216 P
.C. Castilho et al. J. Exp. Mar. Biol. Ecol. 256 2001 215 –227
the coast of Southern Brazil, Uruguay and Argentina Boschi, 1964. In this habitat, it is usually exposed to a broad range of environmental salinities Castello, 1985. Therefore,
ability to cope with salinity changes is essential for the establishment of a population in this habitat. Nery and Santos 1993, studying the ability of C
. granulata to regulate carbohydrate metabolism during osmotic stress, reported that this crab tolerates long-
term exposure to freshwater and hypersaline medium 40‰. In spite of a seasonal difference in osmoregulatory ability, C
. granulata is a good hyper- and hypo-
smoregulator, with an isosmotic point in external media of 30‰ salinity Bromberg, 1992; Miranda, 1994. Also, it was demonstrated that hemolymph osmolality tends to be
maintained at the same level after the hyposmotic stress, both in winter and summer Bromberg et al., 1995.
Sodium and chloride are the major osmotic effectors in the hemolymph of C
.granulata and their concentrations are maintained out of electrochemical equilibrium, both in concentrated and diluted media Bromberg et al., 1995, as do many other
´ osmoregulating crabs Mantel and Farmer, 1983; Pequeux, 1995. In C
.granulata, gills
1 2
are presumed to be the sites of active transport of Na and Cl , in both concentrated
and diluted media. However, the mechanisms of hyper- and hypoosmotic regulation are not well understood.
1 1
The presence of Na ,K -ATPase activity in the gills of estuarine and freshwater decapod crustaceans has been widely reported. Many studies have pointed out a central
1
role of this enzyme in active Na uptake. Studies of the biochemical basis of ion uptake
1 1
have shown that Na ,K -ATPase is present at high specific activities in gills and antennal gland of hyperosmotically regulating species. A larger enzyme activity in
salt-transporting gills posterior pairs than in respiratory ones anterior pairs, as well as changes of this activity as a function of the acclimation salinity, have also been reported
´ Pequeux, 1995.
The present study was undertaken to determine the major kinetic characteristics of the
1 1
Na ,K -ATPase present in anterior and posterior gills of C . granulata, and to verify a
possible correlation between enzyme activity and hemolymph osmolality, during acclimation to diluted sea water or sea water.
2. Materials and methods
Adult male crabs in stage C or early D of the intermoult cycle Drach and Tchernigovtzeff, 1967 were captured at the salt marshes of the Lagoa dos Patos estuary
near Rio Grande RS Southern Brazil. They were immediately transferred to the laboratory, and maintained in tanks with aerated diluted sea water 2‰ salinity or sea
water 30‰ salinity and acclimated for at least 30 days. Photoperiod and temperature were fixed at 12-h light dark and 208C, respectively. Every 2 days during the
acclimation period, crabs were fed ground beef.
After acclimation, crabs were cooled to 2 208C and sacrificed by removing the exoskeleton. Gills were then dissected, isolated and grouped as anterior 3rd; 4th and
5th and posterior 6th; 7th and 8th pairs. They were then rinsed and homogenized in an
P .C. Castilho et al. J. Exp. Mar. Biol. Ecol. 256 2001 215 –227
217
ice-cold medium containing 0.25 M sucrose and 5 mM EDTA. Homogenization was performed with a glass-teflon Potter homogenizer and 10 000 3 g pellets were obtained
1 1
´ by differential centrifugation Pequeux and Chapelle, 1982. Na ,K -ATPase activity
was determined following the method previously described Bianchini and Castilho, 1999. For each assay, 100 ml aliquots of the 10 000 3 g pellet were added to 2.5 ml of
assay medium which contained, for the ‘standard assay’, the following final composition
21
in mmol l 5 77.0 NaCl; 20.0 KCl; 6.0 MgCl ; 3.0 ATP and 0.1 buffer Tris–HCl at pH
2
7.6. It is important to note that the ionic composition of this medium ensured enzyme saturation see Results. Protein concentration in 10 000 3 g pellets and P were
i
determined colorimetrically based on the methods described by Bradford 1976 and Fiske and Subbarow 1925, respectively. Enzyme specific activity was then expressed
as mmol P released per mg protein per h.
i
Each kinetic or inhibition study was performed on gills isolated from five crabs
acclimated to 2‰ salinity. This acclimation salinity was selected considering the lower limit of salinity tolerance of the species Miranda, 1994. The optimum time of reaction
was determined performing assays during 15, 30, 45 or 60 min. In order to determine the effect of protein content in homogenates and temperature of incubation medium on the
maximum enzyme activity, different 10 000 3 g pellets containing protein concen-
21
trations ranging from 0.044 to 0.992 mg ml and different incubation temperatures 10,
20, 30 and 408C were assayed. The pH effect was tested by performing enzymatic assays at pH 3.6, 5.6, 7.6 and 9.6. The pH of the reaction media was adjusted with
Trizma buffer Sigma Co., St. Louis, MO. Data from experiments of incubation time, and of protein content in 10 000 3 g pellets, were subjected to regression analysis, while
those from temperature and pH of incubation media were subjected to two-way ANOVA followed by Newman–Keuls test.
1 1
21
The effect of different Na , K , Mg or ATP concentrations on maximum enzyme
activity was analyzed by replacing them in the ‘standard assay’ medium by choline
1 1
21
chloride. K values for Na , K , Mg
, and ATP were estimated by means of
m
non-linear regression analysis one-site binding. In order to study the enzyme inhibition by ouabain, the total ATPase activity was also
determined. Comparing this activity with that obtained in the ‘standard assay’, the
1 1
maximum Na ,K -ATPase activity was calculated and these values were then consid- ered as 100 in the inhibition assays with ouabain. The final concentrations of ouabain
25 25
24 24
23 22
21
tested were: 10 , 3.3 3 10
, 10 , 3.3 3 10
, 10 , and 10
mol l . Estimation
and comparison of IC for ouabain were performed after data adjustment to a sigmoidal
50
dose–response curve using the GraphPad Prism software GraphPad Software, Inc.. In order to analyze the osmoregulatory performance and gill enzyme activity during
acclimation to diluted and sea water media, 30 crabs were abruptly transferred from 2 to 30‰ salinity hyperosmotic shock or from 30 to 2‰ salinity hyposmotic shock. After
0, 1, 3, 7, 15 and 30 days, hemolymph from five crabs in each treatment was sampled with a 1-ml syringe at the arthrodial membrane at the base of the 4th or 5th pair of
pereiopods. Hemolymph osmolality was then determined by freezing point depression using a semi-micro osmometer Knauer, Berlin, Germany. After this, crabs were
cooled, sacrificed, and their gills were dissected, separated and prepared as previously
1 1
described, in order to determine the Na ,K -ATPase activity in the 10 000 3 g pellets.
218 P
.C. Castilho et al. J. Exp. Mar. Biol. Ecol. 256 2001 215 –227
Hemolymph osmolality and enzyme activity were subjected to two-way ANOVA followed by Newman–Keuls test.
In all statistical analysis, the significance level adopted was 95 a 5 0.05.
3. Results
