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. Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178 165
Na,K-ATPase specific activity of the marine osmoconformers Maja crispata and Dromia personata were examined to find out any adaptive distinction between the Na,K-ATPase
activities of stenohaline-osmoconforming and euryhaline-hyperosmoregulating Crus- tacea.
2. Materials and methods
2.1. Materials Spiny lobsters Palinurus elephas Fabricius, 1787 were caught by local fishermen in
regions of small salinity fluctuations 3861 ppt in the springtime and autumn of 1998 in the South Adriatic near Dubrovnik Croatia. Animals were kept alive in plastic tanks
where seawater was renewed three times weekly T 5 18628C; aeration. Weight of the intermoult animals was 156679 g; length from medial frontal spine to telson was 1763
cm. Stenohaline osmoconforming spider crab Maja crispata Risso, 1827; length of carapace 9.161.2 cm; weight 128630 g and sponge crab Dromia personata Linneaus,
1587; length of carapace 7.961.6 cm; weight 233650 g were kept in the vicinity of town of Rovinj North Adriatic where seawater salinity fluctuates during a year not
more than in narrow range of 36.9560.83 ppt. In preliminary experiments we find that spiny lobsters can survive direct transfer from seawater 38 ppt to 27 ppt seawater, but
direct transfer to lower salinities induces mortality. For experimental purposes spiny lobsters were transferred from 38 to 30 ppt salinity and consecutively salinities were
gradually decreased for 2 ppt each 2 days until 20 ppt salinity, where the spiny lobster were acclimated for 2 weeks. Brachyuran crabs Maja crispata and Dromia personata
were acclimated to DSW by steadily decreasing of the seawater concentration by 2 ppt each 2 days until the salinity where decapods were acclimated for at least 2 weeks.
Animals were fed with fish fillet and a strong tonus of the chaeliped was an indication of successful acclimation to the lowest salinity.
2.2. Blood sampling and ion determination Haemolymph was withdrawn directly from the pericardial sinus. The haemolymph
was allowed to clot at room temperature, the clot was broken up, and the haemolymph was centrifuged at 10 000 rpm for 10 min and frozen at 2 208C until measurements
were performed. For osmometric and chloride measurements, serum was diluted 5 times and for sodium, calcium and magnesium 1000 times with doubly distilled water.
Chloride concentrations were determined using a CMT-10 chloride titrator Radiometer, Copenhagen, sodium by flame photometry and osmolarity by vapour pressure os-
mometry Knauer, Germany. Total Ca and Mg concentrations in serum were measured with inductively coupled plasma atomic emission spectrophotometry ICP-AES, Plasma
IL 200 Thermo Electron, UDA.
After destroying the ventral ganglion by a needle that was pressed through the ventral side of the body, lobsters were killed by removal of the carapace. The trichobranchiate
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C . Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178
gills and epipodites from individual animals were dissected out, blotted dry and frozen for not longer than 2 weeks at 2 808C.
2.3. Protein determination Protein concentration of gill and epipodite homogenates was measured by the
Coomassie Brilliant Blue dye binding technique Bio-Rad protein assay with bovine serum albumin as a standard.
2.4. Na,K-ATPase determination Preparation of the tissue and enzyme determination were performed as previously
described in detail Lucu and Devescovi, 1999. Briefly, tissues were homogenized by 40 strokes in a Dounce homogenizer with a loosely fitting pestle in 10 ml hypotonic
solution g fresh weight of tissue. Hypotonic saline contained 12.5 mmol l NaCl; 1 mmol l dithiothreitol; 0.5 mmol l EDTA with the serine protease inhibitor aprotinin
300 I.U. l. Homogenate was filtered on plastic mesh 200 mm to remove cuticle and branchial septa. The homogenate was kept on ice 08C. Partially purified membrane
vesicles were prepared by first centrifuging the homogenate at 500 g for 5 min. The resulting supernatant was centrifuged for 30 min at 10 000 g to remove mitochondria.
The second supernatant was centrifuged for 1 h at 50 000 g and the pellets were resuspended in saline solution 300 mmol l sucrose; 20 mmol l Hepes; 0.5 mmol l
EDTA; 2.5 mmol l DTT and pH was adjusted at 7.5 by Tris base and frozen at 2 808C before use for not longer than 1 week before enzyme analyses. We used 0.2 mg of
saponin per mg of proteins to unmask enzyme activity in native homogenates of gills and epipodites Flik et al., 1994; Lucu and Devescovi, 1999. Total ATPase activity and
Na,K-ATPase activity were determined as described in details previously Flik et al., 1994; Lucu and Devescovi, 1999. The assay procedure was based on determination of
the P inorganic phosphate released from the substrate ATP in medium containing 100
i
mmol l NaCl, 2.5 mmol l KCl, 30 mmol l imidazole, 3 mmol l ATP, pH 7.5 and the amount of P released in the same medium but without KCl and ouabain to 1 mmol l
i
was added. The difference between the mean values for total and ouabain-insensitive ATPase was noted as Na,K-ATPase specific activity expressed in mmol P h per mg
i
protein. Each experiment was performed in triplicate tubes chilled to 08C where 20 ml homogenate or 5 ml partially purified membranes and 250 ml assay medium was added
and the mixture incubated at 378C for 15 min. Reaction was stopped by 1 ml of trichloracetic acid–ammonium heptamolybdate. Absorption was measured at 700 nm
with a Unicam 8620 UV–Vis spectrophotometer.
2.5. Electrophysiological studies After isolation the epipodite edges were cut off, lifted and separated into two halves of
which one hemiepipodite with supporting cuticle and epithelium layer was mounted in a
2
micro-Ussing chamber aperture 0.0133 cm . The electrophysiological methodology has been described previously in detail Lucu and Devescovi, 1999. The transepithelial
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. Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178 167
potential difference and current pulses were measured by calomel reference electrodes Ingold C., Germany connected by 3 M KCl agar bridge in a micro-Ussing chamber.
The open-circuit potential and short-circuit current were measured using an automatic voltage clamp device Bioengineerenig, The University of Iowa, USA. Tissue conduct-
ance G was calculated from current resulting from a single voltage pulse of 1 mV
t
every 500 s. Cuticular and basolateral sides haemolymph oriented side were continu- ously superfused flow-rate was 0.25 ml min with the following saline identical on both
sides in mmol l: NaCl, 300; KCl, 5; MgCl , 2; CaCl , 4; glucose, 6 and Hepes, 6 and
2 2
adjusted to pH 7.6 with Trizma-base buffer.
3. Results
