J. Exp. Biol. (1971), 54. 355-*&8 2 5 5With 4 text-figura

Printed in Great Britain

SODIUM INFLUX AND LOSS INFRESHWATER AND BRACKISH-WATER POPULATIONS

OF THE AMPHIPOD GAMMARUS DUEBENI LILLJEBORG

BY D. W. SUTCLIFFE

Freshwater Biological Association, The Ferry House, Far Sawrey,Ambleside, Westmorland

(Received 10 August 1970)

INTRODUCTION

Gammarus duebeni 19 commonly found in brackish-water habitats round the coastsof north-west Europe. In western Britain it also occupies fresh water as a series ofmore or less isolated populations living on certain peninsulas and islands where thesodium chloride content of the water is relatively high compared with inland waters(Hynes, 1954; Sutcliffe, 1967 a). In addition G. duebeni is widespread in the inlandfresh waters of Ireland, at sodium concentrations down to about 0-25 mM/1 (Reid,1939; Sutcliffe, 1967a). Following the earlier work of Reid (1939), Beadle & Cragg(1940a, b) and Hynes (1954) on differences in the osmoregulatory abilities of G. duebenifrom brackish and fresh water, several features of sodium regulation in populationsfrom various habitats received more detailed attention (Shaw & Sutcliffe, 1961;Sutcliffe, 1967&, 1968; Sutcliffe & Shaw, 1968). Lockwood (1961, 1964, 1965), Lock-wood & Andrews (1969) has also described aspects of osmoregulation in G. duebenifrom brackish-water habitats.

On the basis of characteristic differences in the sodium influx, and the ability toreduce sodium losses in the urine, Sutcliffe & Shaw (1968) suggested that G. duebeniliving in fresh water in Ireland may represent a distinct physiological race geographic-ally isolated from other populations living in fresh water in western Britain. Thispaper is a further contribution to our understanding of the physiological differencesbetween populations of G. duebeni. It is restricted to an examination of the affinity forsodium in the uptake mechanism at the body surface and the ability to produce adilute urine. The importance of these two features in the maintenance of sodiumbalance at the low sodium concentrations encountered in fresh water was emphasizedin the previous work.

It may be noted that it is difficult to establish whether variations in the osmo-regulatory ability of a species are phenotypic or whether they reside in the genotype.The classic example of racial differences in osmoregulatory ability is the stickleback,Gasterosteus aculeatus where physiological differences between the two forms leiurusand trachurus are closely associated with a number of morphological and behaviouraldifferences shown to be genotypic in origin (Heuts, 1947, 1949; Hagen, 1967). IndeedHagen's remarkable field studies lead him to conclude that leiurus and trachurus arein fact not races but distinct species, at least in North America and possibly throughouttheir almost circumpolar range. Similarly, two morphologically distinct forms of

256 D. W. SUTCLIFFE

Gamrnarus zaddachi with markedly different salinity optima in estuaries were finallyrecognized as separate species, G. zaddachi and G. salinus (Kinne, 1954, 1961; Dennertet al. 1969). Two forms of the polymorphic marine copepod Tisbe reticulata differ intheir responses to salinity fluctuations and the exchange rates of wNa, *2K and 137Cs(Battaglia & Bryan, 1964; Battaglia & Lazzaretto, 1967). Burbanck (1967) suggeststhat raciation is occurring in the euryhaline isopod Cyathura, and physiological racesprobably exist in the isopod Mesidotea entomon (Croghan & Lockwood, 1968). On theother hand Smith (1955) was unable to support previous suggestions that physiologicalraces might exist in Nereis diversicolor.

MATERIAL AND METHODS

G. duebeni was collected from two freshwater localities on the Kintyre and Stranraerpeninsulas, western Scotland, and from a brackish-water locality in northern Ireland.

The population on Kintyre, Argyll, was sampled in the Corachan Burn, a smalltributary of Connie Glen Water, near Southend (Sutcliffe, 1967a, b). In May 1967the sodium concentration of the burn water was o-8 mM/1.

The population on the Rhinns of Galloway, Stranraer, was represented by animalscollected from the Pinminnoch Burn and the loch at Lochnaw Castle. In September1968 these waters contained 0-51 and 0-70 HIM/I sodium respectively. G. duebeni alsooccurs in several other streams on the extreme north-western tip of the peninsula.

A brackish-water population in northern Ireland was sampled on the eastern shoreof Strangford Lough in September 1967. Animals were collected from a bed of Fucusin the intertidal zone of a small stream running over the shore at Ballyhaft Cottage,approximately 3 miles south of Newtownards.

Animals were brought back to Ferry House by van in large bowls containing a smallamount of the natural water. In the case of freshwater animals, sea water was addedto give a salinity roughly equivalent to 2 % sea water. This helped to reduce mortalityduring the journey to Ferry House. In the laboratory animals were acclimatized to atemperature of 9± 1 °C for at least 1 week before experiments were started. Experi-mental media were sea water diluted with de-ionized water to give a range of salinitiesdown to 2 % sea water, and sodium chloride solutions for lower salinities. Sea waterwas obtained from the Dove Marine Laboratory, Cullercoats. The experimentalanimals were normally large males acclimatized to media for at least 48 h before anymeasurements were made. They were fed on leaves of sycamore and elm duringintervals between experiments.

The techniques for determining sodium influx and sodium loss were describedpreviously (Shaw & Sutclifft., 1961; Sutcliffe, 19676,^ Sutcliffe & Shaw, 1968).Determinations of sodium influx using MNa were made with groups of 10-20 animalsdrawn from a large batch of animals acclimatized to either 0-25 or 0-5 mM/1 NaCl.The influx was measured at various external sodium concentrations over periods ofup to 1 h; each group was then returned to the acclimation medium and allowed atleast 24 h to return to the steady state. This procedure is necessary to obtain strictlycomparable measurements of the influx, which is influenced both by the internal stateof the animal and by changes in the external sodium concentration. Since net uptakeor loss of sodium occurs when the influx is measured at any external concentration

Sodium influx and loss in Gammarus duebeni 257

respectively higher or lower than the steady state (acclimation) concentration, the timeallowed for determination of the influx must be kept as short as possible. The internal(blood) sodium concentration must then be allowed to return to the previous steadystate before the influx is measured again.

An experimental population of G. duebeni living in fresh water was established atFerry House in July 1967, from a nucleus of some 200-300 small specimens obtainedfrom the Corachan Burn, Kintyre, in May 1967. They were kept in a small concretepond fed by a continuous flow of Windermere water. A healthy breeding populationwas successfully established in Windermere water containing only 0-20-0-22 mM/1sodium compared with a concentration of about o-8 mM/1 sodium in the natural habitat(Sutcliffe, 1970). This experimental population was not disturbed for 2 years, when130 large males were removed from the pond to determine the sodium influx inJune 1969.

RESULTS

The freshwater population on the Kintyre peninsulaSodium influx

Approximately 150 G. duebeni from the Corachan Burn, with a mean wet weight of53 mg, were acclimatized to 0-25 mM/1 NaCl at 9 °C. This concentration is muchlower than the sodium concentration of the habitat, but in the laboratory the animalssurvived for several weeks in 0-25 mM/1 NaCl at low temperatures. The low externalconcentration used for acclimation was chosen to raise the influx for easier measurement.

1-2 -

. 10

I 08

0-6

0-4

0-2

1 2 3 4 S 6Sodium concentration In medium (mn/l)

Fig. i. The relation between sodium influx and the external sodium concentration in G. duebenifrom Kintyre acclimatized to 0-25 mM/1 NaCl.

The relationship between sodium influx and the external sodium concentration(Fig. 1) is similar to that found previously with G. duebeni from the Connie River onthe Kintyre peninsula. The influx continued to increase at external sodium concentra-tions up to 6 mM/1, and from the previous work on sodium influx in G. duebeni it ispossible that the influx might have continued to rise gradually and reach saturation

17 KXB54

258 D. W. SUTCLIFFE

level at an external sodium concentration approaching 10 mM/1. However, for thepurposes of making comparisons between populations the most useful feature of theinflux is the Km value, denned as the external sodium concentration at which half themaximum influx (K) is reached (Shaw & Sutcliffe, 1961; Sutcliffe, 19676; Sutcliffe &Shaw, 1968). This is a convenient way of estimating the affinity for sodium ions inthe transporting system situated at the body surface, where a high affinity for ions atlow external concentrations of sodium is represented by a low K^ value. From Fig. 1,if K is equal to 0-95 [imjh. (at 6 mM/1 sodium) then Kni = 10 mM/1 sodium. On theother hand, if it is assumed that the influx continues to rise at higher external con-centrations so that it would reach its maximum (saturation) level K at, for example,i-2/m/h then Km would be equal to 15 mM/1 sodium. Thus it seems reasonable toconclude that in G. duebeni from the Corachan Burn the Km value lies between i-oand 1-5 mM/1 sodium. The same Km value was obtained previously with G. duebenifrom the Connie River.

Sodium loss in the urine

This was estimated by comparing the sodium loss rate into de-ionized water withthe loss rate into sucrose made isosmotic with the blood. Loss rates were measuredover a period of 60-90 min using groups of 12-15 animals in 40-50 ml of the appro-priate medium. A recovery period of 24 h was allowed between each set of measure-Table 1. Sodium loss and urine concentrations in Gammarus duebeni from Corachan

Burn, Kintyre, acclimatized to a range of salinities at 9 °C

Na loss intode-ionized water

(/iM/animal/h)

011 (n)±o-oo30-16 (I2)±O-OOQ018 ( u ) i o - o i 2O-2O (6)±O'O2O030 (6)±0039052 (i2)±ooi3

018 (s)±o-oio037 (II)±O-OIO

023 (4)023 (5)±00220 1 9 (s)±ooi2018 (IO)±OOIOOI4 (S)±OOI2

Na loss intoisosmotic sucrose

(/iM/animal/h)

011 (l2)±0'OO2015 (i2)±o-oo9014 (l2)±O'OO7016 (6)±o-onO-2O (6)±O-O2O024 (12) ±0-009

012 (s)±ooo60-14(12) ±0-005

Urinary Na Estimatedloss by urine Na

difference concentration(/iM/animal/h) (min/l)

Acclimationmedium

0-25 mM/1 NaCl2 % SW20%SW4O%SWSO%SW70%SWa % SW40 % SW7o%SW100% SWSO%SW40 % SW2O%SW2 % SW0-25 mM/1 NaCl

ments in de-ionized water or sucrose. The difference between the two sets of loss ratesis attributed to sodium loss in the urine (Sutcliffe, 19676, c; Sutcliffe & Shaw, 1968).The experiments were carried out in the same order as the results presented in Table 1,using animals in which the sodium influx was previously determined. The number ofgroups used for each determination of the sodium loss rate is given in parenthesis,with the standard error (Table 1).

The first set of loss rates obtained with animals acclimatized to 0-25 mM/1 NaCl

O-I9 (s) ±0-012017 (s)±o-oi60-15 (s)±o-oio017 (IO)±O-OIO014 (5)±0009

o-ooO-OI0-040-040 :00-28

0-06023

0-040-06004o-oi000

o7676

190360

" 3360

7611376oo

Sodium influx and loss in Gammarus duebeni 259

gave mean values of o-11 /4M/h for sodium loss into both de-ionized water and sucrose,in agreement with an estimated sodium influx of about 0-12 /JM/h for the same animalsat 0-25 mM/1 sodium (Fig. 1). From Table 1 it appears that no significant sodium lossoccurred in the urine of animals acclimatized to both 0-25 mM/1 NaCl and 2% seawater, a small loss was evident in urine of animals acclimatized to 20 and 40% seawater, and this increased at 50 and 70% sea water.

Sodium concentration in the urine

In the case of animals acclimatized to 70% sea water it may be assumed that thesodium concentration of the urine is the same as that of the blood, equivalent to about360 mM/1 sodium (Lockwood, 1964; Sutcliffe, 19676), and from Table 1 in 23 groupsacclimatized to 70% sea water the mean sodium loss rate in urine was 0*255 /*M/h.Now L = CV, where L is the observed sodium loss rate in urine (/IM Na/h), C is theurine sodium concentration (/*M Na//tl urine) and V is the volume of urine (/il urine/h).Substituting O-36/4M//*1 urine for C, and 0-255/iM Na/h for L, then V = L/C or0-71 /tl urine/h. Now the rate of urine production in gammarids is proportional tothe osmotic gradient between blood and external medium (Werntz, 1963; Sutcliffe,19676). In animals acclimatized to 70% sea water the blood osmotic pressure (andhence the osmotic gradient in de-ionized water) is equivalent to about 400 mM/1 NaCl,whereas in animals acclimatized to the salinity range 50-2 % sea water the blood osmoticpressure is equivalent to about 300 ntM/1 NaCl (Beadle & Cragg, 19406; Kinne, 1952;Lockwood, 1961). For these animals the urine flow rate into de-ionized water will bereduced to $xo"]i or 0-53/41 urine/h. This flow rate is equivalent to 24% bodyweight/day at 9 °C, and agrees very well with the previous estimate of urine flowequivalent to 28% body weight/day at 10 °C in G. duebeni from brackish-waterlocalities (Sutcliffe, 19676).

With the estimated urine flow rate, V = 0-53 /d/h, the sodium concentration in theurine, C, may be calculated for animals acclimatized to the range 50-2% sea water,employing the relationship C = L\V. Appropriate values for sodium loss in urine,L, were taken from Table 1. These estimated sodium concentrations are also given inTable 1. In general these urine concentrations in animals from a freshwater localityare lower than the urine sodium concentrations previously reported for G. duebenifrom brackish-water localities.

The experimental Kintyre population living in Windermere water

Sodium influxOne hundred and thirty G. duebeni with a mean wet weight of 93 mg were initially

acclimatized to 0-25 mM/1 NaCl at a temperature of 18 ± 1 *C for a period of 2 days,but several died and many were inactive. The external concentration was then raisedto 05 mM/1 NaCl and the animals became active and 'normal' in appearance. How-ever, mortality was still rather high; 24% died in the next 5 days, during which periodthe sodium influx was measured at a temperature of 18 ± 1 °C. This temperature,rather than 9-10 °C, was chosen because.it happened to be convenient at the time ofthese experiments. The previous work on populations of G. duebeni from both fresh-water and brackish-water localities showed that although the sodium influx is tem-

17-2

260 D. W. SUTCLIFFE

perature-dependent, the relationship between the influx and the external sodiumconcentration is not affected by changes in temperature.

With respect to the high mortality at 18 °C, the mean daily temperature in theexperimental pond did not rise above 15 °C. During some later measurements (June1970) of sodium and potassium loss rates made at 9 °C, mortality was very low and theanimals were able to maintain sodium balance at an external concentration of abouto-i mM/1 sodium for periods of at least 48 h.

1-6

1-4

1-2

10

E 0-63

0-4

0-2

0 1 2 3 4 5 6Sodium concentration In medium (m^l)

Fig. 2. The relation between sodium influx and the external sodium concentration in an experi-mental population of G. dtubeni kept in Windermere water for 2 years. Animals acclimatizedto O'5 min/l NaCl.

The results of the sodium influx measurements are shown in Fig. 2. The relationshipbetween the influx and the external sodium concentration differs in two importantrespects from that found in the animals taken directly from the natural population inthe Corachan Burn (Fig. 1). First, the influx increased rapidly at external sodiumconcentrations up to about 2 mM/1 where it apparently was already close to themaximum rate. At 2 and 6 mM/1 sodium the mean sodium influxes, with standarderrors, were 1-21 (6)10-072 and 1-33 (6)+o-o6o/tM/h respectively. The differencebetween these two means is barely significant. Second, if the influx was saturated ata rate of 1-33 /*M/h (Fig. 2) then the external sodium concentration at which the influxwas half-saturated is about o-6 mM/1. This Km value is appreciably lower than the K^value found in the natural population, indicating that the affinity for sodium ions hasbeen substantially increased. In fact the affinity for sodium in this experimentalpopulation living in Windermere water is remarkably similar to the affinity for sodiumfound in populations of G. duebeni living in fresh water in Ireland (Sutcliffe & Shaw,1968).

Sodium influx and loss in Gammarus duebeni 261

The freshwater population on the Stranraer peninsulaSodium influx

Eighty large individuals from Pinminnoch Burn and Lochnaw were used to deter-mine the sodium influx. They were acclimatised to 0-5 nrn/l NaCl at 9 °C. The resultsfrom animals with a mean wet weight of 75 mg are shown in Fig. 3. The influx increasedgradually at external sodium concentrations up to at least 6 mM/1, with a value forKm of at least 1*5 mM/1 sodium, although this would probably not exceed 20 ITIM/I.

These results are very similar to the results obtained with the population of G. duebeniliving in fresh water on the Kintyre peninsula (Fig. l). Further measurements ofsodium influx in the Stranraer population, with animals acclimatized to higher externalsodium concentrations in the presence and absence of calcium ions, will be publishedin a following paper.

1-4

1-2

1 0

0-8

0-2

0 1 2 3 4 5 6Sodium concentration in medium (mM/l)

Fig. 3. The relation between sodium influx and the external sodium concentration in G. duebenifrom Stranraer acclimatized to 05 mM/1 NaCl.

The brackish-water population on Strangford Lough, N. Ireland

Sodium balance

The lowest external concentration at which sodium balance could be maintainedover a period of 48 h at 9 °C was determined with animals acclimatized to 0-25 mM/1NaCl. Six groups of nine animals were placed in approximately 50 ml de-ionizedwater and net sodium loss occurred until the sodium concentration in the mediumreached a level where sodium uptake was equal to sodium loss. After 48 h the sodiumconcentration of the medium was determined, and the animals were placed in 0-25 mM/1NaCl for 4 days. The experiment was then repeated. Mean sodium concentrations inthe medium, with standard errors, were o-io± 0-013 mM/1 in the first experimentand o-11 ± 0-008 mM/1 in the second experiment. These balance concentrations areonly one-half the concentration required by brackish-water populations of G. duebeni

17-3

262 D. W. SUTCLIFFE

in Britain and are equivalent to the sodium balance concentrations previously reportedfor Kintyre animals and other populations living in fresh water.

Sodium influx

Sodium influx was determined with about 150 animals, mean wet weight 55 mg,acclimatized to 0-5 mM/1 NaCl at 9 °C. The results are shown in Fig. 4. The influxwas saturated at an external sodium concentration of 6 mM/1, and was half-saturatedat an external sodium concentration of about 1 -9 mM/1. Thus the relationship betweenthe influx and the external concentration in this brackish-water population fromIreland differs from that foundiri four populations of G. duebeni living in fresh waterin Ireland (Sutcliffe & Shaw, 1968), but it is virtually identical with the two populationsliving in fresh water on the Kintyre and Stranraer peninsulas in western Britain.

1-2

1 °'8f 0-6cg 0-43

o1/5 0-2

-

-

-

*

- '/

' I I i

/I

••

1 82 3 4 5 6 7Sodium concentration in medium (nw/l)

Fig. 4. The relation between sodium influx and the external sodium concentration in G. duebenifrom Strangford Lough, N. Ireland. Animals acclimatized to 0-5 mM/1 NaCl.

Table 2. Sodium loss and urine concentrations in Gammarus duebeni from StrangfordLough, N. Ireland, acclimatized to a range of salinities at 9 °C

Acclimationmedium

0-5 DIM/1 NaCl2 % SW20 % SW4o%SW5o%SW7o%SW4O%SW10% SW2 % SW2 mM/1 NaCl1 mM/1 NaCl '0-25 mM/1 NaCl

Na loss intode-ionized water

(/tM/animal/h)

0 1 30 1 80'220240-32

(6) ±0009(6) ±o-o 16(6) ±0-026(6) ±0014(6) ± 0024

0330-28

(6) ±0-017(6) ±0028

o-»2 (6)±0018(6) ±0028(6)±o-oio

o-io0 1 3

Na loss intoisosmotic sucrose

(/tM/animal/h)

0-070 (6) ±0-004

(6) ± 0-008(6) ±0-004(6)±o-oio(6)±o-oo8(S)±O'OI2

(6) ±0018(6) ±0016(6) ±0014(6) ±0019(6) ±0-013

0-075 (6)±0002

0 1 30-160-140-150 1 8

0-230-2OO-I9O-I4O-II

Urinary Na Estimatedloss by urine Na

difference concentrationO*M/animal/h)

0000'O2

0080-09014

O'lO008O-O3

O-O5

O-02

O-OO

O

36145164

255

182

I4S559136o

Sodium influx and loss in Gammarus duebeni 263

Sodium loss and concentration in the urine

The rate of sodium loss into derionized water and isosmotic sucrose was measuredwith groups of 10-12 animals acclimatized to a series of salinities increasing from0-5 mM/1 NaCl to 70% sea water, followed by decreasing salinities down to 0-25 mM/1NaCl. The mean loss rates, with standard errors are given in Table 2. Note that againthere is reasonable agreement between the loss rate (0-13 /iM/h) of animals acclimatizedto 0-5 mM/1 NaCl and sodium influx (c. o-i6/iM/h, Fig. 4) at this concentration.

In general, sodium loss rates from these animals closely resemble the loss ratesfound in the Kintyre population (Table 1) except that sodium losses attributed to theurine tend to be slightly greater in the Strangford Lough population. There was nosodium loss in the urine of animals acclimatized to 0-25 and 0-5 mM/1 NaCl, andurinary sodium losses in animals acclimatized to 1 and 2 mM/1 NaCl, and 2% seawater, might also be regarded as not statistically significant. However, if the differencesbetween the mean sodium loss rates in de-ionized water and sucrose are regarded asrepresenting a real difference in the mean sodium losses via the urine, then somesodium was present in urine of animals in all media except 0-5 and 0-25 mM/1 NaCl.The concentration of sodium in this urine was calculated in the manner described forthe Kintyre population, assuming a urine flow rate equivalent to 24 % body weight/day. These estimates for 55 mg animals are shown in Table 2.

Table 3. Sodium loss and urine concentrations in Gammarus duebeni from a brackish-water population at Warton salt-marsh, Lancashire

Urinary Na EstimatedNa loss into Na loss into loss by urine Na

Acclimation de-ionized water isosmotic sucrose difference concentrationmedium (/iM/animal/h) O*M/animal/h) (/iM/animal/h) (min/l)

40% SW 0-43 (6)±o-oi3 0-10(6)10-003 024 31610% SW 0-31(6)10-020 0-22 (6) ±0-023 °'°9 I I 0

a % SW 0-24(6)10-011 0-13(6)10005 O-II 1452mM/lNaCl 0-25 (6)±o-oai 0-15(6)10-015 010 1321 mM/1 NaCl 0-24(6)10-026 0-13(6)10-014 011 1450-25 mM/1 NaCl 0-14(5)10-006 — — —40% SW 0-41(5)10-047 0-20(5)10-006 021 276

When acclimatized to salinities above 2 % sea water the urine sodium concentrationswere similar to those reported for a brackish-water population of G. duebeni in Britain(Sutcliffe, 19676) but they were much lower when acclimatized to 2% sea water andbelow. In the British population from Warton salt-marsh, Lancashire, the estimatedurine sodium concentration in 2% sea water was 160-190 mM/1, falling to 36-39 mM/1in 0-25 mM/1 NaCl. In contrast, the estimates on the Irish brackish-water populationsuggest that in these animals the urine concentration was already reduced to a lowlevel in 2 % sea water (Table 2). This difference was confirmed by measuring againthe sodium loss in G. duebeni obtained from the brackish-water population on Wartonsalt-marsh. These experiments were carried out at 9 °C in parallel with those on theIrish population, and the same isosmotic sucrose solution was used for both series ofexperiments. The results are presented in.Table 3, where the urine sodium concentra-tion was also estimated from a urine flow rate assumed to be equivalent to 24 % body

^eight/day in animals with a mean wet weight of 76 mg.

264 D. W. SUTCLIFFE

Unfortunately it was not possible to measure the sodium loss rate into sucrose withanimals acclimatized to 0-25 mM/1 NaCl. The animals showed signs of distress aftermeasurement of the loss into de-ionized water, and several died, so they were imme-diately placed in 40 % sea water. This provided an opportunity to check the sodiumloss in urine at 40 % sea water (Table 3). The results confirm that urine with a relativelyhigh mean sodium concentration is produced at salinities well below 2 % sea water inthe case of the brackish-water population from Britain, but not in the brackish-waterpopulation from Ireland.

From a comparison of the loss rates into isosmotic sucrose (Tables 1-3) it appearsthat there is no difference in permeability of the body surface to sodium ions betweenpopulations living in fresh and brackish waters. This again confirms previous measure-ments of sodium loss (Sutcliffe, 1968).

Table 4. Summary of the chief characteristics of populations of Gammarus duebeni/romvarious habitats in Britain and Ireland

Habitat

Brackish waterBritain

Ireland

Fresh waterBritain

Ireland

Windermere water(Experimental

population)

No. ofpopulationsexamined

2

1

4

4

1

Na cone infreshwaterhabitats(mM/1)

—

—

0-60-2-5

0-40-0-46

o-2o-o-aa

External Nacone, at which

influx half-saturated

(/£„, mM/1)

1-5-2-4

1-9

1-0-2-0

0-4-0-7

o-6

Lowest Nabalance

concentration(mM/1)

0-21

O-II

o-io

0-07-0-10

o-io

Source

Shaw & Sutcliffe,1961; Sutcliffe,19676

This paper

Sutcliffe, 19676;this paper

Sutcliffe & Shaw,1968

This paper

DISCUSSION

Taking into account previous work, four populations of G. duebeni living in freshwater in western Britain have now been sampled (Lizard, Kintyre, Stranraer, Isle ofMan), Kintyre on two occasions. In every case the affinity for sodium ions in thetransporting system at the body surface is similar, in that the influx is 50 % saturatedat external sodium concentrations of between 1 and 2 mM/1. Similarly, G. duebeniliving in the inland fresh waters of Ireland have been sampled in four localities (Boyne,Liffey, Melvin, Neagh), the River Boyne on two occasions, and again the sodiuminfluxes of these four populations are very similar to one another. But they are 50%saturated at external sodium concentrations of only o^-o^ mM/1, a feature whichclearly distinguishes these Irish populations from the freshwater populations inwestern Britain. On the other hand, in the Irish brackish-water population on Strang-ford Lough the sodium influx is 50% saturated at about 1-9 mM/1 sodium, closelyresembling the situation found in brackish-water populations of G. duebeni living inBritain. These results provide clear evidence of a direct correlation between the half!

Sodium influx and loss in Gammarus duebeni 265

saturation level (K^ value) of the influx in a population and the external sodium con-centration of the particular habitat occupied by that population (Table 4). Thiscorrelation in natural populations of G. duebeni is reinforced by the results obtainedwith the experimental population originating from Kintyre and exposed to Windermerewater containing a very low sodium concentration. Within a period of 2 years therelationship between sodium influx and the external sodium concentration hadchanged, so that the external concentration required to achieve 50 % saturation in theinflux had fallen to 0-6 ITIM/1 sodium in the experimental population, compared withthe concentration of I#O-I#5 mM/1 sodium required to achieve the same saturationlevel in the natural population living in fresh water on Kintyre.

Associated with these population changes in affinity for sodium ions at the bodysurface are small but distinct changes in the ability of freshwater populations to reducesodium losses in the urine at the low external concentrations characteristic of freshwaters. There is considerable individual variation in G. duebeni from brackish-waterpopulations, with respect to both sodium uptake at the body surface and the abilityto elaborate dilute urine (Lockwood, 1961, 1964, 1965; Lockwood & Andrews, 1969).Furthermore, an internal linkage (probably mediated by a hormone) exists betweensodium uptake at the body surface and in the antennary glands of each individual(Lockwood, 1965; Sutcliffe, 19676). It is therefore probable that the changes inaffinity exhibited at the population level are the result of selection against individualswith a low affinity for sodium ions both at the body surface and in the antennaryglands. This may also explain the marked reduction in sodium losses in urine of theIrish brackish-water population at external concentrations below about 2 % sea water,compared with the brackish-water population from Lancashire. The latter populationlives in pools where the salinity rarely falls below 10%,, (c. 30% sea water), whereasthe Strangford Lough population occupies an 'estuarine' habitat where regular andsevere fluctuations in salinity must occur, with salinities approaching that of streamwater at periods of low tide. Similarly, urinary losses are reduced to a minimum in theKintyre population living in fresh water. Although sodium loss across the body surfaceis reduced at very low external concentrations this occurs in both brackish-water andfreshwater populations of G. duebeni, and there is no marked difference in permeabilitybetween any of the populations in Britain and Ireland. Minimal losses in the urinecan therefore play a significant part in maintaining a steady state when in fresh water,particularly in the British populations where the sodium uptake rate is restricted bythe relatively low affinity for sodium at the body surface. The extent and relativeimportance of changes in both the influx (uptake) and loss rates to achieve sodiumbalance at low external concentrations have been discussed in detail before (Sutcliffe,19676, 1968; Sutcliffe & Shaw, 1968).

The progressive changes in both external and internal affinities for sodium seen inpopulations of G. duebeni living in waters with decreasing sodium concentrations maytherefore be simply the result of increasingly rigorous selection in favour of individualswith a high affinity for sodium ions. This accounts for the rapid change in affinity forsodium in the experimental population living in Windermere water. According toHynes (1954), Kinne (1959) and Solem (1969), the maximum life-span of G. duebeniis normally less than 18 months, so that the mature individuals (males) used to deter-

sodium influx in the experimental population probably represent only the first

266 D . W. SUTCLIFFE

or second generation born in Windermere water. Despite the relatively short exposuretime to Windermere water, with respect to the affinity for sodium ions the experi-mental population phenotypically resembles G. duebeni living in fresh water in Ireland.If this resemblance is substantiated by further work on sodium influx and otherdistinctive features of sodium regulation, then the suggestion that the Irish freshwaterpopulations constitute a distinct physiological race (Sutcliffe & Shaw, 1968) would becalled into question, since the term' race' is properly restricted to population differencesarising from modifications of the genotype (Prosser, 1955; Mayr, 1963).

Lockwood & Croghan (1957) and Croghan & Lockwood (1968) describe a numberof osmoregulatory differences between brackish-water and freshwater populations ofthe isopod Mesidotea entomon. The freshwater population in L. Malaren (Sweden)has a higher blood volume, the sodium influx displays a higher affinity for sodium,and the permeability of the body surface to sodium is reduced to about one-half thepermeability in brackish-water animals from the Baltic Sea. Both M. entomon inMalaren and G. duebeni in fresh water in Ireland are geographically separated fromother populations, and their distinctive physiological characteristics presumably musteventually become invested in the genotype. This may have already occurred in M.entomon since animals from the Baltic population are distinguished by their inabilityto survive in fresh water, whereas the majority of G. duebeni from brackish water cansurvive in fresh water containing more than about 1 mM/1 sodium. The problem ofwhether or not the characteristic features of the Irish freshwater G. duebeni are geno-typic or phenotypic in origin might be resolved by establishing an experimentalpopulation of these animals in water with a raised sodium chloride content. If thedistinguishing characters are retained in the generations born in 'brackish' water,this would eliminate the possibility of phenotypic variation imposed by the sodiumconcentration of the habitat. In any event, it is apparent that G. duebeni is potentiallyable to live in a remarkably wide range of salt concentrations, from the very dilutewaters of Windermere to rock pools where the salinity exceeds that of sea water(Foreman; 1951), and the ability to reproduce in fresh water with a very low sodiumconcentration is not confined to G. duebeni in Ireland (Sutcliffe, 1970).

SUMMARY

1. Sodium influx was examined in Gammarus duebeni from freshwater habitats onthe Kintyre and Stranraer peninsulas in western Britain, and from a brackish-waterhabitat in Ireland. The affinity for sodium ions in the uptake mechanism at the bodysurface was similar in animals from the three localities.

2. Compared with the parent population from Kintyre, an experimental populationestablished for 2 years in water with a lower sodium concentration showed an increasedaffinity for sodium.

3. Sodium losses in the urine of animals from the above localities were negligibleat external salinities below about 2 % sea water. In contrast, urinary sodium losses inanimals from a brackish-water population in Britain were higher at salinities rangingfrom 40% sea water to well below 2 % sea water.

4. The affinity for sodium ions in uptake mechanisms at the body surface and inthe antennary glands of G. duebeni from a wide range of habitate shows a marked

Sodium influx and loss in Gammarus duebeni 267

correlation with the sodium concentration of the habitat. The permeability of thebody surface to outward movement of sodium is similar in G. duebeni from brackish-water and freshwater habitats.

5. It is suggested that most of the observed physiological differences betweenpopulations of G. duebeni are phenotypic in origin. The status of the freshwater ' race'in Ireland is briefly discussed.

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