Variation of apparent permeability to water and sodium transport in Gammarus duebeni exposed to fluctuating salinities

Comp. Biochem. Physiol.Vol. 67B, pp. 465 to 473

0305-0491/80/1101-0465502.00/~

© Pergamon Press Ltd 1980. Printed in Great Britain

VARIATION OF APPARENT PERMEABILITY TO WATER AND SODIUM TRANSPORT IN GAMMARUS DUEBENI EXPOSED TO FLUCTUATING SALINITIES S. R. L. BOLT, M. E. DAWSON, C. B. E. INMANand A. P. M. LOCKWOOD Department of Oceanography, University of Southampton, Southampton S09 5NH, England Abstract--1. The blood sodium concentration, body water content, drinking rate, active uptake of sodium and apparent permeability to water of the amphipod Gammarus duebeni in non-steady state systems are described. 2. The apparent permeability to water of Chaetogammarus marinus has been investigated. 3. Exposure to a salinity cycling between 3 and 97% seawater with a period of 12 hr 25 min results in dramatic changes in the apparent permeability to THO in G. duebeni, but only a small change in the case of C. marinus. 4. The blood sodium concentration of G. duebeni varies by only some 60 mM 1-1 during the cycle and in consequence is hypotonic to the medium for those periods when the external concentration exceeds about 350 mM 1-1 Na. 5. The active influx of sodium by G. duebeni is least when the external salinity is highest and has reached its nadir before blood sodium concentration starts to fall. 6. Total body water and drinking rates show no consistent trends in the cycle. 7. The adaptive features of the response of G. duebeni are discussed.

fluctuation of the body fluid concentration when exposed to cycling salinity regimes of appropriate period (Shumway, 1977a; Shumway & Youngson, 1979; Stickle & Ahokas, 1975). The recent development of mechanical or electronically controlled systems for generating cyclical changes in salinity in the laboratory (Davenport et al., 1975; Atkins & Ritz, 1977; Lockwood & Inman, 1979; Armitage, 1979) have enabled advances to be made in interpreting the responses of various species in such conditions (Hand & Stickle, 1977; Livingstone et al., 1979; Shumway, 1977b, 1978; Shumway et al., 1977; Stickle & Ahokas, 1974; Davenport, 1979a,b; Shumway & Davenport, 1977). The effect on mechanisms normally involved in regulation of blood concentration (active transport of ions, variation of water permeability and variation of urine flow and concentration) has not, however, been extensively studied under such condition~ It is the purpose of the present paper to illustrate that a euryhaline crustacean species which displays a considerable range of blood concentration when acclimated to different media may regulate the blood concentration almost constant when in a cyclical salinity regime. The changes observed in sodium uptake and apparent permeability to water during a cycle are discussed.

INTRODUCTION

As a result largely of the pioneer studies of Marcel Florkin and his school, there is general appreciation of the fact that invertebrates inhabiting regions of variable salinity have wide powers of osmoregulation at the cellular level. Estuarine forms acclimated to dilute media respond to reduction in blood concentration by diminution of both the free amino acids and inorganic ion content of their cells with the result that osmotic balance is maintained between blood and cells without the necessity for major water shifts between b o d y compartments. Such ability to effect rapid and substantial regulation of cell free amino acids appears to play a fundamental role in the tolerance of extensive salinity fluctuations of the medium. On a quantitative basis, it is one of the features which most markedly differentiates euryhaline from stenohaline organisms. Necessarily the process of effecting changes in cell free amino acid levels is likely to disturb cell metabolism, if for no other reasons than the associated changes in ionic level and the demands made upon organic acid supply and the N A D - N A D H system. It is perhaps for this reason that mechanisms permitting more rigid control of blood concentration in the face of fluctuations of the medium salinity have evolved in those species where permeability of the body surface is sufficiently reduced. Certain crabs, prawns, mysids and isopods display the ability to regulate the haemolymph hypotonic to strongly saline media and hypertonic to dilute media. For a number of these species the maintenance of a relatively constant haemolymph concentration can be achieved over long-term acclimation to a range of salinities. However, even forms not noted for their capacity to regulate the concentration of the blood may also show relatively limited

MATERIALS AND METHODS

Salinity cycle generation

A mechanical system was used to generate the cycle during studies on drinking rate, sodium uptake rate, total body sodium and some of the measurements on blood sodium concentration. The principal element of the system involved two opposed blocks of teflon one of which was

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S.R.L. BOLT et al.

rotated on a 12-hr cycle. Slits cut in both teflon blocks permitted the passage of either seawater or freshwater to pass to the animal chamber at different phases of the cycle. The salinity profile produced by the system was essentially a series of successive exponentials (el. Fig. 9). Later experiments on blood sodium concentration and water fluxes were made using a cycle generated by a microprocessor built from a Cosmac CDP 1802 kit operated in conjunction with a feedback salinity monitor based on the salinometer designed by Coghill & Bowers (1975, and personal communication). Software developed for the microprocessor permits comparison every 30 see of the salinity in an experimental tank with the appropriate byte of a preprogrammed curve in memory. Any difference between the observed salinity and the memory value is corrected by the microprocessor operating solenoids which control the entry of seawater or freshwater from constant head tanks. Any shape and period of salinity curve may be generated but for the experiments here described the curve was a sine wave with a period of either 12 hr 50 min or 12 hr 25 min. The limit salinities used were 3% seawater and 97%seawater (where 100% seawater is 3 2 ~ salinity).

cryoscopic method based on the principle of Ramsay & Brown (1955). Drinking rate 5~CrEDTA was used to monitor drinking rates. It is assumed (a) that this material is not metabolized during short term experiments and (b) that ?-emission from animals previously placed in a medium containing 51CrEDTA results from medium having been taken up by oral or anal drinking. The ~,-emission from 51CrEDTA was measured by a Panax well-crystal scintillation counter. Tritiated water Tritiated water samples were counted by the method of Lockwood et al. (1973) with a Beckman 3100 series scintillation counter. Animals Gammarus duebeni were collected from the Totton salt marsh at the head of Southampton Water. Chaetogammarus (Marinooammarus) marinus came from the foreshore of Hayling Island. Most G. duebeni used were in the 50-80 mg weight range and Chaetogammarus were in the range 50-150 mg. In experiments involving cycling salinities the animals were exposed to the cycle for at least eight cycles prior to the start of measurements. Food was not provided in the preliminary period or during the experimental runs.

Concentration of blood and medium Sodium concentration of blood and medium were measured on a Unicam SP. 900 flame spectrophotometer except that in the mechanically generated cycle the concentration of the medium was monitored by an Orion sodium electrode, the accuracy of which was checked at intervals by flame spectrophotometric measurements. Osmotic pressure measurements were made using a

Temperature All flux and drinking rate experiments were undertaken at 15 + I°C. Heart rate was measured at 11 4- 0.5°C.

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Blood Concentration

The blood sodium concentration of G. duebeni acclimated to salinities in the range 10 mM l-~ NaCl (-----2% seawater) to 713mMI - I (-----140% seawater) varies between 285 mM l- 1 Na and 713 mM l- 1 Na (Lockwood & Inman, 1979) (Fig. 1). By contrast, G. duebeni maintained in a salinity cycle of sine wave form varying between 97% seawater and 3% seawater with a cycle periodicity of 12 hr 25 min showed blood sodium concentration changes only in the range 295 mM l- ~Na to 340 mM l- ~ Na (Fig. 2). The blood concentration is thus maintained in the cycle over the same range as it is actively regulated in dilute media (Fig. 1) and does not rise to the higher levels observed during long-term adaptation to media with concentrations in excess of 50% seawater. Blood sodium concentration is still more effectively regulated if the salinity of the medium cycles only between 0 and 50% seawater (Fig. 3). The limits of blood concentration are then only 1 8 m M l - ~ N a apart. When the medium fluctuates between 100% and 50% seawater the blood concentration varies by about 60 mM l- ~ Na and the mean concentration is shifted upwards (Fig. 3). In the two cycles which have upper salinity limits exceeding 50% seawater the body fluids of G. duebeni are hypotonic to the medium for several hours. Regulatory processes

Three components may be involved in regulation of the blood concentration, control of water entry, active transport of ions and urine production. Apparent permeability to water G. duebeni acclimated to media in the range 2%

seawater to 150% seawater at 18 + I°C show half times (T~/2) for tritiated water (THO) outflux varying

between 16.8min at the lower concentration to 5.8 min at the higher salinity (Fig. 4). Somewhat larger variations in half time for exchange are apparent in individuals exposed to a salinity cycle with concentration limits at 3 and 97% seawater (Fig. 5.). In the cycle the minimum value for T~/2 is 6 min, a level comparable with the exchange in 150% seawater in acclimated individuals. However, the phase of high apparent permeability (low T~/2) does not relate to the time of peak salinity in the cycle, as might be expected from the steady state results. Instead it occurs when the external salinity is falling. Similarly the period of lowest apparent permeability in the cycle does not correlate with the salinity nadir as would be found if the individuals were behaving like animals which are fully acclimated. A dramatic change in apparent permeability is seen shortly after the cycle reaches its peak salinity (Fig. 5). More detailed examination of the blood sodium concentration and T1/2 for THO influx in the period following the peaking of the salinity cycle (Fig. 6) permits certain conclusions to be drawn. (1) Apparent permeability reaches its minimum value close to the time when the highest external concentration and maximum hypotonic gradient between blood and medium occur. (2) Increase in apparent permeability commences more than an hour before the sodium concentration of the medium falls to a level isionic with the haemolymph and reaches its lowest level at about the time blood and medium are isionic with respect to sodium. It may be concluded that neither the absolute concentration of the medium nor of the blood directly or solely control the apparent permeability to water. There is, however, a suggestion of a correlation between the permeability to THO and the concentration gradient between blood and medium since the apparent permeability is greatest when the blood and medium are isionic and decreases when the body fluids are either hypertonic or hypotonic to the medium (Figs 5 and 6).

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Fig. 3. Blood sodium concentration of G. duebeni (a) experiencing ca. 12 hr cycles of salinity changes, and (b) acclimated to media in the same range. ZX, acclimated animals; x, in cycle of 50% seawater to freshwater, ©, in cycle of 100% seawater to freshwater; O, in cycle of 50% seawater to 100% seawater; ~, seawater. Determination of the influx of THO in the less euryhaline amphipod C. (Marinogammarus) marinus in the same salinity regime gives no indication of (a) any increase in half time for exchange when the animal is hypotonic to the medium, (b) rapid decrease in T1/2 as the concentration of the medium declines from its peak, or (c) temporal association between the lowest T1/2 and isionicity between blood and medium (Fig. 5). The features (a), (b) and (c) are all present in the G. duebeni profile of apparent water exchange and it seems, therefore, that this pattern is not general to all crustaceans exposed to salinity cycles of this period. Heart rate variation on transfer of G. duebeni between salinities Consideration of the meaning of apparent permeability data derived from THO flux experiments prompts questions with regard to the validity of interpreting the observed effects in terms of actual changes of water permeability. Indeed the question as to whether flux changes represent genuine alterations in permeability of cell or body surfaces or arise from experimental artifacts has been a matter of some debate (Oschman et al., 1974; Smith, 1976). Cornell (1973) showed that in the crab Libinia emarginata decrease in the apparent permeability to water could be related to a decrease in heart rate. In this case, therefore, the possibility arises of changes in flow patterns in the gills creating the illusion of decreased permeability, or alternatively, that an actual decrease in

net uptake of water may occur as an indirect result of diminished blood flow past the respiratory surfaces. Direct observation has been made of the heart rate of G. duebeni exposed to successive changes in salinity from freshwater to seawater and vice versa. Control animals show little change in heart rate if maintained in constant temperature at constant salinity (Fig. 7). Similarly, individuals pre-acclimated to 100% seawater do not display much variation on subsequent salinity changes. Greater variability is shown by specimens initially acclimated to 2% seawater and, though the individual variability is considerable (Fig. 7b, c) there is a tendency for the heart rate to be slower in 100% seawater than in 2% seawater. Drinking rate

When G. duebeni is subjected to a 12-hr cycle in which salinity varies between 3% seawater and 97% seawater there is a period of some 4 hr during which the blood sodium concentration is hypotonic to that of the medium (Fig. 2). During this period a net loss of water from the body would be anticipated even assuming that urine production ceases. Estimates based on the mole fraction differences between blood and medium when the animal is hypotonic to the medium and the apparent permeability suggest that some 2 to 3% of the total body water should be lost by osmosis during this period. Measurement of the total body water of animals taken from the cycle at different times gives no indication of any systematic

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When aquatic organisms are exposed to an alteration in external salinity it is self-evident that any subsequent adjustment of body fluid lags behind the change in the concentration of the medium. In cycling salinity regimes this has the effect of reducing the degree of change of body fluid concentration so that the limits reached by the medium are not experienced internally. The extent to which such damping of variation in haemolymph concentration occurs is dependent on the permeability of the body surface and

regulatory responses of the organism. Most studies of such effects have been concentrated on molluscs (Stickle & Ahokas, 1975; Hand & Stickle, 1977; Shumway, 1977a; Shumway & Youngson, 1979; Livingstone et al., 1979; Davenport, 1979a,b), though echinoderms (Stickle & Ahokas 1974), a polychaete (Shumway & Davenport, 1977) and crustacea (Shumway, 1978; Lockwood & Inman, 1979) have also been studied. Behavioural responses associated with shell closure limit the concentration changes in bivalves (Davenport, 1979b; Davenport et al., 1975); similar salinity related valve closure occurs in barnacles (Davenport, 1976). The valve closing response of different species of barnacle can be related to the salinity they inhabit (Davenport, 1976) and is clearly adaptive. Much less is known about the dynamic responses to salinity change in species which are unable to isolate themselves effectively from the medium though Lockwood & Inman (1979) have found that the relative constancy of the blood sodium shown by G. duebeni in a cyclical salinity regime can in part be attributed to appropriate variation in the rate of active uptake of sodium. The present results provide additional support for the view that fluctuations in blood sodium concentration of G. duebeni are heavily damped when the animals are exposed to ca. 12-hr cycles with limits at 39/0 and 97% seawater. Furthermore, it is shown that changes in total body water during the different phases of the cycle, if present, are within the limits of accuracy of the method of determination. Drinking rate variations are not consistent with the assumption that oral or anal intake of medium is increased during the period when the animals are hypotonic to the medium. With regard to the apparent permeability to water (APW) as determined by THO influx, striking differences are observed between earlier results obtained on

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Fig. 9. Sodium influx by G. duebeni in a ca. 12-hr cycle of salinity change in relation to total body sodium and blood sodium concentration (after Lockwood & Inman, 1979). G. duebeni acclimated to particular salinities and those from animals in a salinity cycle. Acclimated animals have a lower APW when in 2% seawater than in 100% seawater, the difference between the two values being c. 2.5-fold (Lockwood et al. 1973). By contrast, animals in the cycle display a high APW when isotonic with the medium and an intermediate value when the hyperosmotic gradient between blood and medium is maximal. The lowest APW occurs when the animals' body fluids are most hypotonic to the medium. These finding permit the immediate conclusion that the APW values are not correlated with either the absolute concentration of the medium or the absolute concentration of the blood. Their significance with respect to net movements of waters across the body surface remains uncertain at present. In G. duebeni acclimated to particular salinities the influx and outflux of THO are similar. The calculated diffusional permeability, unlike the situation in Carcinus maenas (Smith, 1976) and Rhithropanopeus harrisi (Smith, 1967), is not dissimilar to the osmotic permeability calculated from urine production rates (Lockwood & Inman, 1973). However, the relationship between osmotic permeability and diffusional permeability in G. duebeni does not necessarily obtain when the animals are no longer in a steady state. As pointed out by Oschman et al. (1974) if water is moving via the pores the osmotic permeability may be expected to vary with the square of the pore area whereas diffusional permeability varies in direct proportion to the area. Unstirred layers in association with the permeability barrier may have the potential to cause discrepancies between calculated osmotic and diffusional permeabilities (Dainty & House, 1966). We do not know how

water crosses the surface in these animals but clearly any factor which might influence either pore dimensions or unstirred layers could differentially affect osmotic and diffusional permeabilities. One such factor has been suggested by Cornell (1973) and Cornell (in Smith, 1976). Working with the Atlantic spider crab Libinia emarginata he showed that reduction of heart rate on exposure of the crab to 80% seawater could be correlated with reduction in apparent permeability to water. Furthermore, the rate at which fluid perfused through isolated gills becomes saturated with D H O from the medium is positively related to the flow rate of the perfusate. Decrease in circulation through the gills following osmotic shock may, therefore, contribute to the change in apparent permeability in Libinia emarginata and Smith suggests that such a circulatory change may represent a temporary adaptive response. By contrast with Libinia, emarginata, sudden dilution of the medium from 100% seawater to 2% seawater does not normally result in much change in heart rate of G. duebeni though it does influence the APW. In this case therefore it seems unlikely that heart rate affects APW, though possible rerouting of the blood in the gills following such osmotic shock has not been ruled out. Rapid increase in external salinity from 2% seawater to 100% seawater does produce a small decline in heart rate but the change in percentage terms is less than the corresponding decline in APW. We may conclude that the substantial variation in APW seen in G. duebeni in cycling salinity systems cannot be readily related to heart rate changes alone. Whether or not they represent artefacts of measurement in non steady state systems or genuinely reflect underlying, if not directly proportional, variation in

Water and sodium transport in G. duebeni net water flow must await detailed analysis of urine flow rates throughout the period of the cycle. Pending such a study it would be specious to speculate further on the potential value of reduction or increase in permeability to water in this species. It remains only to note that if the A P W changes do not have some adaptive significance it is remarkable that the highly euryhaline G. duebeni and the less tolerant but closely related C. marinus differ so strikingly in their APW values. In contrast with the uncertainties relating to control over water permeability, the response of the ion transport system in cycling salinity conditions shows a clear adaptive response directed towards the maintenance of a constant blood concentration. In the cycle uptake falls during the periods of rising salinity, the intake of sodium reaching its nadir close to the peak of the external concentration. Uptake increases again during the falling phase of the cycle. An interesting feature of the response is that even before blood concentration begins to decline there is a suggestion that sodium intake is beginning to rise. Such an effect confirms the earlier finding of Lockwood (1964) that ion uptake rate is not necessarily governed by the absolute concentration of the body fluids. The advantage of such a system is that response can be directed at slowing the rate of change of body fluids and hence restricting the rate at which osmotic correction must be effected at the cellular level. Acknowledgements--We are grateful to Mr N. Jenkinson for his skilful assistance in assembling the microprocessor and redesigning and building the salinometer. We would also like to acknowledge the aid of Mr J. Cross in constructing the mechanically generated salinity cycling device. Parts of this work were undertaken with assistance from the Natural Environment Research Council under research grants G3/2552 and GR3/3560 to A. P. M. Lockwood. S. R. L. Bolt is supported by a studentship from N.E.R.C.

REFERENCES ARMITAGEM. E. (1979) Amino acids and their metabolism in two species of mysid. Ph.D. thesis, Southampton University. ATKINSB. 8~ RtTZ D. A. (1977) New instrument for supplying water of fluctuating salinity. Lab. Practice, 26, 99-100. BOWERSR. & COGHILLG. G. (1975) J. Phys. E. 8, 150-152. CORNELLJ. C. (1973). A reduction in water permeability in response to a dilute medium in the stenohaline crab Libinia emarginata (Brachyura, Majidae). Biol. Bull. mar. biol. Lab. Woods Hole 145, 430~31. DAINTY J. & HOUSE C. R. (1966) An examination of the evidence for membrane pores in frog skin. J. Physiol., Lond. 185, 172-184. DAVENPORTJ. (1976) A comparative study of the behaviour of some balanomorph barnacles exposed to fluctuating seawater concentrations. J. mar. biol. Ass. U.K. 56, 889-907. DAVENPORT J. (1979a) Is Mytilus edulis a short term osmoregulator? Comp. Biochem. Physiol. 64A, 91-95. DAVENPORT J. (1979b) The isolation response of mussels (Mytilus edulis L.) exposed to falling seawater concentrations. J. mar. biol. Ass., U.K. 59, 123-132. DAVENPORTJ., GRUFFYDDEL. D. t~ BEAUMONTA. R. (1975) An apparatus to supply water of fluctuating salinity and

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