Effects of long-term exposure to different salinities on the location and activity of Na +–K +ATPase in the gills of juvenile mitten crab, Eriocheir sinensis (original) (raw)
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Marine Biology, 2004
We studied Na+/K+–ATPase activity and ultrastructure in gills of the hyper-hypo-regulating crab Chasmagnathus granulatus Dana, 1851 acclimated to different salinities: 10, 30 and 45‰, known to be hypo-, iso-, and hyper-osmotic to the hemolymph, respectively. After centrifugation of homogenates at 11,000 g, Na+/K+–ATPase activity was almost entirely found in the pellets from the posterior (6–8) and anterior (3–5) gills, whereas very little was detected in the supernatant liquid. Specific activity of gill 6 was 41.3, 30.2, and 28.2 µmol Pi h–1 mg prot–1 for crabs acclimated to 10, 30, and 45‰, respectively, the result for 10‰ being significantly higher than those at 30 and 45‰. Although the concentration of sodium at which the reaction rate is half-maximal (K M) was similar in the three acclimation salinities, only the enzyme from crabs acclimated to 10‰ was inhibited by high sodium concentration. Specific activity of gill 5 increased with the increment in external salinity (10.1, 15, and 18.1 µmol Pi h–1 mg prot–1 for 10, 30, and 45‰, respectively), the only significant difference being that between the extreme salinities. The epithelium thickness of the dorsal portion of gill 6 showed a variation among salinities: 21.7, 15.8 and 17.2 µm for 10, 30 and 45‰, respectively. There were significant differences in epithelium thickness between the 10‰ and the other salinities. In all three salinities, the ultrastructure of gill 6 epithelium showed a high density of mitochondria, estimated by their volume fraction (Vv m=0.307–0.355). These mitochondria were packed between extensive basolateral membrane interdigitations in ionocytes and pillar cells. Gill 5 showed three cell types: pillars which possess mitochondria packed between membrane folds only in their interdigitations with neighbouring cells; type-I cells 8.0 µm thick with low density of mitochondria (Vv m=0.088), and type-II cells, 9.9 µm thick and rich in mitochondria (Vv m=0.423), but lacking basolateral interdigitations. Vv m of type-I cells of gill 5 was significantly lower than those of type-II cells of the same gill and the ionocytes of gill 6. No significant difference in Vv m was detected between the latter cell types.
Background: Invasive mitten crabs of the genus Eriocheir, are catadromous species. As in other decapod crustaceans, their hyperosmoregulation during and after migration into fresh water has been studied, but information about the physiology during seawater acclimation of adults is limited mostly to the dynamics of free amino acids. Therefore, the purpose of this study was to investigate the possible osmo/iono-regulation in adult mitten crabs (E. japonica) during adaptation to seawater. Methods: Adult crabs collected in fresh water were transferred to 30-ppt seawater for 3 and 10 days, and the osmolality and ionic status of the hemolymph as well as gill Na+/K+-ATPase, which has been implicated in ion transport in various crustaceans, were analyzed. Results: Analysis of the hemolymph osmolality and Na+ status indicated that adults were able to hypo-regulate these parameters in seawater. On the other hand, the free Ca2+ concentration was two-fold hyper-ionic to seawater in both fresh water and seawater, but relatively maintained compared with Na+ and osmolality in seawater, while complexed calcium (total minus free calcium) increased after 3 days in the hemolymph. Examination of Na+/K+-ATPase activity revealed that seawater acclimation decreased the activity in the most posterior gill (gill number 8), where immunoreactive Na+/K+- ATPase was localized to the basolateral membrane region of gill epithelial cells in fresh water. Conclusions: Adult mitten crabs can hypo-regulate the hemolymph concentrations of ions and their osmolality during seawater adaptation, partly by reducing the Na+/K+-ATPase activity involved in ion absorption in fresh water and using the hemolymph complexed calcium as an internal reserve. Keywords: Crustacean, osmoregulation, salinity, gills, Na+/K+-ATPase
2008
Embryos, larvae, and adults of Palaemonetes argentinus tolerate a wide range of salinities (1 to 25 ‰). While osmoregulatory capacities have previously been demonstrated in all postembryonic stages, little is known about the occurrence of osmoregulation during the embryonic phase. We examined ontogenetic and salinity-induced changes in the activity of a key enzyme involved in osmoregulation, Na + ,K + -ATPase. Its activity was studied in: (1) eggs at an early (SI), an intermediate (SII), and a late stage of embryonic development (SIII); (2) in newly hatched larvae (Zoea-I, ZI); and in homogenates of (3) whole adults and (4) isolated gill tissue. All stages were directly exposed to 1, 15, or 25 ‰, and Na + ,K + -ATPase activity was chemically determined 24 h (embryos, larvae) or 48 h later (adults). Enzyme activity was detected in all developmental stages, being low in SI and SII, maximum in SIII, and intermediate in ZI and adults. Maximum salinityinduced activity changes prior to hatching (SIII) suggest that hyper-osmoregulatory functions are expressed by the end of the embryonic phase. The ontogenetic activity maximum at this stage, however, may also be related to the hatching process. Comparing different salinities, Na + ,K + -ATPase activity in SIII was always highest at 15 ‰, whereas the activity in gills was higher at both 15 and 25 ‰ than at 1 ‰. While gills are absent in the embryonic and early larval stages, ion-transporting cells must be located elsewhere during these early ontogenetic stages, probably in the brachiostegites.
Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2006
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. J. Exp. Zool. 305A:233–245, 2006.© 2006 Wiley-Liss, Inc.
Journal of Experimental Biology, 2001
SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na++K+-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3′ and 5′ untranslated regions of the mRNA coding for the α-subunit of Na++K+-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The α-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81–83 % identity to those of other arthropods and 74...
Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2005
The kinetic properties of a microsomal gill (Na + , K + ) ATPase from the blue crab, Callinectes danae, acclimated to 15 % salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na + , K + )-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V¼102.974.3 U.mg À1 with K 0.5 ¼1.770.1 mmol.L À1 , while stimulation by magnesium (V¼93.772.3 U.mg À1 ; K 0.5 ¼1.4070.03 mmol.L À1 ) and potassium ions (V¼94.973.5 U.mg À1 ; K 0.5 ¼2.970.1 mmol.L À1 ) was cooperative. K + -phosphatase activity was also stimulated by ammonium ions to a rate of V¼106.272.2 U. mg À1 with K 0.5 ¼9.870.2 mmol.L À1 , following cooperative kinetics (n H ¼2.9). However, K + -phosphatase activity was not stimulated further by K + plus NH 4 + ions. Sodium ions (K I ¼22.771.7 mmol.L À1 ), and orthovanadate (K I ¼28.171.4 nmol.L À1 ) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K I ¼142.077.1 mmol.L -1 ). Western blotting analysis revealed increased expression of the (Na + , K + )-ATPase asubunit in crabs acclimated to 15% salinity compared to those acclimated to 33% salinity. The increase in (Na + , K + )-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na + , K + )-ATPase a-subunit; phosphatehydrolyzing enzymes other than (Na + , K + )-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.
The Journal of experimental biology, 2001
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other ...