Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers

LJournal of Experimental Marine Biology and Ecology246 (2000) 163–178

www.elsevier.nl / locate / jembe

Gill Na,K-ATPase in the spiny lobster Palinurus elephas andother marine osmoconformers

Adaptiveness of enzymes from osmoconformity tohyperregulation

a , a b bˇ * ˇ ˇCedomil Lucu , Massimo Devescovi , Bosko Skaramuca , Valter Kozula ˇ ´Center for Marine Research Rovinj, 52210 Rovinj, Institute Ruder Boskovic, Croatia

bInstitute of Oceanography and Fisheries, 20000 Dubrovnik, Croatia

Received 18 July 1999; received in revised form 1 November 1999; accepted 29 November 1999

Abstract

Haemolymph inorganic osmolyte changes and Na,K-ATPase activities in trichobranchiate andepipodite tissues were examined in the spiny lobster Palinurus elephas gradually acclimated fromseawater (SW; 38 ppt, salinity; 1291 mOsmol / l) down to dilute seawater (DSW; 20 ppt, salinity;679 mOsmol / l). During acclimation to DSW haemolymph was only transiently hypoosmotic,becoming isosmotic to the medium over a 24-h period of acclimation. Na,K-ATPase specificactivities in homogenates of the trichobranchiate gills from SW- and DSW-acclimated spinylobsters were in the range of 2–3 mmol Pi /h /mg protein and were not significantly different. Ithas also been confirmed for the marine stenohaline crustaceans Maja crispata and Dromiapersonata that gill Na,K-ATPase maintains the same level of specific activity in SW- andDSW-acclimated crabs. The saponin-treated fraction of Na,K-ATPase activity in trichobranchiategills was 67–89% and epipodites 63–64% over the native homogenates’ activity and nodifferences in enzyme activities upon saponin treatment between SW- and DSW-acclimated spinylobsters were found. Recovery of 6% and enrichment factor (1.6) of Na,K-ATPase in partiallypurified plasma membrane fractions of epipodites was relatively low and not different in SW- andDSW-acclimated spiny lobsters. In the hemiepipodite, negative short-circuit current was in the

22range from 2 16.7 to 2 22.7 mA cm and conductance varied in the range of 205–290 mS22cm , values which were not significantly different in spiny lobsters residing in SW or DSW.

Very high conductance suggests leakiness of the hemiepipodite epithelium–cuticular complex. Incontrast to the group of euryhaline hyperosmoregulating Crustacea in which activation of thespecific activity of Na,K-ATPase upon acclimation to dilute seawater occurs, in marineosmoconformers there is no activation of the enzyme in dilute seawater. Based on the literaturedata and our own results, we have reported a correlation coefficient of 0.65 between specificactivity of Na,K-ATPase and the sodium gradient (mmol Na/ l; haemolymph–seawater) between

*Corresponding author.

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 99 )00179-3

ˇ164 C. Lucu et al. / J. Exp. Mar. Biol. Ecol. 246 (2000) 163 –178

12 species of osmoconforming and osmoregulating Crustacea. During evolution, hyperos-moregulating Crustacea have achieved internal osmolyte gradients generated by Na,K-ATPase andlowering the gill surface permeability. However these adaptive characteristics are not present inmarine osmoconforming Crustacea, restraining them to migrate in the brackish water habitats. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Isosmotic haemolymph; Na; K-ATPase activity; Trichobranchiate gills; Epipodite; Voltage clamp

1. Introduction

In spite of the fact that representatives of the superfamily Palinuroidea (Latreille1802) has great economic value on the market, there is a dearth of publications on theirphysiology and biochemistry (Dall, 1974). No substantial progress, except in the field ofthe nervous system and its physiology (Schmidt and Ache, 1994), has been attained upto this point. The Western rock lobster Panulirus longipes is an isosmotic crustaceantolerating seawater salinity in the range 25–40 ppt (Dall, 1974). The osmotic adjustmentof principal monovalent ions is made by the gills and Ca and Mg regulation by the gut(Dall, 1974; 1977).

Marine osmoconforming Crustacea have haemolymph in osmotic equilibrium withseawater and all show very limited ionic regulation. Differences between plasma andseawater in these species are mostly due to the indiffusibility of haemolymph proteinsand formation of complexes between proteins and inorganic osmolytes. In isolatedperfused gills of the osmoconforming decapod Maja squinado, transepithelial potentialwas close to 0 mV and no net sodium fluxes were found over the concentration range20–100% seawater (King and Schoffeniels, 1969). In these osmoconforming specieswhich allow their blood osmolarity to parallel their environment over a narrow salinityrange, intracellular volume regulation occurs controlling organic osmolyte content in thecells (Gilles, 1987).

In the hyperosmoregulating Crustacea, gill Na,K-ATPase activity increases pro-portionally with the increase in the number of chloride cells (Neufeld et al., 1980) and asa key enzyme for Na/K exchange indirectly regulates activities of the Na/H exchanger(Shetlar and Towle, 1989; Towle et al., 1997) and Na/K/2Cl cotransporter (Riestenpattet al., 1996) on the apical membrane side, and the Na/Ca exchanger on the basolateralmembrane side (Flik et al., 1994). In the marine osmoconforming Crustacea Calappahepatica (Spencer et al., 1979), Cancer and Nephrops (Harris and Bayliss, 1988),haemolymph is isosmotic with surrounding seawater and Na,K-ATPase specific ac-tivities were reported to be at the detection limits. Accordingly, specific activity of thegill Na,K-ATPase in marine Crustacea is lower than in the brackish water and freshwaterCrustacea and sodium gradients between haemolymph and medium are in positivecorrelation with Na,K-ATPase activity (Harris and Bayliss, 1988).

In the spiny lobster Palinurus elephas the role of the Na,K-ATPase in gills andepipodites and short-circuit current and conductance (in epipodites) were studied inrelation to this species’ limited osmoregulatory ability in dilute seawater. In addition,

C. 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 Dromiapersonata were examined to find out any adaptive distinction between the Na,K-ATPaseactivities 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 inregions of small salinity fluctuations (3861 ppt) in the springtime and autumn of 1998in the South Adriatic near Dubrovnik (Croatia). Animals were kept alive in plastic tankswhere seawater was renewed three times weekly (T 5 18628C; aeration). Weight of theintermoult animals was 156679 g; length from medial frontal spine to telson was 1763cm. Stenohaline osmoconforming spider crab Maja crispata (Risso, 1827; length ofcarapace 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 oftown of Rovinj (North Adriatic) where seawater salinity fluctuates during a year notmore than in narrow range of 36.9560.83 ppt. In preliminary experiments we find thatspiny lobsters can survive direct transfer from seawater (38 ppt) to 27 ppt seawater, butdirect transfer to lower salinities induces mortality. For experimental purposes spinylobsters were transferred from 38 to 30 ppt salinity and consecutively salinities weregradually decreased for 2 ppt each 2 days until 20 ppt salinity, where the spiny lobsterwere acclimated for 2 weeks. Brachyuran crabs Maja crispata and Dromia personatawere acclimated to DSW by steadily decreasing of the seawater concentration by 2 ppteach 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 ofsuccessful acclimation to the lowest salinity.

2.2. Blood sampling and ion determination

Haemolymph was withdrawn directly from the pericardial sinus. The haemolymphwas allowed to clot at room temperature, the clot was broken up, and the haemolymphwas centrifuged at 10 000 rpm for 10 min and frozen at 2 208C until measurementswere performed. For osmometric and chloride measurements, serum was diluted 5 timesand 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 measuredwith inductively coupled plasma atomic emission spectrophotometry (ICP-AES, PlasmaIL 200 Thermo Electron, UDA).

After destroying the ventral ganglion by a needle that was pressed through the ventralside of the body, lobsters were killed by removal of the carapace. The trichobranchiate


gills and epipodites from individual animals were dissected out, blotted dry and frozenfor not longer than 2 weeks at 2 808C.

2.3. Protein determination

Protein concentration of gill and epipodite homogenates was measured by theCoomassie Brilliant Blue dye binding technique (Bio-Rad protein assay) with bovineserum albumin as a standard.

2.4. Na,K-ATPase determination

Preparation of the tissue and enzyme determination were performed as previouslydescribed in detail (Lucu and Devescovi, 1999). Briefly, tissues were homogenized by40 strokes in a Dounce homogenizer with a loosely fitting pestle in 10 ml hypotonicsolution /g fresh weight of tissue. Hypotonic saline contained 12.5 mmol / l NaCl; 1mmol / 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 andbranchial septa. The homogenate was kept on ice (08C). Partially purified membranevesicles were prepared by first centrifuging the homogenate at 500 g for 5 min. Theresulting 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 wereresuspended in saline solution (300 mmol / l sucrose; 20 mmol / l Hepes; 0.5 mmol / lEDTA; 2.5 mmol / l DTT and pH was adjusted at 7.5 by Tris base) and frozen at 2 808Cbefore use for not longer than 1 week before enzyme analyses. We used 0.2 mg ofsaponin per mg of proteins to unmask enzyme activity in native homogenates of gillsand epipodites (Flik et al., 1994; Lucu and Devescovi, 1999). Total ATPase activity andNa,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 ofthe P (inorganic phosphate) released from the substrate ATP in medium containing 100i

mmol / l NaCl, 2.5 mmol / l KCl, 30 mmol / l imidazole, 3 mmol / l ATP, pH 7.5 and theamount of P released in the same medium but without KCl and ouabain to 1 mmol / li

was added. The difference between the mean values for total and ouabain-insensitiveATPase was noted as Na,K-ATPase specific activity expressed in mmol P /h per mgi

protein. Each experiment was performed in triplicate tubes chilled to 08C where 20 mlhomogenate or 5 ml partially purified membranes and 250 ml assay medium was addedand the mixture incubated at 378C for 15 min. Reaction was stopped by 1 ml oftrichloracetic acid–ammonium heptamolybdate. Absorption was measured at 700 nmwith a Unicam 8620 UV–Vis spectrophotometer.

2.5. Electrophysiological studies

After isolation the epipodite edges were cut off, lifted and separated into two halves ofwhich one hemiepipodite with supporting cuticle and epithelium layer was mounted in a

2micro-Ussing chamber (aperture 0.0133 cm ). The electrophysiological methodology hasbeen described previously in detail (Lucu and Devescovi, 1999). The transepithelial


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 automaticvoltage clamp device (Bioengineerenig, The University of Iowa, USA). Tissue conduct-ance (G ) was calculated from current resulting from a single voltage pulse of 1 mVt

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 bothsides (in mmol / l): NaCl, 300; KCl, 5; MgCl , 2; CaCl , 4; glucose, 6 and Hepes, 6 and2 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 fromseawater (SW; 38 ppt, salinity; 1291 mOsmol / l) to intermediate 27 ppt, salinity and bygradual acclimation shows increased tolerance to a more extreme salinity dilution at 20ppt (DSW; 679 mOsmol / l). After transfer of spiny lobsters from 38 ppt to 27 ppt, thenew steady state in haemolymph osmolarity and sodium and chloride was reached within24 h. Gradual acclimation from 27 to 20 ppt followed the same pattern, i.e. osmolarityand 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 andslightly increased to a new steady state. A steady state of serum calcium was reached at9.17 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 inDSW (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 seawater salinities of 20 and 45 ppt (Dall, 1974). Under the same conditions, bloodmagnesium was about 33% less than the external seawater concentration. After abrupttransfer from seawater (36–38 ppt) to lower or higher salinities, a new steady state wasreached in 10 h.

3.2. Na,K-ATPase and seawater osmoconcentration changes

In trichobranchiate gills, specific activity of the native homogenates ranged from 1.9to 3.2 mmol P /h per mg protein and for saponin treated homogenate ranged from 3.4 toi

5.4 mmol P /h per mg protein and exhibited no significant change from SW- andi

DSW-acclimated spiny lobsters (Fig. 2). In the osmoconforming crabs studied we havefound equal distribution of the enzyme in anterior and posterior gill pairs. Saponintreatment increased Na,K-ATPase in homogenates isolated from trichobranchiate gills by67–89% and in epipodites by 63–64% (Fig. 2; Table 1).

The total and specific Na,K-ATPase activities in homogenates and partially purified


Fig. 1. Time course of changes in osmolarity and ion concentration in the haemolymph of spiny lobsterPalinurus elephas acclimated to seawater (0 h; 38 ppt, salinity; 1291 mOsmo/ l) and transferred to diluteseawater (27 ppt; 895 mOsmo/ l) and gradually decreased seawater concentration to 20 ppt, salinity (679mOsmol / l). Closed circles represent means6S.D.; (n55–7). The line parallel to the horizontal axis indicatesconcentrations in seawater.


Fig. 2. Na,K-ATPase specific activity of the native homogenates (white bars) and of saponin-treatedhomogenates (black bars) in pleurobranchia (pleuro), arthrobranchia (arthro), and podobranchia (podo) fromspiny 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. Thedifferences between values for SW- and DSW-acclimated animals (native homogenates and saponin treatedhomogenates) 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 lobsterswere not significantly different (Table 1). Similar results were obtained with partiallypurified membrane vesicles from trichobranchiate gills (results not shown). Thepercentage of recovery (V /V ) for gills was 2.7–3.1% (results not shown) andtotal-M total H

for epipodites isolated from SW- and DSW-acclimated osmoconforming spiny lobstersabout 6% (Table 1), much less than for gills from the osmoregulating shore crabCarcinus (13–15%; Lucu and Flik, 1999). The enrichment factor (V membranes /Vsp 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. Theshort-circuit currents of hemiepipodites show a negative electrical charge flow fromcuticular to the basolateral (haemolymph side). In hemiepipodites isolated from seawater


Table 1Specific Na,K-ATPase activity of native and saponin-treated homogenates and membrane vesicles from theepipodites isolated from spiny lobster Palinurus elephas acclimated to seawater (SW) and dilute seawater

a(DSW)

Homogenates Vesicles Recovery EnrichmentV V (%) factorSPEC SPEC

Seawater (38 ppt)Native 3.7560.44 6.1562.68 5.7563.54 1.6360.62Native1saponin 6.1260.73 25.5165.71 13.865.4 4.1460.51Increase (%) 63.367.5 314.6646.5

Dilute seawater (20 ppt)Native 5.4061.3 8.7061.84 6.3361.59 1.6560.30Native1saponin 8.8662.31 29.8367.12 13.1562.19 3.416e 0.62Increase (%) 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 thespec i

total Na,K-ATPase activity of the vesicles (V 3total protein content; mg) and the total Na,K-ATPasespec

activity of homogenate (V 3 total protein content; mg)3100; enrichment factor is the V (vesicles) /Vspec spec spec

(homogenate); values are means6S.D. calculated from 4–7 individual experiments; ppt5salinity of theseawater; no significant differences between SW and DSW were detected at P,0.05 using the paired Student’st-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

22transepithelial conductance ranged from 205.0 (DSW) to 289.8 mS cm (SW) andwere not statistically significantly different when measured in SW- and DSW-acclimatedepipodites.

4. Discussion

Besides aquatic Crustacea, there are few systematic categories of invertebrates withsuch a variety of osmotic behaviour. Marine osmoconforming Crustacea have no abilityof osmoregulation, surviving limited dilutions in the osmoconcentration of seawater.Their haemolymph ion composition and osmoconcentration vary with external seawaterconcentration. According to this study, after acclimation of the spiny lobster to SW andDSW, haemolymph osmolarity was, respectively, 17.3 and 19.4% higher than the

1 2predicted osmolarities calculated from the sum of respective Na and Cl con-

Table 2Short-circuit current (I ) and conductance (G) across single split epipodite isolated from spiny lobstersc

aPalinurus elephas acclimated to seawater (38 ppt, salinity) and to dilute seawater (20 ppt, salinity)

Seawater Dilute seawater22I (mA cm ) 222.764.2 216.765.1sc22G (mS cm ) 289.8647.9 205.0668.9

a Values are means6S.D. calculated from 4–5 individual experiments. No significant differences betweenseawater and dilute seawater were detected at P,0.05 using the paired Student’s t-test.


centrations alone; the remainder of the osmolarity may be related to an accumulation oforganic osmolytes (Pequeux, 1995). The Western rock lobster Panulirus longipesacclimated in this range showed slight hyperionic regulation of sodium and slighthyporegulation of chloride in haemolymph (Dall, 1974; Malley, 1977). Sensillar lymphof the spiny lobster Panulirus argus has a composition of Na, K, Ca and Mg similar tothat of seawater and haemolymph, however Cl is present at a reduced level (Gleeson etal., 1993).

Hyperosmoregulating Crustacea distributed within estuaries show regulation of bodyfluids osmoconcentration that protect the intracellular space from drastic changes in cellhomeostasis. The moderate hyperosmoregulator Carcinus can withstand externalsalinities close to 10 ppt, and the weak osmoregulator Homarus can tolerate 20 ppt,keeping blood osmolarities, respectively, 300 and 150 mOsmol hyperosmostic to theirmedium (Zanders, 1980; Lucu and Devescovi, 1999). Crustacean gills in hyperosmo-regulating crabs act as a selective interface actively absorbing Na and Cl from dilutedexternal medium. The gills of osmoregulating Crustacea are a convenient model inwhich to study Na,K-ATPase activation (Lucu and Flik, 1999).

In the next sections we will discuss the differences in responses of Na,K-ATPase ofthe osmoregulatory tissues located in branchial cavity from spiny lobster and a fewbrachyuran osmoconformers with osmoregulating Crustacea.

4.1. Na,K-ATPase activity and short-circuit current (SCC) of epipodite inosmoconforming and weakly regulating lobsters

Apart from the gill epithelium, a candidate for osmoregulation in the branchial cavityof the lobster Homarus gammarus is the epipodite (Lucu and Devescovi, 1999),showing in DSW-acclimated animals more developed apical microvilli with a network ofmicrotubules and basolaterally numerous elongated mitochondria (Haond et al., 1998).The following characteristics of the epipodites in the osmoconforming spiny lobsterPalinurus elephas and weakly hyperosmoregulating lobster Homarus gammarus (Lucuand Devescovi, 1999) may be pointed out: (a) the specific activity of Na,K-ATPase innative homogenates of epipodites of SW lobsters Homarus gammarus was no differentfrom the spiny lobster P. elephas. In DSW-acclimated H. gammarus, Na,K-ATPaseactivity of homogenate was 2.8- and in isolated membranes 2.5-fold of activitiesreported in SW. However, the corresponding specific activities were no different for SW-and DSW-acclimated P. elephas (Fig. 3). Moreover, by Western blotting of solubilizedepipodite tissue isolated from the spiny lobster, a monoclonal mouse raised against thealpha subunit of the avian Na,K-ATPase (Hybridoma Bank, The University of Iowa,USA) bind specifically to a single band and densitometric ratio of DSW/SW were notfound to be statistically significantly different (results not shown). (b) In Palinurusepipodites, the percentage of recovery of Na,K-ATPase in membrane plasma fractionscompared to that in homogenates was 5.8 (SW) to 6.3 (DSW) per cent (Table 1), lessefficient than 23% (SW) and 26% (DSW) for lobster H. gammarus (Lucu andDevescovi, 1999). For comparison, the ratio of activity in membrane fraction to that inoriginal homogenates (enrichment factor) of Homarus epipodites from DSW and SWwas, respectively, 6.8 to 7.6, significantly increased compared to the enrichment factor


Fig. 3. Comparison of specific activity of the enzyme Na,K-ATPase in epipodite homogenates and of partiallypurified membrane fraction from lobster Homarus gammarus (Lucu and Devescovi, 1999) and of spiny lobsterPalinurus elephas (this study). Na,K-ATPase specific activities from native homogenates (H) and partiallypurified membranes (M) from animals acclimated to seawater (white bars) and to dilute seawater (black stripedbars) is presented. Statistical significance of differences between seawater (38 ppt) and dilute seawater (20 ppt,salinity). Na,K-ATPase activities were tested by paired Student’s t-test. Homarus: H, P,0.001; M, P,0.01(n55–6). Palinurus: In H and M seawater enzyme activities were not statistically significantly different at thelevel P,0.05 using Student’s t-test from those in dilute seawater (n56).

from the spiny lobster (1.6). (c) In the hemiepipodite epithelium isolated from the spinylobster the SCC was used as a good alternative estimation of the active transport(negative charge flow driven from apical to basolateral side of preparation). The SCC ofhemiepipodite from SW and DSW acclimated spiny lobsters are very low and do not

22differ, ranged from 216.7 to 222.7 mA cm (cuticle1epithelium layer; Table 2)10–14 times less than in hemiepipodite isolated from the weakly hyperosmoregulatinglobster Homarus gammarus. Moreover, conductance of spiny lobster hemiepipodite

22(205–290 mS cm ; Table 2) was 3–4.5 fold higher than the Homarus hemiepipodite22(65 mS cm ; Lucu and Devescovi, 1999). From an electrical point of view (high

conductance), it can be deduced that the epipodite epithelium of the spiny lobsterbelongs to the group of leaky epithelia. These findings, therefore, would strongly suggest


that in epipodites of the spiny lobsters acclimated to DSW there are no substantialchanges in turnover rate or densities of sodium pump.

4.2. Gill Na,K-ATPase activity in Crustacea — adaptivness from osmoconformity toregulation

Upon acclimation of the spiny lobsters to dilute seawater, Na,K-ATPase specificactivity of native and saponin-treated trichobranchiate homogenates was not changedcompared to gill enzyme activity from the seawater acclimated animals (Fig. 2).

Table 3 depicts the relationship between Na,K-ATPase specific activity in gillhomogenates and the haemolymph osmoconcentration gradient (mOsmol / l;haemolymph-medium) during acclimation to the changed osmoconcentration of theiraquatic environment in the osmoconforming and regulating Crustacea. Because of thelack of evidence regarding Na,K-ATPase activity in other taxonomic groups of marineosmoconforming Crustacea upon modest dilution of the seawater, besides the presentstudy on spiny lobsters, we have examined gill Na,K-ATPase activity in the otherosmoconformers, i.e. marine stenohaline sponge crab Dromia personata and spider crabMaja crispata gradually acclimated for 2 weeks in SW and DSW (20 ppt salinity; Table3). Haemolymph osmolarity was like for the spiny lobster, only slightly below SW, andno changes in Na,K-ATPase in gill homogenates were found between SW- andDSW-acclimated groups (Table 3). The lack of osmoregulatory ability of thehaemolymph confines life of the osmoconformers to limited sea water osmodilution,preventing their migration into extremely dilute waters. We predict similar responses fora large group of stenohaline marine osmoconforming Crustacea not yet studied.

In the hyperosmoregulating Crustacea, Na,K-ATPase activity is increased duringacclimation to dilute seawater (more expressed in posterior than anterior gills), likelyleading to the generation of a hyperosmotic haemolymph osmoconcentration gradient inlow salinities (Table 3). Studies on hyperosmoregulators have shown that increased gillNa,K-ATPase activity is related to the increase in the haemolymph osmoconcentrationgradient (mOsmol / l, haemolymph-medium; Table 3). There are numerous data sug-gesting that the pattern of osmoconcentration is quite similar to the pattern of sodiumregulation for aquatic Crustacea. Analysis of the relationship between gill Na,K-ATPaseactivity (see Table 3) and the sodium gradient (mmol / l Na; haemolymph-medium) ofosmoconformers and regulators during a course of acclimation to different osmoconcen-trations of medium has shown correlation of these parameters (regression coefficient of0.65; P,0.01; Fig. 4).

When acclimated to SW crabs C. sapidus, C. similis, H. gammarus, C. maenas, M.olfersii, H. nudus and U. cordatus are slightly hyperosmotic; upon acclimation to diluteseawater they become hyperosmoregulators (Fig. 4). Most of the above mentionedCrustacea are moderate hyperosmoregulators (sodium gradient increases from 150 to 170mmol / l) and they tolerate dilute sea water down to 9 ppt. The weakly hyperosmo-regulating lobster H. gammarus tolerate salinities as low as 20 ppt and Na,K-ATPaseincreases when a sodium gradient of 59 mmol / l is established. Extremely goodhyperosmoregulators (Uca minax, U. pugnax) tolerate life from brines and seawater to aslow as 3 ppt seawater salinity, keeping the haemolymph osmoconcentration gradient


Table 3Gills Na,K-ATPase in aquatic Crustacea in relation to osmoconcentration differences between haemolymph

aand medium (h-m; mOsmol)

Medium Na,K-ATPase Gradient Remarks Referencessalinity (ppt) (mmol P /h per mg prot.) (h-m; mOsmol)i

ISOSMOTIC1Palinurus elephas SW→DSW

Trichobranchiate 38 2.1–2.9 38 14 days This study20 2.1–3.2 16

Epipodites 38 3.660.4 3820 5.361.3 16

2Maja crispata 38 2.161.7 221 SW→DSW This study20 1.260.2 3 14 days

3Dromia personata 38 1.561.0 214 SW→DSW This study20 0.960.5 5 14 days

HYPEROSMOTIC4Callinectes sapidus 30–35 12.960.6 42 SW→DSW Neufeld et al. 1980

24 17.4 150 2–3 weeks15 20.560.9 3606 24.11 562

5Callinectes similis 30 8.762.4 20 SW→DSW Piller et al., 199520 15.863.5 94 2 weeks10 29.862.4 235

6Carcinus maenas 30 7.062.4 50 SW→DSW Siebers et al., 198720 8.860.6 130 4 weeks10 12.461.4 220

7Homarus gammarus SW→DSW Lucu and Devescovi, 1999Trichobranchiate 38 1.4 to 1.8 226 10 days

20 3.1 to 3.8 154Epipodites 38 4.561.1 226

20 12.663.4 154

8Macrobrachium olfersii 28 7.9 40 FW→SW Lima et al., 199721 7.3 128 10 daysFreshwater 11.6 352

Eriocheir sinensis 34–36 1.460.3 FW→SW Pequeux and Gilles, 19842 weeks

Freshwater 4.560.536 2.060.1 FW→SW Bigalke, 1986Freshwater 2.060.1 Few weeks

9Hemigrapsus nudus 30 13.461.9 20 SW→DSW Corotto and Holliday, 199622.7 20.462.2 16115.1 26.461.9 3067.6 21.461.9 287

HYPER-HYPOSMOTIC10Uca minax 17–18 5.760.4 156 Few weeks Wanson et al., 1984

4–5 5.760.4 44411Ucides cordatus 26 7.261.7 3.4 SW→DSW Harris and Santos, 1993

9 9.661.2 336 Few weeks

12Uca pugnax 15.2 28.2 296 SW→DSW Holliday, 19853.0 40.0 622 3 weeks

a Remarks indicate direction and duration of acclimation to SW (seawater) and DSW (dilute seawatersalinities) (ppt) at which animals were acclimated. Indexes above species are related to Fig. 4.


Fig. 4. Summarized literature sources and present results on dependence of Na,K-ATPase specific activity ingill homogenates on the sodium haemolymph gradient (mmol Na/ l; haemolymph-medium) of regulating andosmoconforming Crustacea during acclimation to dilute seawater. The regression line fitted by the equationy 5 0.071x 1 6.90 (regression coefficient R50.65; P,0.01, n531). Individual specific activities of Na,K-ATPase indicated above full circles are related to appropriate specimens from Table 3 (indexes above

11descriptions of species). Additional data for sodium gradients for Ucides cordatus (Martelo and Zanders,6 51984); Carcinus maenas (Zanders, 1980) and Callinectes similis (Piller et al., 1995) and Callinectes

4sapidus (Colvocoresses et al., 1974) were used.

from 200 to 300 mmol / l above their aquatic environment. Representatives of euryhalineregulators exhibit maximum Na,K-ATPase activities in freshwater and live over acomplete range of salinities from seawater to the freshwater habitats (M. olfersii, C.sapidus). In the strong euryhaline osmoregulating Chinese crab Eriocheir sinensis,unmasking latent gill Na,K-ATPase activity with sodium dodecyl sulphate led to largeincreases which were not different between SW and DSW acclimated crabs (Bigalke,1986). In contrast to these results, Chinese crabs transferred from freshwater to seawater,a 3-fold decrease of Na,K-ATPase activity after 2 weeks acclimation was found


(Pequeux and Gilles, 1984; Table 3). Moreover, the gill epithelium of Eriocheir is 30times less permeable (Onken et al., 1991) when compared to the regulating Uca(Schwarz, 1990), providing an additional adaptive mechanism that makes these crabsfunctionally divergent. These controversies should be cleared up in the future andtherefore we have not introduced these results in Fig. 4. Thus sodium losses throughleaky permeable surfaces in dilute medium are replaced by active sodium pumpingacross branchial cavity cells into the haemolymph space (Lucu and Siebers, 1987). TheNa,K-ATPase located basolaterally (Towle and Kays, 1986) is responsible for activetransport of sodium and for generation of the sodium gradients that energize secondaryactive transporters important for maintaining other ion gradient in euryhaline hy-perosmotic Crustacea (Riestenpatt et al., 1996).

Besides Na,K-ATPase activation, one additional physiological adaptive peculiarity forthe invasion of the brackish water and freshwater habitats is a reduction of body surfacepermeabilities in order to restrict inward water flux and diffusive losses of electrolytes.The cuticle, which covers the gill and epipodite epithelium, has to be considered as aselective barrier for cation and anion movement and this selectivity is more expressed inhyperosmoregulating Crustacea such as Eriocheir and Carcinus and less than forosmoconforming Maja squinado and Nephrops norvegicus (Pequeux and Lignon, 1991).In the spiny lobster acclimated to DSW, the isolated gill cuticle conductance was 1961

22mS cm , about 2.6–4 times more than reported for cuticle of the euryhaline shore crab22Carcinus acclimated to dilute sea water (500–750 mS cm , Onken and Riestenpatt,

1998). There is little doubt that the branchial structures are more leaky in osmoconfor-mers than in osmoregulating euryhaline Crustacea. Accordingly,the conductances for gill

22epithelium (cuticle1epithelium layer) of osmoconforming P. elephas (289 mS cm ;22this study) are larger than for moderate hyperosmoregulator Carcinus (40 mS cm ;

22Onken and Siebers, 1992) and Uca (90 mS cm ; Schwarz, 1990) and much larger thanfor the tight branchial epithelium of the extremely euryhaline Chinese crab Eriocheir

22sinensis (3 mS cm ; Onken et al., 1991).One of the major steps in the evolution of homeostatic control in osmoregulating

Crustacea is the development of ionic and osmotic composition of the extracellular fluidsdifferent from seawater. In the most primitive osmotic conformers, the internalenvironment is almost totally dependent upon the external environment. Under theseconditions the Na,K-ATPase is not involved in anisosmotic regulation of thehaemolymph during acclimation in the narrow salinity ranges but rather plays a role inmaintaining intracellular homeostasis.

Interestingly, marine osmoconformers are groups with a geological history which canbe traced back to Cretaceous (Majidae) and Jurassic (Palinuridae, Dromioidea) and forthe weak hyperosmoregulator Homarus even to the Permian period. The history of thebrackish-water Crustacea is more recent (Moore, 1969), i.e. pliocene (Carcinus) andoligocene (Callinectes). Among different Na,K-ATPase isoforms of different speciesincluding vertebrates and invertebrates, the sequence homology is not below 70%,indicating a highly conservative molecule during a course of evolution (Vasilets andSchwarz, 1993). Hypoosmotic medium was perhaps a signal for acquisition of the newgenetic information which codes for new biochemical adaptation. Evolutionary adaptivetrends begin when Na,K-ATPase activation occurs, supplying ion regulation to maintain


an osmoconcentration gradient between haemolymph and aquatic environment, aprerequisite for invasion of Crustacea into brackish and freshwater habitats.

Acknowledgements

This research was supported by the Ministry of Science and Technology of theRepublic of Croatia. Thanks the Research School M&T, Wageningen, The Netherlandsfor financial support. We would like to thank David W. Towle for helpful comments and

´ ´to B. Jagio and I. Korenic for technical help. [SS]

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Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers