J. exp. Biol. 114, 477-491 (1985) 4 7 7

Printed in Great Britain © The Company of Biologists Limited 1985

ELECTRICAL-POTENTIAL DIFFERENCE AND SODIUM IONFLUXES ACROSS THE INTEGUMENT OF

COROPHIUM VOLUTATOR (CRUSTACEA; AMPHIPODA),A EURYHALINE HYPEROSMOTIC REGULATOR

BY PETER M. TAYLOR*

Department of Zoology, University of Leicester, University Road,Leicester, LEI 7RH

Accepted 1 August 1984

SUMMARY

1. Measurements of electrical-potential difference (TEP) and Na+

efflux, across the integument have been made for Corophium volutatoracclimated to 85 or 15%SW.

2. The TEP of acclimated animals in 85 or 15%SW is l-4mV or11-1 mV respectively (haemolymph negative). For acclimated animals,unidirectional Na+ efflux is 154-3 nmol mg"1 body weight h"1 in 85 %SW[efflux rate constant (k) = 0-70 h"1] and approximately 35 • 5 nmol mg~' h~'in 15%SW (k = 0-50h"1 in 10%SW).

3. The results indicate that Na+ and Cl~are passively distributed acrossthe ion-permeable (gill) integument of acclimated animals in 85 %SW, butthat active uptake of Cl~, and possibly Na+ also, occurs across the gills ofacclimated animals in 15%SW. The ion transport mechanisms appear toeffect electroneutral transfers across the gill integumental epithelium.

4. Corophium volutator gill integument has a high permeability to ions;permeability to Na+(PNa) is 7-5x 10~8ms~1, and the ratio PCI/PNB is 0-45,for animals acclimated to either salinity. The resistance of the gill epi-thelium of acclimated animals has been calculated to be 5'3xlO~3rim2 and2-0x l0" 2 am 2 in 85 and 15%SW respectively.

INTRODUCTION

The majority of euryhaline crustaceans are aniso-osmotic regulators over at leastpart of their salinity tolerance range. The strategies employed by these species tomaintain salt and water balance whilst retaining permeable areas of integument(principally that of the gills) for exchange of dissolved gases and certain metaboliteshave been well documented (Lockwood, 1977; Kirschner, 1979). Osmoregulatorsgenerally maintain a transintegumental concentration gradient of NaCl betweenextracellular fluid (haemolymph) and medium. A significant electrical-potentialdifference (p.d.) also is developed across the integument of many species (Croghan,

•Present address: Department of Zoology, Bedford College, University of London, Regent's Park,London, NW1 4NS.

Key words: Crustacean, epithelial potential, sodium flux.

478 P. M. T A Y L O R

Curra & Lockwood, 1965; Lockwood & Andrews, 1969; Smith, 1969a; Lucu,1977); this p.d. may be described in terms of the diffusional movements of Na+ andC P across the permeable surface of the gills (Croghan, 1976). Transintegumentalelectrochemical-potential gradients of Na+ and C P are maintained by utilizingactive ion transport mechanisms situated within the epithelium of the gill integu-ment (Kirschner, 1979).

Many studies on the nature of ion fluxes across the gill integument of osmo-regulating crustacean species have considered ion movements with regard to certainelectrical characteristics of the integumental epithelium (e.g. Croghan et al. 1965;Smith, 1969a,b), although few of these investigations relate to a euryhaline hyper-osmotic regulator. Corophium volutator (Pallas) is a euryhaline amphipod (salinitytolerance range 2%oto 50%o; McLusky, 1968) displaying considerable tolerance torapid changes of environmental salinity (Taylor, 1984). Corophium volutatorexhibits marked hyperosmotic regulation when acclimated to salinities below 20%o(60% seawater); at higher salinities haemolymph and medium tend increasinglytowards iso-osmoticity (McLusky, 1968). A burrowing species, it characteristicallyinhabits muddy estuaries. This study investigates the p.d. and Na+ fluxes acrossthe body surface of Corophium volutator, particularly in relation to the environ-mental salinity, in an attempt to outline the electrochemical nature of Na+ (and,indirectly, CP) movements across the (gill) integument.

MATERIALS AND METHODS

General methods

Corophium volutator was collected from a small North Sea estuary near Skegness(Lincolnshire). The species was identified after Ingle (1969).

Experiments were performed at 120±0-5°C. Animals were acclimated at thistemperature to dilutions of Skegness seawater (85 %SW or 15%SW; where100 %SW = 34-5%o salinity) for at least 7 days prior to the experiments. Intermoultanimals over 3 mm in body length (excluding ovigerous females) were usedthroughout. Pleopod ventilatory activity of all experimental animals was noted; ifthis became impaired the experiment was terminated and the results discarded.

Individual animals were weighed to ±0-05 nig on a DKP torsion microbalance.Mean experimental animal weight was 7'0mg.

Haemolymph samples (0-2-0-5/u.1) were collected using fine-tipped glass micro-pipettes; Na+ concentration of a sample was determined by atomic absorptionspectrophotometry (Techtron AA6) or C P concentration by the first method ofRamsay, Brown & Croghan (1955).

The ionic compositions of the major experimental media are shown in Table 1.The osmotic pressure (OP) of all experimental media was determined on 0-2 mlsamples using an Advanced 3W osmometer. Dilutions of artificial sea water (ASW)were used in experiments. In certain media major cations (Na+, K+, Ca2+, Mg2+)or anions (CP) were substituted by choline (Cho+, as ChoCl) or benzenesul-phonate (BeS~, as NaBeS), ions respectively. Amiloride (Merck, Sharp & DohmeLtd; donated by the Physiology Department of Leicester University) was used(0-lmmolP1) to inhibit Na+ transport (Kirschner, 1979). SCN~ (as NaSCN;

Amphipod Na+ fluxes and TEP 479

Table 1. Composition of experimental media

Ion

Na+

K +

Ca2+

Mg2+

Cho+

crSO4

2"HCO-T

ASW

460101053

53628-752-5

ChoASW

66101053

39453628-75

2-5

Ion concentration

BeSASW

460101053

14228-752-5

(nimol 1 ')

NaASW

460

13653628-752-5

AFW

20-252-50-5

3-02-50-25

FWB

0-252-50-5

102-50-25

BcS" 394

ASW, artificial sea water (after Pantin, 1948) (osmotic pressure = 1060 mosmol kg" ' ) .AFW, artificial fresh water (osmotic equivalent of 10 %AS\V; 12 mosmol kg" ' ) .FWB, freshwater buffer.

0-05mmoll ') was used as a Cl transport inhibitor (Kirschner, 1979). Allexperimental media were adjusted to pH7-6 as necessary.

Data are expressed a s ± l standard error of the mean. The number of experi-mental animals (N) is given in parenthesis. The difference between sample meanswas tested for significance using Students f-test, differences were consideredsignificant if P< 0-05.

Theoretical considerations

In this study it is assumed that the silver-staining integument of Corophiumvolutator (0-99mm2mg~' animal wet weight; Taylor, 1984), which is principallythat of the gills, represents the only low resistance pathway for ion movementsbetween haemolymph and external medium, and that the limiting barrier to suchmovements is the epithelium of this integument. Ion activity coefficients areassumed to be all equal to one another. The sign given to the p.d. across theintegumental epithelium (transepithelial potential; TEP) is that of the haemo-lymph with respect to the medium.

At equilibrium there is equality of the electrochemical potential of an ion species(j) in the two phases [denoted here; in (haemolymph), out (medium)] separated byan epithelium (treated here as a single membrane); the Nernst equilibrium poten-tial for that ion (Ej, in V) is

Ej=(RT/ZF)ln([j]o u l / [ j ] i n ) , (1)

where [j] = ion concentration in denoted phase (mmoll"'), R = gas constant(8-31 J mol~ K~ ), T = absolute temperature (K), Z = ion valency (includingsign), F = Faraday constant (96 500 C moP1).

The system of ion movements across and within the apical and basal membranesof an epithelium may be considered in terms of a theoretical model (Croghan, 1976;Schultz, 1979). In simple models all transepithelial ion movements (or at least allion movements contributing to the development of the TEP) are assumed to be

480 P. M. TAYLOR

passive and independent of the movement of other ions and of solvent water. Twosuch models are considered here (in terms of the major ion species Na+ and Cl~only) as alternative descriptions of ion movements across Corophium permeableintegument.

The Goldman (constant field) model

A TEP may be developed across an epithelium displaying differential perme-ability between Na+ and Cl~ (PNa, PCi - the epithelial permeability to Na+ or Cl~respectively). If the epithelium is regarded as a single membrane the TEP [adiffusion potential; E (in V)] may be related to the ion gradients across theepithelium, thus (in terms of PN8; after Hodgkin & Katz, 1949)

EMRT/F)ln[Na+],n +a[Cr]o u t

where a= ratio of Cl~ to Na+, permeability (Pci/PNa)- The model assumes thatthere is a constant electric field in a homogeneous epithelium significantly perme-able to Na+ and Cl~ only, and that P^a and Pci are constant.

The equivalent circuit model

Ions may be regarded as passing across a membrane through ion-selectivechannels, the current carried by the ion being proportional to the channel con-ductance and to the difference between the transmembrane p.d. and the ionequilibrium potential (Hodgkin & Horowicz, 1959). In a steady-state the sum of therespective ion channel currents in a membrane is zero, thus:

Eni = tNaENa + tCi Eci, (3)

where tNa, t^i = transport number of Na+ or Cl~ in the membrane (i.e. theproportion of total membrane conductance attributable to the Na+ or Cl~ channel;here it is assumed that tNa+tCi = 1), Em = transmembrane p.d. (V), ENa, E Q =equilibrium potential for the membrane of Na+ or Cl~ (V). tNa and tCi in the outermembrane of an integumental epithelium (tlNa(ci)) may be estimated from therelationship:

tUNa(ci) = AEmo/AE?3°(C1), (4)

where AEmo, AEN°(CI) = finite 'instantaneous1 changes in measured p.d., andcalculated Na+ (Cl~) equilibrium potential, respectively across the membrane.

A known change in E^li or E™" may be made experimentally by reducing theconcentration of that particular ion species in the external medium by substitutionwith a non-permeating ion (i.e. one having t° = 0) (Croghan et al. 1965; Smith,1969a). In this study Na+ was substituted by Cho+ and Cl" by BeS"; t' a or tciwas estimated, using equation 4, from the change in Corophium TEP (AE)following animal exposure to Cho ASW or BeS ASW respectively, assuming

Amphipod Na+ fluxes and TEP 481

AE = AEmo. It is assumed that intracellular ion concentrations remained constantover the experimental period. Transport numbers determined in this manner arebest termed 'apparent transport numbers' (Croghan et al. 1965).

Measurement of TEP

The TEP was measured using a pair of glass capillary microelectrodes. Themicroelectrodes were pulled from 0i65mm bore tubing to a tip diameter of10-20/urn, and were filled with 3 moll"1 KC1 by ethanol replacement (Tasaki,Polley & Orrego, 1954); tip resistance was approximately 106ft. The experimentalanimal, cemented to a sharpened match head using a drop of superglue (LoctiteLtd), was mounted ventral side uppermost in a small Perspex chamber. Mediumflowed through the chamber by gravity from one of several aerated reservoirscontaining different media. Introduction of a new medium into the chamber wascomplete within 30 s of connecting the chamber inflow to a different reservoir. Onemicroelectrode, mounted on a micromanipulator, was manoeuvred so that its tippierced the ventrolateral cuticle of the animal through an intersegmental mem-brane, and was positioned in the haemolymph. The tip of the second (reference)microelectrode was immersed in the medium surrounding the animal. Each of themicroelectrode pair was connected to a calomel (Hg/Hg2Cl2/saturated KC1) half-cell via a salt (3 moll"1 KC1) bridge. The p.d. between the half-cells was measuredusing an electrometer with a high input resistance (Vibron 33B-2, ElectronicInstruments Ltd; input resistance exceeds 1015 O) attached to a chart recorder. Theasymmetry (tip) potential between the two microelectrodes was measured for eachmedium used in an experiment, then subtracted from the respective p.d. to give theTEP. Microelectrodes having ion-selective tips were rejected. Results where theasymmetry potential exceeded 5-0mV were discarded. A brass plate in the base ofthe Perspex chamber was grounded; this held the microelectrode circuit at arelatively constant low potential relative to earth. The apparatus was grounded andenclosed by a Faraday cage.

The sequence of media used in a TEP measurement experiment followed one oftwo patterns.

(i) A progressive series of dilutions of ASW (100%, 50%, 20%, 10%ASW)plus artificial fresh water (AFW).

(ii) A sequence of media containing substitute ions (Cho+, BeS~) or trans-integumental ion transport inhibitors.

Generally a TEP was recorded over a 2-min period immediately after it hadstabilized following a change in medium. In all experiments TEP was initially andfinally measured in a reference medium (ASW for 85 % SW acclimated animals and10% ASW for 15% SW acclimated animals); initial and final values were alwayswithin 1 -0 mV of one another. Reference medium TEPs recorded over periods of upto 30min were stable (±0-2mV), indicating that there was no significant short-circuiting through the microelectrode penetration wound. Measurement accuracywas approximately ± 5 % of the recorded TEP.

482 P. M. TAYLOR

Measurement of Na+fluxes

Unidirectional sodium efflux (JNa; nmol Na+ mg~' animal wet weight h~') wasdetermined by washout of a Na-loaded animal into unlabelled experimentalmedium, the radioactivity remaining in the animal being recorded against time.

Na was obtained as NaCl in aqueous solution from the Radiochemical Centre,Amersham. Radioactivity was assayed using a well-crystal scintillation counter(Panax Instruments Ltd). Cylindrical plastic minivials (8 ml capacity) were used asexperimental chambers. All radioactive samples were counted in such vials, a singlevial exactly fitting the well of the counter. 22Na counting efficiency was 20'5%throughout.

Animals were 22Na-loaded for 24 h in 5 ml of aerated acclimation mediumcontaining the isotope at a specific activity of 3-7 Bq nmol"1 Na+. After this periodanimals were fully 22Na-loaded; i.e. animal specific activity was equal to that of themedium (Taylor, 1982).

Individual fully 22Na-loaded animals were rinsed in unlabelled medium andtransferred to a minivial. A 0-5ml sample of experimental medium was pipettedinto the chamber, and the animal counted (0-05 ml samples of 22Na-loading mediawere also counted). Further medium was added to give a total volume of 8 ml. Theexperimental medium was changed (by careful pipetting) at time intervals, at whichpoints the residual animal radioactivity was determined, the animal being countedin an initial 0-5 ml aliquot of the fresh medium. The interval between each mediumchange was timed from the addition of this aliquot. The experimental medium wasaerated periodically.

Two types of experiment were performed:(i) complete 22Na washout of an animal in a single type of medium (ASW,

85 %ASW or 10 %ASW);(ii) alternation of experimental media (at changes) between 85 %ASW/

10%ASW (85 %SW/15 %SW acclimated animals respectively) and either sucrose,or NaCl + sucrose, in FWB (the FWB media being iso-osmotic to animal haemo-lymph; see Table 4).

RESUL IS

TEP measurements

The TEPs of Corophium exposed to various media are summarized in Fig. 1 andTable 2. Here it is regarded that TEPs recorded under non-steady-state conditionsrepresent 'instantaneous' (formally steady-state) p.d.s; i.e. it is assumed that suchTEPs are recorded before any significant changes in Corophium body fluid com-position occur. The TEP of 15%SW acclimated animals in 10%ASW containingamiloride or SCN~ was identical to the TEP in 10%ASW (amiloride experiments,A ' = 8 ; SCN~ experiments, A '=6) . Similarly, the TEPs of 85 %SW acclimatedanimals in ASW, and ASW plus either inhibitor, were identical to one another(amiloride experiments, A'= 4; SCN~ experiments, AT=2). These TEPs ininhibitor-containing medium did not change significantly over experimental ex-posure periods of 15— 20min.

Amphipod Na+ fluxes and TEP 483

O-i

W - 1 2 -

- 1 6 -

- 2 0 -

(18)

• - >

i(6) .

y(3) . - " '

1(4)

20 40 60 80

Salinity (% ASW)

100

- 3 2 -J

(8)

20

, = 0-45

40 60

Salinity (% ASW)

100

Fig. 1. TEP of Corophium volutator in relation to environmental salinity (where 10%ASW =AFW): (A) 85 %SW acclimated animals; (B) 15 %S\V acclimated animals. A closed circle ( • )indicates the mean TEP [± 1 S.K.M. (A')] measured immediately following exposure to the denotedsalinity. An open circle (O) indicates the TEP in the acclimation salinity, estimated by inter-polation from adjacent experimental sample means. The solid bar indicates the point at whichhaemolymph and medium are iso-osmotic. The dotted line indicates the TEP-salinity relationpredicted by the Goldman model (equation 2) where 0 = 0 4 5 ; this value of a gives the line of bestfit (estimated by eye) of the model for animals acclimated to cither salinity.

[Na+];n and [Cl~];n of Corophium, together with the equilibrium potentials (E\ia,E Q ) across the integument, are shown in Table 3; the values are similar to thosereported by McLusky (1968). In 85 %SW ENia and Eci of acclimated animals areclose to the TEP, suggesting that both ion species are passively distributed (i.e. inequilibrium) across the integument. However, it appears that Cl~, and possiblyNa+, are out of electrochemical equilibrium across the integument of animalsacclimated to 15%SW.

Tab

le 2

. C

hang

es in

Tb'

P o

f 85

%SW

acc

lim

ated

C

orop

hium

vol

utat

or/o

//oT

Om

jr exp

osur

e to

a r

ange

of

expe

rim

enta

l m

edia

Exp

erim

enta

l m

ediu

mC

hang

e in

TE

P (m

V),

rel

ativ

e to

TE

P in

ASW

Mea

n T

EP

(mV

) in

ASW

cfer

ence

med

ium

; se

e F

ig.

1A

Chu

AS

WB

cSA

SW

Ion

tran

spor

tnu

mbe

r*

-9-4

+ 0

-7(1

3)+

2-6

±0-3

5(8

)-3

5±

O-4

(8)

All

TE

P ch

ange

s si

gnif

ican

tly d

iffe

rent

fr

om z

ero

(pai

red

sam

ple

/-te

st).

* E

stim

ated

fro

m e

quat

ion

4.

For

abb

revi

atio

ns s

ee T

able

I.

Tab

le 3

. Io

n eq

uili

briu

m p

oten

tial

s (E

~) a

cros

s th

e in

tegu

men

t o

f Cor

ophi

um v

olut

ator

acc

lim

ated

to

85

%SW

or

15 %

SW

Acc

limat

ion

sali

nity

(%S

W)

85 15

[Na+

] (m

mo'

[Na+

],,,

435

±1

4 (1

5)17

8 ±

15

(11)

IP')

[Na+

]

393 67

EN

. (m

V)t

-2-5

-24

-0

[crj

[cr]

in

496

± 19

(6)

169

±2

2 (6

)(mm

ol r

1)

[cr]

,,,,,

458 79

Ec

l(m

V)t

+ 2

-0+

18-

7

TE

P (m

V)

-1-4

-11

1

[Na+

] in,

[Na+

] ou

l, co

ncen

trat

ion

of N

a+ in

hae

mol

ymph

and

med

ium

res

pect

ivel

y; [

Cl

] in,

[Cl

]„„,

, sa

me

for

Cl

*T

EP

esti

mat

ed f

rom

F

ig.l

.f

Ez

calc

ulat

ed f

rom

equ

atio

n 1.

H

Amphipod Na+ fluxes and TEP 485In Corophium the TEP becomes increasingly negative as external ion concen-

trations (i.e. environmental salinity) are progressively lowered. The Goldmanmodel (equation 2) approximates most closely to this relationship between TEP andexternal salinity, in both 85 %SW and 15 %SW acclimated animals, when cv= 0-45(Fig. 1); [Na+]i,n and.[CF] i n are assumed to remain constant at the levels ofacclimated animals (Table 3).

The magnitude and sign of TEP changes following ion substitutions weredependent on the ion species substituted. The transport numbers t' a and t'ci wereestimated for 85 %SW acclimated Corophium (Table 2). The change in TEPfollowing animal exposure to NaASW was small compared to the TEP changefollowing exposure to ChoASVV, indicating that the transport numbers t'k, t'ca andtMg are considerably lower than t%.d. The largest values of ion transport numbers inthe outer membrane of Corophium integumental epithelium appear to be t^j, andtci, although it is conceivable that a significant proportion of the membraneconductance is attributable to ion channels selective for H + , HCO3~ or OH~.These 'apparent' transport numbers may underestimate considerably actual values,as 2tj (here, tNa+tci: = 028) should equal unity. This large discrepancy might beascribed to limitations of the experimental system; it is possible that intracellularion concentrations changed significantly during the experiments (tj is strictly afunction of [j] in the membrane), and also that the substitute ions were not entirelynon-permeating or inert (Smith, 1969a).

Unidirectional Na+ efflux (JXa)

Measurement of Na washouts of Corophium in a single medium type showedthat the decrease in animal radioactivity was described by a single exponentialfunction;

Q, = Qoe-k t , (5)

where Qo = initial animal radioactivity (counts min~'; c.p.m.), Q, = animal radio-activity at time t (c.p.m.), t = time (h), k = efflux rate constant (h~'). The rateconstant (k) was estimated as the slope of a plot of ln(Qt/Qo) against time. Typicalplots of this type are shown in Fig. 2. In experiments where the medium com-position was changed at intervals, k in each medium was estimated from equation 5with Qo and Qt representing animal radioactivity at the commencement and theend, respectively, of exposure to each experimental medium.

The magnitude of k was dependent on animal wet weight (W; in mg); kdecreased with increase in animal weight. Therefore, rate constants were weight-corrected to those of a 70-mg animal (k70) according to the relation (after Taylor,1982):

k7.0 = 0-61 k/exp(-0-641n(W) +0-75). (6)

The weight-corrected Na+ efflux rate constants for Corophium exposed to variousmedia are summarized in Table 4.

Tab

le 4

. 22 N

a ef

flux

rat

e co

nsta

nts

(k)

of C

orop

hium

vol

utat

or i

n ex

peri

men

tal

med

ia

Acc

lim

atio

nsa

lini

ty

85 IS

* FW

B m

edia

n

ASW

0-76

+ 0

-09

(16)

0-88

±0

-08

(3)

Rat

e co

nsta

nt (

h ')

85 %

ASW

10

%A

SW

0-70

±0-

07

0-89

±0-

12(1

2)

(4)

0-50

±0

03

(12)

Iso-

osm

otic

suc

rose

*

0-36

±0

-03

(16)

0-34

±0

-05

(15)

Cha

nge

in r

ate

cons

tant

re

lati

veto

tha

t in

iso

-osm

otic

suc

rose

(h

~')

150

mm

ol 1

"'

NaC

I+su

cros

e*

+ 0

10

± 00

35f

[+0-

10]t

(6)

+ 0

-35±

0-07

t [+

0-26

]t(6

)

iade

iso

-osm

otic

to

hacm

olym

ph;

85 %

SW

acc

lim

ated

ani

mal

s -

910

mos

mol

kg

~',

15 %

SW

acc

lim

ated

ani

mal

s -

340

mos

mol

kg~

'

y M. TAYL 0 JO(M

cLus

ky,

1968

).t

Sign

ific

ant

chan

ge i

n k

(pai

red-

sam

ple

(-te

st).

| V

alue

s in

par

enth

eses

rep

rese

nt c

hang

esii

n k

pred

icte

d by

Gol

dman

mod

el (

equa

tion

7);

pre

dict

ed c

hang

es a

rc n

ot s

igni

fica

ntly

dif

fere

nt f

rom

obs

erve

d ch

ange

s.A

ll ra

te c

onst

ants

arc

wei

ght-

corr

ecte

d to

tho

se o

f a

7-0-

mg

anim

al (

equa

tion

6).

Amphipod Na+ fluxes and TEP 487

Time (h)

Fig. 2. Semi-logarithmic plots of decreasing Cotvphium volutator 22Na radioactivity against time.Each plot relates to a single animal, where Qo is initial animal radioactivity (c.p.m.) and Q, isanimal radioactivity at the denoted time (c.p.m.). Open circles (O); 85 %SW acclimated animal,efflux into ASW. Closed circles ( • ) ; 15 %SW acclimated animal, efflux into 10 %AS\V.

The rate constant k has two major components, transintegumental Na+ effluxand Na+ loss in the urine. In iso-osmotic, and hyperosmotic, media (in Table 4, allexcept 10%ASW), urine production rate of Corophium (and hence urinary Na +

loss) is likely to be comparatively low. In these media k may be regarded asrepresenting mainly transintegumental Na+ efflux (kim). The kiiu of 85 %SW and15 %SW acclimated animals increases significantly when [Na+]()Ut rises; suchchanges in unidirectional Na+ flux across the integument may be related toconcomitant changes in TEP (House, 1963; Smith, 196%). If Na+ movement isdiffusional, the ratio of kint in two different media [denoted (i) and (ii)] may bedescribed as a function of the respective TEPs using the Goldman model (hereassuming [Na+],n is constant)(after House, 1963).

= D(i) (7)

where (for each medium) D = (FE/RT)/[1—exp( —FE/RT)]. Here, medium (ii)may be identified as a sucrose solution iso-osmotic to the haemolymph, and medium(i) as 150 mmolF1 NaCl with sucrose to the same osmolarity (see Table 4). Theexpected TEP in these two media may be calculated for 85 %SW and 15%SW

488 P. M. TAYLOR

acclimated animals from equation 2 (a= 0'45), assuming sucrose has no indepen-dent effect on the TEP. Values of k(i)—k(ii), predicted from equation 7 usingexperimentally-determined k(ii), are shown in Table 4; these are not significantlydifferent from the observed changes in k (kint). Values of kint in ASW may bepredicted similarly if medium (i) is reidentified as ASW; predicted kint in ASW is0-58 h"1 and 0-68 h~' for 85%SW and 15%SW acclimated animals respectively(neither is significantly different from observed k, Table 4).

Total body sodium ion content of individual Corophium (B ; nmolNa+mg~1

animal wet weight) was estimated from the relation:

BNa = Qo/(WXn^a), (8)

where Qo = initial animal radioactivity (i.e. that of a fully 22Na-loaded animal)(c.p.m.), Xm = specific activity of loading medium (c.p.m. nmoP1 Na+). B a is220-5 ± 7-9 nmolmg"1 for 85 %SW acclimated animals (Ar=14) and 71-0±2i2nmolmg~1 for 15%SW acclimated animals (./V=ll). The values are signifi-cantly different from one another, and reflect the difference between [Na+]in of theacclimatory types (Table 3). JNa may be determined as:

Thus steady-state JNa may be estimated for Corophium; for 85 %SW acclimatedanimals JNa (in 85 %ASW) is 154-3 nmolmg"1 h~ \ and for 15 %SW acclimatedanimals JNa (in 10%ASW) is 35-5 nmolmg"1 h"1.

PNa may be estimated, using the Goldman model, from the following relationship(House, 1963):

P T _ J , N n a [ l -ex P (-FE/RT)]Na Ap(FE/RT)[Na+] , n

where J ," = diffusional transintegumental flux (molmg"'s" ' ) , Ap = surfacearea of permeable integument (9-9X 10~7m2mg~'), ([Na+]i n; molm~3). Identi-fying J- j1 as Na+ efflux into iso-osmotic sucrose, PN3 may be estimated as7-5xlO"8ms~' for Corophium acclimated to either 85 %SW or 15%SW. P a / P N a

appears to be approximately 045 for both acclimatory types, thus PCi may beestimated as 3-4X 10~8ms~'. Although k appears to be weight-dependent, there isevidence to suggest that Ap is not (Taylor, 1984); thus PN3 may be higher in smallerindividuals than larger ones.

DISCUSSION

The Goldman model provides a reasonable description of experimentally inducedchanges in TEP of Corophium volutator, suggesting that it may be a simplediffusion potential generated principally by independent passive Na+ and Cl~movements across a gill integument of constant PN3 and Pci- [Na ]out-dependentchanges of Na+kim appear to reflect changes in the nature of diffusional Na+

Amphipod Na+ fluxes and TEP 489

movements across the integument, with no significant residual flux componentswhich might be ascribed to an 'exchange-diffusion' (Ussing, 1952) Na+ transfermechanism. Electrochemical-potential differences of Na+ and Cl~ across Coro-phium volutator integument (apparent here in the case of 15%SW acclimatedanimals, where such differences favour passive ion loss from the haemolymph,across the integument) are likely to be maintained by ion transport (uptake)mechanisms situated within the integumental epithelium, as is the case for otherhyperosmotically-regulating crustacean species (Kirschner, 1979). Externally-applied ion transport inhibitors have no significant effect on the TEP of animalsacclimated to either 85 %SW or 15 %SW. This supports the suggestion that theTEP is a diffusion potential with any Na+ and Cl~ transport mechanisms (at leastthose located on the outer epithelial membrane) effecting overall electroneutral iontransfers.

G (Sm~2), the ion partial conductance of the integumental epithelium, may beestimated using the Goldman model from the relation (for ion j) (Dawson,Croghan, Atwater & Rojas, 1983):

r _ PjCZF{([j]out - [j]in)Cexp[C] + ([j]out - [j]inexp[C])(-l - exp[C])}G ) " E( l -exp[C]) 2 ( H )

where C = ZFE/RT ([j]; molm"3).For acclimated Corophium volutator; in 85 %SW, GNa is 123 Sm~2 and GGI is

64Sm~2, and in 15%SW GNa is 33 S m~2 and G a is 17Sm~2. Epithelial resist-ance of acclimated animals [as l/(GNa+Gci); probably a minimum estimate] is5-3xl(T3nm2 and 2-0xl(T2nm2 in 85%SW and 15%SW respectively. This is ofthe same order of magnitude as resistance reported for certain other epithelialtissues, which are regarded as 'leaky' (as opposed to 'tight') epithelia; i.e. they are ofcomparatively low resistance (Diamond, 1962; Smith, 1969a; Fromter, 1972).Smith (1969a) suggested that in such situations, where transintegumental ion fluxesare comparatively rapid, 'apparent t0' should be regarded as a gross estimate oftransport number for the whole epithelium (te). G may be formally related to te,where for any ion te = G/(SGj); thus tci/tNa should equal GCi/GNa. In fact for85 %SW acclimated Corophium volutator tci/t^a ('n ASW) is 0-4, a value notdissimilar to the respective Gci/GNa (in 85 %SW) of 0-52 calculated using theGoldman model. The limiting junctional complexes of the epithelial cells, asopposed to the cell membranes, may be the major site of diffusional ion movementsacross low-resistance epithelia (Fromter, 1972). These junctional complexes mayconstitute a single barrier for ions to cross, which might tentatively be considered assupporting evidence for the application of the Goldman model to describe passiveion movements across Corophium volutator integument.

Corophium volutator kint is markedly higher than comparable values for othereuryhaline amphipod species (these are of the order 0-04-0-12 h"1: Sutcliffe, 1968,1975), suggesting that PNa of this species is unusually high. PNa of Corophiumvolutator is higher than Pa, as appears to be the case for crustacean integumentalepithelia in general (Croghan et al. 1965; Smith, 19696; Lucu, 1977).

490 P. M. TAYLOR

There is no evidence in this study to suggest that Corvphium volutator is able toalter its integumental permeability to Na+ or Cl~ after acclimation, or immediatelyfollowing exposure, to different salinities. However, JNa does alter followingacclimation as a function of changes of [Na+]jn (i.e. BNa). These changes would belikely to result in an adaptive reduction of any passive integumental (and urinary)Na+ loss during periods of hyperosmotic regulation at low environmental salinity.Corophium volutator exhibits only a limited capacity to reabsorb ions from primaryurine (McLusky, 1968), and also appears to have a highly water-permeable integu-ment (Taylor, 1982). Hence, a significant proportion of JNa when haemolymph ishyperosmotic to the medium (e.g. animals in 10 %ASW) might be apportioned tourinary Na+ loss.

This investigation was undertaken under the tenure of Science Research CouncilStudentship No. 78307501. I am indebted to Dr R. R. Harris for his supervision ofthe study and for helpful criticism of the manuscript. I thank Mr M. B. Andrewsfor technical assistance and a referee for directing me to the general Pj—Gjrelationship.

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