larger surface-area-to-body weight ratio, juvenile crabs could experience osmo- and ionoregulatory costs well in excess of that of adults. To test this hypothesis, crabs ranging over three orders of magnitude in body weight were acclimated to either sea water (1,000 mOsm) or dilute sea water (150 mOsm), and gill surface area, water and sodium permeabilities (calculated from the passive efflux of 3H2O and 22Na+), gill Na+, K+ -ATPase activity and expression were measured. Juveniles had a relatively larger gill surface area; weight-specific gill surface area decreased with body weight. Weight-specific water and sodium fluxes also decreased with weight, but not to the same extent as gill surface area; thus juveniles were able to decrease gill permeability slightly more than adults upon acclimation to dilute media. Crabs < 5 g in body weight had markedly higher activities of gill Na+ ,K+ -ATPase than crabs > 5 g in both posterior and anterior gills. Acclimation to dilute medium induced increased expression of Na+, K+ -ATPase and enzyme activity, but the increase was not as great in juveniles as in larger crabs.The increased weight-specific surface area for water gain and salt loss for small crabs in dilute media presents a challenge that is incompletely compensated by reduced permeability and increased affinity of gill Na+, K+ -ATPase for Na+. Juveniles maintain osmotic and ionic homeostasis by the expression and utilization of extremely high levels of gill Na+, K+ -ATPase, in posterior, as well as in anterior, gills.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.     

Characteristics of (Na+ + K+)-ATPase in the gills of bass

A partial characterization of bass gill (Na+ + K+-ATPase is reported in the present paper. Microsomal preparation from gill homogenate showed optimal (Na+ + K+)-ATPase activity at pH 6,5 in the presence of 100 mM Na+, 20mM K+ and 5mM Mg2+. Under these conditions maximal activity was shown at 45 degrees C, even if an increased lability of the enzyme was shown at temperature greater than 30 degrees C. A complete inhibition of the enzyme occurred in the presence of 1 mM ouabain. The break in the Arrhenius plot occurred approximatively at the temperature of adaptation of these fish (18 degrees C). The energies of activation above and below the break were scarcely different from each other and lower than those reported in other Poikilotherms. Furthermore similar values of Km for Na+ were evidenced at 18 degrees C and 30 degrees C. The whole of data are discussed in comparison with other teleost gill (Na+ + K+)-ATPase reports and related to the physiological role of the enzyme in osmoregulation.     

ATPase stimulated by Na+ or K+ in gills of the freshwater mussel Anodonta.

1. Microsomal preparations from the gills of the freshwater mussel anodonta cygnea cellensis show Mg2+ -dependent Na+ - or K+ -stimulated ATPase activity, which is not inhibited by ouabain. 2. Na+ - or Ka+ -ATPase activity is decreased by Ca2+, acetylcholine, choline, and tetramethylammonium, but slightly increased by ethyl alcohol. 3. It is tentatively suggested that Na+ - or K+ -ATPase is involved in the mechanism of active monovalent cation uptake through the gills of freshwater mussels.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Relationship between gill Na+,K+-activated ATPase activity and osmotic stress in the plecopteran nymph, Paragnetina media

A relationship between osmotic stress and gill Na+,K+-activated ATPase was observed in plecopteran nymphs acclimated to diluted creek water and a hypertonic medium. An increase of 84% in diluted creek water is presumably related to an active uptake of sodium ions from the hypotonic medium. Whereas a 21% decrease in the enzyme activity may be related to the morphological changes in the specialized cells in the gills. The Na+,K+-activated ATPase activity was also compared with the Malpighian tubules and the rectum. The highest ATPase activity of 32.6 +/- 2 mumoles Pi mg protein-1 30 min-1 was observed in the Malpighian tubules. The activity in the gills (19 +/- 1.2 mumoles Pi mg-1 30 min-1) was slightly lower than the rectum. Since the ATPase activity in the gills is quite high, the gills can be considered to play an active role in hyperosmotic regulation in plecopteran nymphs.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: effects on gill (Na+,K+)-ATPase alpha-subunit expression and K+-phosphatase activity

The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.     

Effects of soybean lipoxygenase on Na+/K+-ATPase activity in vitro

Oxidized metabolites of polyunsaturated fatty acids produced by lipoxygenase are among the endogenous regulators of Na+/K+-ATPase. The direct effect of lipoxygenase on Na+/K+-ATPase activity was assessed in vitro using soybean lipoxygenase. Treatment of 4.2 microg/mL Na+/K+-ATPase (from dog kidneys) with 4.2 microg/mL of soybean lipoxygenase caused 20+/-2% inhibition of ATPase activity. A 10-fold increase in lipoxygenase concentration (41.6 microg/mL) led to 30+/-0.3% inhibition. In the presence of 12 microg/mL phenidone (a lipoxygenase inhibitor) and 15.4 microg/mL glutathione (a tripeptide containing a cysteine residue) inhibition of Na+/K+-ATPase activity was blocked and an increase in ATPase activity was observed. The presence of lipoxygenase enhanced the inhibition of Na+/K+-ATPase activity caused by 20 ng/mL ouabain (31+/-2 vs. 19+/-2) but had little or no effect with higher concentrations of ouabain. These findings suggest that lipoxygenase may regulate Na+/K+-ATPase by acting directly on the enzyme.     

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Response of rainbow trout gill Na+-ATPpase to T(3) and NaCl administration

The effect of the administration of commercial diets supplemented with 9 mg kg(-1) 3,5,3'-triiodo-l-thyronine (T(3)) or 10% (w/w) NaCl was evaluated on the ouabain-insensitive Na+-ATPase activity in rainbow trout gill microsomes. The trial, carried out following the seasonal trend from March to mid-May, included a treatment phase in freshwater and a subsequent transfer to brackish water (22 per thousand salinity) where trout were not treated. pH dependence, apparent Km values for Mg(2+) and Na+, and Hill coefficients evaluated throughout the trial for Na+-ATPase were generally not affected by the treatments and habitat change. In comparison with the control group, in both treated groups, Na+-ATPase activity was lower during the freshwater phase and higher after brackish-water transfer. As compared with untreated trout, gill (Na++K+)-ATPase activity during the freshwater phase was stimulated by NaCl treatment and also by T(3) treatment after transfer to brackish water. The results indicate that NaCl and T(3) administration act differently on the two ATPase activities involved in Na+ regulation and suggest a prevalent role of Na+-ATPase activity in hypoosmotic conditions.     

Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14?μm2 membrane per μm3cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5?μm2?μm?2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6?μm2?μm?2), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.