On the anion-cation mobility ratio in the body wall of the shore crab, Carcinus maenas

Netherlands Journal of Sea Research 11 (2) : 200-207 (1977)

ON THE ANION-CATION MOBILITY RATIO IN THE BODY WALL OF THE SHORE CRAB, CARCINUS MAENAS by D. H. SPAARGAREN (Netherlands Institute for Sea Research, TexeI, The Netherlands)

CONTENTS I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Material and Methods . . . . . . . . . . . . . . . . . . . . . Results and Interpretation . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

200 201 202 207 207

I. I N T R O D U C T I O N Differences exist in the chemical composition of dissolved substances across the body wall of all aquatic animals. The differences maintained are related to cellular metabolic processes which can only function within narrow Iimits of the intracellular ionic composition and which in turn require a certain composition of the body fluids bathing the cells. Physical processes, diffusion and osmosis, tend to equalize the differences between internal and external ionic composition. The fact that these concentration differences are maintained in living organisms must be ascribed to active transport of ions against a concentration gradient at the expense of metabolic energy. One of the problems of active transport concerns the question whether all ionic species are transported actively by ion specific transport systems. If for instance only cations are transported it is possible that electric potentials occur which force the anions to follow passively down the electrochemical gradient. The amount of energy needed to maintain a concentration gradient across the body wall will depend on the overall permeability of the interface (PoTTS, 1954; SPAARGAREN,1975a, 1975b). If only the cations are transported actively it would be of energetic advantage if the cation mobility in the body wall (especially the gills) could be suppressed; when the concurrent anion transport proceeded passively, a change in the anion mobility would have no energetic advantage. A simultaneous reduction of the anion mobility may inhibit the cation transport by formation of an anion gradient. In this paper attempts are made, by measuring the electrical poten-

ION

MOBILITY

BODY

WALL

CARCINUS

201

tials over the body wall, to find out whether the anion-cation mobility ratio in the body wall of an aquatic animal is affected in the process of maintaining a concentration gradient. Acknowledgements.--I am much indebted to Dr R. E. Weber, Zoophysiological Laboratory, University of Aarhus, Denmark, for giving valuable comments. Thanks are also due to M r A. Nienkemper for technical assistance. II. MATERIAL AND METHODS Intact shore crabs, Carcinus maenas (L.), showing strong hyper-regulation of the blood concentrations in brackish water and osmoconformity at normal and supranormal salinities (TnEF.D~,, 1969), were chosen as experimental objects, The choice of intact animals, instead of isolated gills, has the advantage that the preparation can be used for a considerable time--usually several days--while no precautions are necessary for a glucose and oxygen supply to the tissues. This also avoids the danger of artefacts introduced by unnatural conditions. A (marine) crustacean species has the additional advantages of (1) a large, open circulatory system in direct contact with the tissues and the body wall, (2) a simple, monocellular gill epithelium and (3) a rigid exoskeleton in which electrodes can easily be mounted. Adult animals with fresh weights ranging between 20 and 40 grammes, were collected in the spring of 1976 near the island of Texel. In the laboratory the animals were kept in aquaria containing water of various salinities ranging from 5~oo to 40~oo S (obtained either by dilution of normal sea water or by evaporation through aeration). The aquaria were provided with a sand bottom and a filter for aeration and purification and placed in a constant temperature room of 20 ° C. Values for the anion-cation mobility ratio were derived from measurements of the electrical potential between an internal, hook-shaped, Ag-AgC1 electrode (lenth ca 10 m m ; diameter 1 mm) and an earthed reference electrode in the medium (either an Ag-AgC1 electrode, length 10 era, diameter 1 m m or a calomel electrode). For implantation of an internal electrode a small hole was drilled in the centre of the carapax, in front of the heart region, which was closed off with black wax ("Pyeseal") after insertion of the electrode. Electric potential differences were recorded directly on a potentiometric millivolt recorder (Kipp, type BD 9); because the circuit resistance is low (about 2 k~) a voltmeter with a high input impedance is not necessary.

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D.H. SPAAROAREN

III. R E S U L T S AND I N T E R P R E T A T I O N Between an internal Ag-AgC1 electrode and an Ag-AgC1 reference electrode in the medium stable potentials were found that are negative with respect to the earthed reference electrode. Generally values ranged between zero and --0.6 Volts. Fig. 1 gives an example of a Vm,mV .60-

] ]

I

I

J •40 -

I I I

I

f

-20 -

131.9

0 "J

~¢

44.4

22.4

12.2

34,9 %o

Fig. 1. Long-term registration of the potential difference between an Ag-AgC1 electrode in the blood of Cardnus ~ s and an Ag-AgC1 electrode in the medium. The salinity of the sea water medium was changed as indicated. Temperature 18° C.

long-term registration during which the salinity of the medium was frequently altered. Alteration of the salinity results in a sudden change of the potential measured followed by a gradual change in the opposite direction. The potential differences are strongly dependent on the internal and external ion concentration (and also of the properties of the electrodes used--damage to the AgC1 film covering the surface may cause a high bias potential). When the reference Ag-AgC1 electrode is placed directly in the medium, the measured potential, Vra, is given by the sum of 3 terms, an electrical (bias) potential, E0, the diffusion potential, and the concentration potential (e.g. GLASSTONE & L~WIS, 1965) :

ION MOBILITY

u--v Vm =

Eo +

- -

u+ v 2u

=Eo+--

BODY WALL

RT - -

ae In

RT +

nF

a¢

RT

ae

nF

ae In - -

nF

--ln-u+ v

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CARCINUS

a~

(1) a~

where I'm and E0 are potential differences in Volts, u and v represent respectively the cation and the anion mobility in the body wall in m2.V-X.sec -1, R is the gas constant of 8.314Joule.°K-l.mole -1, n is the valency of the ions, F is the Farady constant of 96497 Coulomb and ae and a~ are the ion activities in medium and blood respectively. When the reference Ag-AgCI electrode is placed in a solution having the same ion composition as the blood (a'e = a~), and this solution is connected with the medium via a KCl-bridge, then the concentration potential is eliminated. As K+ and C1- have approximately the same ion mobility the diffusion potential across the KC1 bridge is zero. Hence: u--v

V,~ = Eo +

RT

ae

--Inu+ v

nF

(2) a~

In experiments in which both electrodes were placed in two plexiglass chambers containing different sea water dilutions and connected with each other via a semipermeable collodion membrane, it was found that the voltage measured could be predicted with the above equations using the specific conductivities (re, K~) of the solutions as a measure of the ion activities (ao a~) in both compartments. The specific conductivity of the blood (K~) of Carcinus maenas at 20 ° C at various medium salinities is known (Fig. 2). At 20 ° C the term R T / n F reaches a value of 25.2 10 -a Volts. Hence, u and v are the only unknown values in equations (1) and (2). Fig. 3 shows examples of the potential differences measured over the body wall in 2 specimens of Cardnus maenas following rapid changes in salinity using a reference electrode in the medium (Fig. 3a), as well as a reference electrode in sea water approximately isotonic to the blood and connected with the medium via a KCl-bridge (Fig. 3b). Both animals were acclimated to roughly the same salinity of about 28%0 S. As it takes several hours for the blood to become adjusted to a changed salinity (see also Fig. 1) it is assumed that during the rapid experimental salinity changes (within 5 minutes) the blood ion con-

204

D. H. S P A A R G A R E N

contration remained constant. Plotting the measured potentials against In Ke/K, yields straight lines. In "direct" measurements (Fig. 3a) the slope of the lines has values of 0.480 and 1.08, corresponding with anion-cation mobility ratios of Ki 20 m ~ -t crn- t o

oO//

50

40,

30-

o/U °

20-~

,;

2'0

;o

.,'o

°,,o°S

Fig. 2. Specific conductivity (K~) of the blood ofCarcinus maenas acclimated to various

salinities. Temperature 20 ° C (data from ENGELSMA,1973). 3.17 and 0.85, respectively, and indicating large differences in this ratio. The "indirect" measurements (Fig. 3b), taken simultaneously with the direct measurements, yield slopes of --0.396 and +0.125, corresponding with anion-cation mobility ratios of 2.31 and 0.78. These ratios are both lower than those found in the direct measurements but confirm the existence of large differences. As Na+ and C1- represent both in the blood and in the medium the dominant ions, the diffusion potential wilt be mainly determined by the mobility of these ions, cf. equation (2). In free aqueous solutions the mobility o f N a + and C1- are 4.5 × 10 -s and 6.8 × 10 -s m~.V-l.sec -1, respectively, representing a ratio of 1.51. Plotting potential against In (Ke/~,) should yield in direct measurements (Fig. 3a) a slope of 2 u/(u+v) = 0.796; and in indirect measurements (Fig. 3b) a slope of (u--v) / (u+v) = --0.20. It can be seen that the changes in potential measured at various salinities clearly deviate from the changes which

ION MOBILITY

BODY

WALL

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CARCINUS

should be f o u n d w h e n the m o b i l i t y o f N a + a n d C1- in the b o d y wall w a s the s a m e as in free a q u e o u s solution. Similar experiments as illustrated in Fig. 3 were carried o u t in 20 different specimens a c c l i m a t e d to various salinities b e t w e e n 5 a n d Vm

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Vm

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-120,

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-BO-

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a/t

.

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o"

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a

of /

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o ~o,.~o~..~..~-o~8

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/d

..'''~ ag~ ~,.,"

-680

sI

mV

oo~O -o~ "~" G

o

o

a oQ...

.eeo,

8 ..o

.

/a o

-700

-'tO0

-~o

-4'o

-3'o

-2'o

-,~

b

k,

-~'o

-go

-3'o

-~'o

-,~

~

,~

I -3 RT In ~

0 ~

Iq

Fig. 3. a. Potential differences between an Ag-AgC1 electrode in the blood o f Cardnus maenas and an external Ag-AgCI electrode in a sea water medium that changes rapidly (within 5 rain) in salinity; the measured potentials are plotted against In re/K, (re and r~ representing the specific conductivity of medium and blood, assuming that r, remains constant during the rapid salinity changes). Measurements in one specimen, fresh weight 32.4 g, acclimated to 29.4~o S, r~ = 39.3 mfl-l'cm -t, by one internal electrode (A), and in another specimen, fresh weight 30.1 g, acclimated to 26.1~oo S, 1¢, = 37.7 m~-Z.cm -1, by 2 internal electrodes mounted (O and D), together with expected relations for free NaCI movement through an aqueous boundary layer (dashed lines). Temperature 20 ° C. b. The same using an external Ag-AgC1 electrode in a sea water dilution isotonic to the blood and connected with the medium via a KCl-agar bridge. 45~oo S (Fig. 4). T h e values for the a n i o n - c a t i o n m o b i l i t y ratio a p p e a r e d to be highly variable, the o b t a i n e d values ranging b e t w e e n 0.57 a n d 3.17 ( m e a n 1.31; s.d. 0.60; n ---- 20), indicating that t h e ratio in the crab c a n vary b e t w e e n m o r e t h a n h a l f as l o w as to a b o u t t w i c e as high as the v a l u e to be e x p e c t e d for free m o v e m e n t o f N a + a n d C1- in an a q u e o u s solution. G e n e r a l l y the m o b i l i t y ratio is a b o u t the s a m e or

206

D. H. S P A A R G A R E N

lower than the expected value. A lower ratio may result from either an increase in cation mobility or a decrease in anion mobility. The latter possibility is most likely explained by structural properties of the body wall inhibiting free anion movement. Lowered values are only found at ~¢,values between about 28 and 38 mf~-l.cm -1 that is to say at blood concentrations that are strongly regulated (see Fig. 2). No correlation could be found between anion-cation mobility ratio and animal weight.

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Fig. 4. Anion-cation mobility ratios in the body wall ofCarcinus maenasplotted against the conductivity of their blood; determinations in animals acclimated to various salinities. Mobility ratio of Na + and C1- in free aqueous solution indicated (dashed line).

As at isotonic salinities (~, = ~ ) the diffusion potential across the body wall will be zero, values for the bias potential, E0, can be derived. Highly variable (see also the scales of the upper and lower diagrams in Fig. 3), but negative values were always found for Eo. Using two electrodes in the same animal shows that properties of the electrodes (probably of the AgC1 surface layer) influence the Eo value as is evident from the difference in lines O and [] in Fig. 3. The disturbing influence of electrode properties cannot be avoided when using internally implanted electrodes which do not allow proper zero calibration. However, under all circumstances--even at low, hypotonic salinities when the diffusion potential should be expected to be higher (positive) - - t h e blood was always found to be negative compared to the medium. This suggests that anions are transported against an electrical as well

ION MOBILITY

BODY WALL CARGINUS

207

as a chemical concentration gradient. A similar active transport of anions at hypotonic conditions is, among crustaceans, also established in the shrimps Crangon crangon (GRIMM, 1969) and Palaemonetes varians (PoTTS & PARnY, 1964). The results lead to the following interpretation: Carcinus maenas, having internal concentrations usually hypertonic or isotonic to the medium, performs an active transport of anions from the medium against an electrochemical gradient towards its blood; at the same time the animal reduces the relative mobility of these actively transported anions at their passage across the body wall. The reduction of the mobility in the body wall of ions that are actively transported, saves energy by the maintenance of a high internal anion concentration in the often hypotonic medium. Although the cations may follow passively, as a result of the electrical gradient formed, the results do not exclude the possibility that part of the cation transport also proceeds actively. IV. SUMMARY From measurements of the differences in electrical potential across the body wall of Carcinus maenas it could be deduced that the anion-cation mobility in the interfaces between blood and medium is often reduced compared to the ratio expected for free ion movement. Blood potential was always found to be negative compared to the medium. The findings are interpreted as resulting from an active transport of anions; a concurrent reduction of the anion mobility in the body wall inhibits the passive loss of these actively transported ions. V. REFERENCES F. J., 1973. Osmoregulatie van de strandkrab (Cardnus maenas (L.)). Intern Verslag Nederlands Instituut voor Onderzoek der Zee, Texel 1973-8: 1-37 (mimeo). GLASSTONE,S. & D. LEWXS,1965. Elements of physical chemistry. Macmillan, London: 1-758. GRrMM,A. S., 1969. Osmotic and ionic regulation in the shrimps Crangon vulgaris Fabricius and Crangon allmanni Kinahan. University of Glasgow: 1-88 (thesis). POTTS, W. T. W., 1954. The energetics of osmoregulation in brackish- and freshwater animals.--J, exp. Biol. 31-"618-630. POTWS,W. T. W. & G. PARRY, 1964. Sodium and chloride balance in the prawn, Palaemonetes varians.--J, exp. Biol. 41 ~591-601. SPAAROAREN,D. H., 1975a. Energy relations in the ion regulation in three crustacean species.--Comp. Bioehem. Physiol. 51A: 543-548. ----, 1975b. Heat production of the shore-crab Carcinus maenas (L.) and its relation to osmotic stress. In: H. BARNES.Proc. 9th Europ. mar. biol. Symp., Aberdeen University Press: 475-482. Tn~EDE, H., 1969. Osmoregulation yon Cardnus maenas.--Mar. Biol. 2 (2) z 114-119. ENGELSMA,