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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
3.1. Haemolymph osmoconcentration and ionic changes The osmoconforming spiny lobster Palinurus elephas survives sudden transfer from
seawater SW; 38 ppt, salinity; 1291 mOsmol l to intermediate 27 ppt, salinity and by gradual acclimation shows increased tolerance to a more extreme salinity dilution at 20
ppt DSW; 679 mOsmol l. After transfer of spiny lobsters from 38 ppt to 27 ppt, the new steady state in haemolymph osmolarity and sodium and chloride was reached within
24 h. Gradual acclimation from 27 to 20 ppt followed the same pattern, i.e. osmolarity and sodium concentration of the haemolymph remain in equilibrium with DSW Fig. 1.
Haemolymph chloride concentration in SW and DSW was slightly hyporegulated.
In P . elephas, serum calcium and magnesium 12.5 mmol Ca l; 34.2 mmol Mg l
decreased 26 and 42, respectively, 2 and 4 days after transfer from SW to DSW and slightly increased to a new steady state. A steady state of serum calcium was reached at
9.17 mmol l, about 2.9 mmol l above the calcium concentration in DSW 6.2 mmol l, and magnesium concentration was 17.56 mmol l, about 14 mmol l below magnesium in
DSW 31.2 mmol l; Fig. 1. In the spiny lobster Panulirus longipes calcium con- centration in the blood was 53 and 17 over the respective ion concentration in sea
water salinities of 20 and 45 ppt Dall, 1974. Under the same conditions, blood magnesium was about 33 less than the external seawater concentration. After abrupt
transfer from seawater 36–38 ppt to lower or higher salinities, a new steady state was reached in 10 h.
3.2. Na,K-ATPase and seawater osmoconcentration changes In trichobranchiate gills, specific activity of the native homogenates ranged from 1.9
to 3.2 mmol P h per mg protein and for saponin treated homogenate ranged from 3.4 to
i
5.4 mmol P h per mg protein and exhibited no significant change from SW- and
i
DSW-acclimated spiny lobsters Fig. 2. In the osmoconforming crabs studied we have found equal distribution of the enzyme in anterior and posterior gill pairs. Saponin
treatment increased Na,K-ATPase in homogenates isolated from trichobranchiate gills by 67–89 and in epipodites by 63–64 Fig. 2; Table 1.
The total and specific Na,K-ATPase activities in homogenates and partially purified
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C . Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178
Fig. 1. Time course of changes in osmolarity and ion concentration in the haemolymph of spiny lobster Palinurus elephas acclimated to seawater 0 h; 38 ppt, salinity; 1291 mOsmo l and transferred to dilute
seawater 27 ppt; 895 mOsmo l and gradually decreased seawater concentration to 20 ppt, salinity 679 mOsmol l. Closed circles represent means6S.D.; n 55–7. The line parallel to the horizontal axis indicates
concentrations in seawater.
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. Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178 169
Fig. 2. Na,K-ATPase specific activity of the native homogenates white bars and of saponin-treated homogenates black bars in pleurobranchia pleuro, arthrobranchia arthro, and podobranchia podo from
spiny lobsters Palinurus elephas acclimated to seawater 38 ppt salinity; 1291 mOsmol l and dilute seawater 20 ppt salinity; 679 mOsmol l. The mean values were calculated from 5–6 individual measurements. The
differences between values for SW- and DSW-acclimated animals native homogenates and saponin treated homogenates were not significant at the level P ,0.05 using Student’s t-test.
membranes from the epipodite epithelium of SW- and DSW-acclimated spiny lobsters were not significantly different Table 1. Similar results were obtained with partially
purified membrane vesicles from trichobranchiate gills results not shown. The percentage of recovery V
V for gills was 2.7–3.1 results not shown and
total-M total H
for epipodites isolated from SW- and DSW-acclimated osmoconforming spiny lobsters about 6 Table 1, much less than for gills from the osmoregulating shore crab
Carcinus 13–15; Lucu and Flik, 1999. The enrichment factor V membranes V
sp sp
homogenate was 1.6 in both SW- and DSW-acclimated animals Table 1. 3.3. Short-circuit current and conductance of hemiepipodite
The transepithelial–cuticular electrical parameters are summarized in Table 2. The short-circuit currents of hemiepipodites show a negative electrical charge flow from
cuticular to the basolateral haemolymph side. In hemiepipodites isolated from seawater
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C . Lucu et al. J. Exp. Mar. Biol. Ecol. 246 2000 163 –178
Table 1 Specific Na,K-ATPase activity of native and saponin-treated homogenates and membrane vesicles from the
epipodites isolated from spiny lobster Palinurus elephas acclimated to seawater SW and dilute seawater
a
DSW Homogenates
Vesicles Recovery
Enrichment V
V factor
SPEC SPEC
Seawater 38 ppt Native
3.7560.44 6.1562.68
5.7563.54 1.6360.62
Native1saponin 6.1260.73
25.5165.71 13.865.4
4.1460.51 Increase
63.367.5 314.6646.5
Dilute seawater 20 ppt Native
5.4061.3 8.7061.84
6.3361.59 1.6560.30
Native1saponin 8.8662.31
29.8367.12 13.1562.19
3.416e 0.62 Increase
64.1616.0 242.9654.5
a
V specific activity of Na,K-ATPase mmol P h per mg protein; recovery per cent is the ratio of the
spec i
total Na,K-ATPase activity of the vesicles V 3total protein content; mg and the total Na,K-ATPase
spec
activity of homogenate V 3 total protein content; mg3100; enrichment factor is the V
vesicles V
spec spec
spec
homogenate; values are means6S.D. calculated from 4–7 individual experiments; ppt5salinity of the seawater; no significant differences between SW and DSW were detected at P ,0.05 using the paired Student’s
t-test.
22 22
222.7 mA cm and dilute seawater 216.7 mA cm
acclimated spiny lobsters, short-circuit currents were not significantly different from each other. The corresponding
22
transepithelial conductance ranged from 205.0 DSW to 289.8 mS cm SW and
were not statistically significantly different when measured in SW- and DSW-acclimated epipodites.
4. Discussion
