Comparative studies of sodium transport and its relation to hydrostatic pressure in deep and shallow water gammarid crustaceans from Lake Baikal

Corn,,. Brochrm Phwol.. Vol 65.4. pp II9 to I27 Perpamon Press Lfd IYBO PrInted I” Great Britain

COMPARATIVE STUDIES OF SODIUM TRANSPORT AND ITS RELATION TO HYDROSTATIC PRESSURE IN DEEP AND SHALLOW WATER GAMMARID CRUSTACEANS FROM LAKE BAIKAL R. W. BRAUER’, M. Y. BEKMAN’, J. B. KEYSER’, D. L. NESBITT’, S. G. SHVETZOV~,G. N. SIDELEV~and S. L. WRIGHT’ ‘Institute of Marine Biomedical Research, University of North Carolina at Wilmington, Wilmington, NC, U.S.A., ‘Institute of Limnology, Siberian Branch, USSR Academy of Sciences, Listvyenichnoye na Baikale, Irkutsk Oblast U.S.S.R. and %iberian Institute of Plant Physiology and Biochemistry, Siberian Branch, USSR Academy of Sciences, Irkutsk 664033, U.S.S.R. (Received 6 April 1979) Abstract-l. Freshwater gammarids from 90&14OOm depths lose Na at 1 atm, 4”C, while related shallow water gammarids are near neutral Na balance. 2. Na+ influx rates are similar at 1 atm, 4”C, for abyssal and shallow water gammarids of similar weight. 3. Na+ efflux is faster for abyssal gammarids than for comparable shallow water gammarids. 4. Compressing abyssal gammarids to 90-140 atm increases Na + influx rates enough to restore neutral Na balance, while in shallow water crustaceans, compression decreases Na+ influx. 5. Na+ influx rates in Baikalian gammarids vary with the 0.55 power of weight. 6. The equation F,,, = 1.3 x W”.55 pEq/hr/animal applies to freshwater crustaceans over the weight range from 0.03 tc! 35 g.

INTRODUCTION Abyssal environments, marine or limnic, constitute a unique challenge to the adaptive powers of living systems. Along with special conditions of cold, lightlessness and generally low density of biomass, these regions are set off from all others on earth by the high hydrostatic pressures prevailing there. While it would appear that there is a rather sharp break between fauna inhabiting the upper 200&3OOOm and those living at depths in excess of 3000m [from which few, if any, animals can be retrieved to the surface alive, (Menzies et al., 1972; MacDonald, 1975; George, 1979; Yayanos, 1978)], there is evidence to suggest that, even at depths of lOOOm, substantial adaptation to high pressure regimes characterizes many resident crustaceans and other invertebrates (George & Marum, 1974; MacDonald & Teal, 1975; MacDonald & Gilchrist, 1978). In a recent study using the unique topography and the unique faunistic characteristics of Lake Baikal (Brauer, et al., 1979), this hypothesis could be tested more rigorously. It was established that gammarids from depths between 900 and 1400 m display pressure tolerances significantly in excess of those of related shallow water forms; furthermore, the pressures at which severe behavioral disturbances occurred in the two groups made it clear that adaptation to a high pressure regime is a prerequisite for the establishment of gammarid populations in the abyssal regions of Lake Baikal. In animals living at depths exposing them to pressures of 5O@lOOOatm, a wide variety of cellular constituents must have undergone adaptive modifications

to enable them to function (e.g. Hochachka & Somero, 1975; Zimmerman, 1970; Arronet & Konstantinova, 1969; Josephs & Harrington, 1968; Guthe, 1969). It would appear, however, that such extreme changes are hardly required for survival under pressures of lOtX200 atm. Under these conditions, the biological functions first affected and, hence, first requiring adaptive or acclimative modification, may well be confined to permeability and transport properties related to membrane function (e.g. Campenot, 1975; Pkqueux, 1972). The existence of pressure adapted fresh water crustaceans living in Lake Baikal side by side with closely related shallow water species provides test material for the exploration of at least one aspect of membrane related transport mechanisms, the accumulation of sodium ions from the fresh water into extracellular body fluids against nearly lOOO-fold concentration gradients. A preliminary study of sodium metabolism in closely related shallow and deep water gammarid crustaceans from Lake Baikal in Eastern Siberia was undertaken during the summer of 1978, in the course of a collaborative study between the Institute of Limnology of the USSR Academy of Sciences, the Siberian Institute of Plant Physiology and Biochemistry, and the Institute of Marine Biomedical Research. While experimental designs that could be implemented during this first effort were limited by time, logistics, and equipment constraints, the results appear to us, nonetheless, sufficiently conclusive and sufficiently interesting to warrant their publication at this time. In the nature of things, such results of expeditionary research pose a number of questions which

119

require further study. It IS hoped that the data can bz amplified and the majority of these remaining points elucidated

during

plan to conduct

a second joint during 1980.

campaign

which

we

METHODS

Collectton and maintenance procedures used in this effort have been described in a previous communication (Brauer PI dl.. 1979). In general. benthic gammarids were collected by trapping near the lake floor: were brought to the surface under conditions minimizing thermal stress: were classified and transferred to aquarium trays which vvere maintained as near as possible to habitat temperature (3.S 4.C for the deep water animals and 610°C for the shallow water forms). Survival of deep water forms under these conditions was satisfactory, 90”1,, of the animals surviving in the laboratory for I week or longer. The species utilized in the work to be reported here are summarized in Table I.

Because of the low ionic content of Lake Baikal water (sodium concentrations of 0.17mEqJ (Votyntzev, 1961). it was practicable to conduct sodium balance experiments by placing freshfy captured animals in unpolluted water (collected from the surface of Lake Baikal) using sodium-free plastic containers closed by screw caps. The ratio of biomass to water in such experimental set-ups was kept near I : 150. Thus. approx 509,;, of the total exchangeable sodium ion in any given set-up was contained initially within the test animals and any change in the animals’ Na reserves wsould elicit an approximately equal, hut opposite relative change in the Na c(~ncent~tion of the water. These assemblies were. in turn. placed in the refrigerator at a temperature as close as practicable to the respective habitat temperatures. Water samples were withdrawn at 1, 2. 4. 12 and 24 hr and analyzed for sodium content by flame photometry. Overall accuracy of the flame photometry allowed reproduction of water analyses to within fO.S#Zq.l, corresponding to net sodium movement of 0.075 /tEqg animal or about kO.25”” of the exchangeable Na stores in the test animals during any given test period. At the end of each experiment. the animals were removed from the container. blotted dry, and weighed rapidly on a precision torsion balance. Such weight determinations were reproducible to within t0.01 g.

Table

NO

Genus

All of thcsc proccdursa. L’t~ltn~tr, were conducted aboard the R V G. .S ‘Iirrat~ \o ;I’. trl norm mire the interval between capture and thi. ~nit~atron II! tl~ balance experiments. Water ~anlplc\ wI!rc \torcti !il t11<, refrigerator. aboard ship. and \ubsequ~nt)! taken aslro~c I%% be transported to Irkutsk for flame photornctrt~~ an:rl~ \t\ for total Na content. The result\ of ~>drum bal~nie \tatiic\ are expressed as microequivalent\ of ~oJium lo\t or .entncd per hour per gram net weight of anttmri

These were performed hv measurrng the rate of &sappearancc of radioactive Na” from a bath contaming Na _ of known concentr~~ti~n and \prcific ;tcttvity into ‘3 predetermined mass of gammarids. The expcrtrnental set-up and test conditions were identtcal with thoyc utilized for the balance experiments. After collecting the initial water sample for determination of Na concentration. the tracer NaZ’ was introduced in the form of carrier-free I\ia”c‘l tn 3.33 f/l of a solution containmy I /Kit Na” and Its< than 30 x lO~“~rg Na per /(I .~dditl~~nal water samples were collected I. 2 and 4 hr after intr[~~iucti~~l~ of the isotope. and SO ~tl aliquots of each sample placed in 21) ml of scintitlant solution [6.25 g 2.5 diphenyloxaxole and 0.625 g elf his- t5 phenyloxazolyl) benzene.1 toluenr triton IO0 ialkyl phenoxy polyethoxyethanol)] These prsparation\ wcrc then stored aboard ship unttl tt bccamc fcasihie to transfer them to Irkutsk for further :tnalvsts. YaL’ content aaz determined by sc~ntill~ition c~,~,~~~ng to an accuracy of i:?“,,. Direct determination by Ramc photometry of tntai Na content of water sample\ containing Na” wa\ precluded by considerations of radiologtc safety. However. sufficient data were available on the basis of the Ka balance studies to permit confdcnt &sign of influx measurements in which the sodium ton c,<*ntent of cxtcrnai and internal compartnlent~ did not var) ~lgnitic~lntl~ over the course of the 2 hr test periods. cmplo>ed. a conclusion confirmed by the constancy of obser\td intluw rate\ over that period.

The effect upon sodium intlux of returning ab!rsal antmals to habitat pressure was measured hq’ placing the animals into plastic 150 ml vials, tilled to I he top with lake water containing the required amount of Na” and covrrcd with a flexible membrane seal to permit pressure transduction The entire assembly was immersed in water contained within a vertical cylindrical pressure chamber 5 bx 9 cm (cf. Brauer c’f (rl.. 1979). Compression was effected b\- means of

1. ~.har~~cteriz~iti~~n of species of Baikalian

and species

gammarids

used in prcient

Number of animals used in Balance Fltlx Exps Exps

stud\

Mean hod! %etght *SF. rgt

Depth of capture (m)

Reported depth range (m)

beach 50

littoral I- 175

6 8

4

0.36 ‘- 0.04 0.28 * 0.01

5-820

4

5

I .6 * 0.1

so@I300

4

4

0.13 r 0.01

5@ 1000

4X

so 935

I (x)0 935

1Mm

0 IX t 0.01

935

* Calculated

from freezing

point depression

data of Basikalova

Ed a/., 1946.

Hemoi) mph osmolarity* (m OsmJ

121

Na+ transport in Lake Baikal gammarids a precision pressure gauge. Compression rates were SOOatm/hr. Animals were brought to a pressure corresponding to that of their habitat and held at that pressure for either 1 or 4 hr. The entire set-up procedure could be completed in less than 30 min. Temperature of the chamber was controlled during the test exposures at 4°C by circulating refrigerated water through coils surrounding the chamber. The particular chamber utilized did not permit collection of samples under pressure and, thus, each set-up provided data concerning sodium influx only for a single test period. Experimental design Quantitative comparisons were made of a total of seven species of Baikalian gammarids with respect to net sodium balance and sodium influx rates. Sufficient numbers of abyssal animals could be tested for one particular species to permit assessment of individual variability. That same species was utilized for studies of the effect of pressure on sodium influx. For the remaining species, results for each type of test are restricted to small numbers of individuals (2-8) so that any conclusions derived here must be viewed as tentative to be verified by more extensive future studies addressing specific questions posed as a result of the exploratory work accomplished in 1978. Statistical treatment of the data was achieved by pooling all of the analytical results available for a given set of constraints, computing mean values, standard deviation, and standard error. These statistics, therefore, refer to a compound of analytical and biological parameters, and set limits to the confidence to be placed in the measurements, but in general do not provide a measure of biological variability within the several subpopulations of animals. Non-parametric comparison of balance data for the two large Acanthogammarus species was achieved by means of Wilcoxson’s rank order (Wilcoxson, 1945).

RESULTS Species ofgammarids

tested

Table I contains data characterizing the seven species of gammarids utilized in the present study.

“Depth of Capture”, Column 3, refers to the actual conditions under which our specimens were collected, while “Depth Range”, Column 4, refers to the total range reported on the basis of longterm observations by investigators at the Limnological Institute. The seven species were subdivided on the basis of the

depth of actual capture into two shallow water groups-littoral (L) and open lake (S), and one deep water group (D), Column 1. The data for mean body weight, Column 7, illustrate both, the relatively uniform size of the animals of any given species, and the wide range of sizes covered by the several species collected (0.134.2 g). Column 8 presents estimates of hemolymph osmolarity calculated on the basis of freezing point determinations for various Baikalian gammarids as reported in 1946 by Basikalova et al. (1946). Sodium balance

experiments

Net sodium gain or loss was measured for representatives of the different species shown in Table 1 by placing the animals in known values of fresh lake water at habitat temperature immediately after capture and measuring the change in sodium concentration in the bath over the course of I2 hr. The rate of sodium

gain or loss was greatest

during

the first

2 hr, continued at a slightly reduced rate between 2 and 4 hr (Table 2, Columns 3 and 4) and had decreased to low levels barely distinguishable from zero by 12 hr. Since the net flux rates observed are of such magnitude that they entail substantial changes in sodium content within 4 hr in most of the species studied, further consideration of these results will be limited to the data derived during the first 2 hr period. Among shallow water gammarids, a positive Na balance is well secured for species L-I and S-I (P < O.OOS), while Acanthogammarus, S-II, is probably in neutral Na balance (P > 0.2). All of the abyssal gammarids, on the other hand, show net Na+ losses under our test conditions. These are well secured statistically for species D-I and D-III (P < 0.025 for the null hypothesis in each case). For species D-IV, the data even up to 4 hr are too limited to permit valid inference by parametric analysis (P = 0.1 for the null hypothesis). Non-parametric analysis (rank order, Wilcoxson, 1945), shows that the Na+ balance for the abyssal Acanthogrammarus is clearly more negative than that for the shallow water Acanthogammarus, S-II (P < 0.025). Since, as suggested above, species S-II is probably in neutral Na balance, the ranking analysis supports the hypothesis

Table 2. Sodium balance and sodium ion fluxes in various Baikalian gammarids Net Na balance (&/glhr)

No

Genus and species

L-I

Eulimnogammarus

S-I S-II

verrucosus Pallasea grubei Acanthogammarus

D-I

albus Odontogammarus margaritaceus Ommatogrammarus albinus

D-II

Parapallasea lagowski Acanthogammarus grewingki

D-III D-IV *

t&2 hr

2-4 hr

Na flux (2 hr) Sq/g/hr Influx (4) Efflux (6)

1.13 * 0.22

0.87 ) 0.17

0.57

*

0.13

0.52

0.078

f

0.062

0.043

-0.96

f

0.26

- 1.00

-0.14

*

0.11t

-0.04 f 0.08

-0.74

+ 0.13

-0.78

+ 0.13

0.83

k 0.04

1.57

+ 0.082

-0.13

+ 0.09

0.34

*

0.44

-0.087

k 0.17

(2.6)* 1.78 f 0.09

)

0.70

0.026

+ 0.26

By extrapolation from Fig. 1. t Mean for first 12 hr = 0.11 + 0.05.

+ 0.04

1.39 + 0.17

(1.5)* 1.17 0.61 2.3

-

0.04

R.

1”_.. Table 3. Effect of hydrostatic

Genus

pressure

and Species

W. BRAN-R CI 4.

on so&urn

influx rates in abyssal

Depth of capture (m)

of a negative Na* balance for the abyssal Acanthogammurus. The sign test applied to the data points for this species likewise leads to the inference that the Na balance is smaller than zero (P < 0.035). Finally, increasing the number of data points available for analysis by including in the series for species D-IV the results up to 12hr, rather than only up to 4 hr. the mean Na balance for this expanded series is found to be -0.11 f 0.05 ,uEq/g,ihr and again establishes to an acceptable degree of probability that this value is indeed smaller than zero (Z = 2.3, 0.025 < P < 0.05). Among species caught in deep water only species D-II fails to show a negative Na balance which can be accepted on statistical grounds (P = 0.5 for the null hypothesis). This species. however. is a eurybathial, almost ubiquitous, one and, hence. may not show characteristics typical of deep water gammarids.

In our gammarids, as in other gammarid and decapod crustaceans (cf. e.g. Shaw PI ~11..1959a.b) apparent Na+ influx rates as determined by tracer methods remain constant for well in excess of 4 hr since the rise of internal Na+ specific activity which might affect the measured apparent isotope uptake rates is slow. Nonetheless, we restrict the following analysis to values obtained during the first 2 hr in the NaZ2 containing environment to avoid any effect related to the observed decrease in the numerical values of net Na balance (cf. Table 2, Columns 3 and 4). Effect of’ time ujier capture. Using Parapallusea lagowski, species D-III, a systematic study was made of the effect of time from capture to the beginning of the flux studies. Animals tested 2 hr after capture had a mean Naf influx rate. #J%~+. of 0.83 * 0.04 mEq/g/hr compared with 0.65 + mEq/g/hr for animals tested 12 hr after capture: the decrease is not statistically secured (0.05 < P < 0. I ). In shallow water forms. such effects appear to he missing altogether. &, . = 0.7 1& 0.03 and 0.77 2 0.04 mEq&hr for species S-If tested 2 and 12 hr. respectively, after capture: corresponding values for species S-f are: 1.78 i 0.09 and 1.51 + 0.05 mEqjg/hr. In both cases. the differences are devoid of statistical significance (P > 0.1). Nonetheless, except where stated otherwise. the results reported below are based upon tests started I?hr after capture. fZfict o~‘c~)~?pressjo~.Representatives of species D-I and D-III were studied not only at 1 atm but also at a pressure corresponding to their respective capture depths. Species D-I was tested 2 hr after capture. both at I atm and at 137 atm. Species D-III was similarly tested I2 hr after capture at pressures of I and 9.5 atm.

gammarlds

from

Lake Balkal

Na Influx rate (pEq/g’hr) at habitat pressure (1 atml

The results are summarized in Table 3. In both species, recompression resulted in substantial and statistically significant increases in the rate of Na’ influx. The increase observed at 95 atm was by 33:;. and that at 137 atm by 7896, suggesting the possibility that the pressure enhancement of Na’ influx increases to some extent with increasing pressure. Relative berween Na + infrux rutes und uet %a balance. The relation between these two functions in the several species where both were measured is illustrated by Fig. I. The curves reflect the difference in the sign of Na balance for deep and shallow water species and suggest that high Na* influx rates correspond to high absoiute magnitudes for the deviation of Na balance from neutrality. By extrapolation. Fig. 1 further suggests that the high observed positive Na balance for the one littoral species, L-I, should correspond to a high Na+ influx rate, possibly near 2.6 mEq/g/hr. Data to substantiate or refute this point by direct measurement were not obtained in 1978. Relafi~n to body ~veig~t. Mean body weights for the species studied here ranged from 0.13 to 4.2 g, sufficient to test the relation between body weight and Nat influx rates at a common external Nat concentration (0.17 mEq/‘l). A plot of log (Na” influx rate) against log (body weight) (Fig. 2) shows that weight specific Na + influx rate is not constant but decreases systematically with increasing body weight. Since the flux rates for deep water and shallow water gammarids were determined at different temperatures (4°C for the deep and 9’C for the shallow species) more precise comparison requires reducing these data to a common test temperature, for which 4°C was chosen. A QZo of about 2. corresponding to an activation energy of about IO.5OO~al~m~~lfor Na + transport, was accepted as the most probable value (see Discussion) and used to recalculate Na’ influx rates

c

05

IO

15

2

2.5

Nd Influx-yEq&‘h

Fig. 1. Net sodium balance at 1atm for Baikalian gammarids as a function of weight specific sodium influx rate. l Deep water species, o shallow water species. I-range of balance values for littoral species (L-I).

Na+ transport in Lake Baikal gammarids

123

with a correlation coefficient of 0.96. Clearing logarithms and multiplying through by Wgives:

of

eNa+ = 0.84 x W0.53pEq/hr

log (Body

weIghi)

Fig. 2. Na+ influx rates at 1 atm (&J of Baikalian gammarids as a function of body weight. 0 Deep water species, 4”C, 0 shallow water species, 9”C, x shallow water species-recalculated to 4°C (see text). The line shown corresponds to the regression equation shown in the text. for the two shallow water species for 4°C. The series of values which represent Na+ influx rates for all gammarid species tested now closely define a com-

mon line in the log-log plot of Fig. 2 and allow the computation of an equation representing the regression of Na+ influx rate on body weight in 0.17 mEq/l Naf solution for five species of gammarids: log k = -0.43 log CM ( I (. > where &a is Na+ influx rate in pEq/g/hr and W is weight in g. The correlation coefficient is 0.93, and the slope is clearly less than 0 (P << 0.001). Clearing of logarithms and multiplying through by w one obtains the relation between body weight and Na+ influx rate per whole animal: &a+ = 0.63 W”.“. Na+ eflux rates

Direct determination of sodium efflux rates was impracticable during this first joint study for reasons of radiologic safety which precluded the necessary sodium determinations on radioactive hemolymph samples. Estimates of mean efflux rates were obtained, however, by subtracting the mean Na+ influx rate from the mean net sodium balance for each species at the test temperature actually employed in the experiments. The resulting values for Na+ efflux rate per gram animal plotted on log/log coordinates as a function of body weight for each species can be represented by a single regression equation for Na+ efflux on body weight (Fig. 3): = -0.47.log

log E$

(.

I

I

-0 8

I

I

I

-0 4

log (Body

I 0

Relation between Na+efJlux and Na’ inJlux

While Fig. 3 shows the relation between the regression lines for log &at and log eNa+ on body weight for the several species, a clearer picture of the relations implied can be obtained from a linear representation of Na’ efflux rate as a function of Na+ influx rate (Fig. 4) for the five gammarid species for which such estimates can be derived from the present data, On such a graph, the line &,+ = eNa+ separates the territories of positive and negative Na* balance. For a given influx rate, the efflux rates in Fig. 4 are higher for deep water than for shallow water species-corresponding to the difference in sign of the net sodium balance for the two groups. The maximum possible impact of correcting for test temperature is shown by the double arrowheads in Fig. 4, computed upon the assumption that lowering the test temperature for the shallow water species has no effect upon efflux rates. If efflux rates were also lowered by cooling, the curve representing the resulting situation would fall between the double arrowheads and the curve representing the data for the shallow water species at 9°C. Figure 4 suggests that the temperature effect alone may suffice to restore the shallow water open lake gammarids to neutral sodium balance.

& (

>

for the relation between Na efflux rate for the whole animal and its body weight. Examination of Fig. 3 shows that the efflux rates for the shallow water species fall systematically below the calculated regression line while the values for deep water species fall on or above that line. This cannot be explained by the different test temperatures for the two series: observed changes in Na balance as a function of temperature (cf. Lockwood, 1960) are compatible with the assumption that only the energy coupled Na+ influx in freshwater crustaceans has a sufficiently large temperature coefficient to be taken into account under circumstances such as the present. If efflux had a QIo between 1 and 2, application of the resulting correlation to the data from the shallow water gammarids would merely further separate their Na+ efflux rates from the larger ones for the deep water gammarids of comparable weight.

I

>

I

04

1

f

08

weight)

Fig. 3. Naf efflux (eNa= net balance -Na+ influx) of Baikalian gammarids as a function of body weight. 0 Deep water species, 0 shallow water species. The solid line corresponds to the regression line for lNPon W-regression line for 6~~.

”

I 0

1

I

No+Influx

-

;Eq/q/h

:

Fig. 4. Relation between Na+ influx and Na+ efflux rates for Baikalian gammarids. l Deep water species, 1 atm, 4°C; + Deep water species, habitat pressure, 4°C; 0 shallow water species, 1 &m, 9°C; -ct shallow water species, recalculated to 1 atm, 4°C. - Influx = efflux.

The deep water gammarids, on the other hand, are seen to fall on a line clearly different from that for the shallow water forms, even after the maximum temperature correction has been applied to the latter: all points for the abyssal gammarids fall above the line 4% = e\,,. separating negative from positive Na * balance conditions. The previous analysis of Fig. 2 led to the conclusion that at a common test temperature. and at 1 atm, Nat influx rates for deep and shallow water gammarids of common body weight do not differ substantially from one another. Conversely. Fig. 3 led to the inference that Na etflux rates of deep water gammarids tend to be greater than those for shallow water forms of similar weight, The data, then. suggest that the separation of deep and shallow water forms obsctved in Fig. 4 should be attributed largely to a higher Na* efflux rate for the abyssal species at 1 atm and 4 c‘.

Table 3 showed that pressure increase to levels characteristic of the habitat from which the animals were captured entails increases in the rate of Na’ intlux. This effect has been applied to the data of Fig. 4 and the result is indicated by the position of the single arrowheads. It would appear that this effect by itself could suffice to restore the abyssal gammarids to neutral Na * balance. The possibility of a pressure effect upon Na’ efflux cannot be discounted but no data were obtained on this point during 1978. DISCIIWON The first paper of this series (Brauer ef ul., 1979) reported data which established the fact that among the diversified gammarid fauna of Lake Baikal species inhabiting depths of 900 or more meters show definitivc evidence of adaptation. or of acclimatization, to the relatively high pressure regimes that prevail in the abyssal regtons of Lake Baikal. The present communication takes this subject further by demonstrating that when such abyssal gammarids are compared to shallow water gammarids with respect to sodium balance at 1 atm, there is a qualitative difference between shallow and deep water groups: abyssal gammarids. shortly after capture. are found to be in negative sodtum balance while shallow water forms are in neutral or in positive sodium balance. Analysis of the mechanisms underlying this characteristic of the abyssal gammarids requires comparison of the sodium lhtx data for species of widely different weights. A second significant discovery coming out of the present series of observations is that the relation between body weight and sodium influx rates takes the familiar exponential form &.,, = .4 x W’. where A and B are constants and B has a value of 0.57. substantially smaller than 1.0. Published data for maximum Na+ influx rates in other fresh water crustaceans (Shaw. 1959a,b: Shaw & Sutliffe. 1961) can be shown to correspond to the same basic form of equation. The actual equation relating to the published in values is: F,,,,,, = 1.32. W”.55 pmol.!hranimal. which the exponent is substantially identical with that deduced for the present series. The similarity can be pushed further: for those freshwater crustaceans for which this factor has been investigated so far, the relation between external Na+ concentration and Na+

influx rates is adequately the form:

described

hy an cquarlon

$1)

The magnitude of the constant C‘ for different frcshwater crustaceans was found to range from 0.15 to 0.2 mmol (cf. Lockwood. 1967). Since the concentration of sodium in Lake Baikal water. in which the present studies were carried out. averaged 0.17 mmol/l, and since it appears reasonable to surmize that our animals might be described by a relation similar to that which applies to the other fresh water crustaceans, under our test conditions the Baikalian gammarids should be transporting sodium at approx one-half their maximum capacity. Using this inference to compute hypothetical maximum sodium transport rates for the Baikalian gammarids. the scaling equation becomes F,,,, = 1.26. W”.57. which is all but identical with that deduced from the data of Shaw, and Shaw & Sutliffe. Pooling all available data. including the present ones. and computing the regression equation for F,.,,(Na) on body weight. we obtain : (Na) = 1.30. U”’ ”

F,,,

with a correlation coefficient for the logarithmic form of the equation of 0.98 (Fig. 5). Taken together, then, the totality of the data available indicates that over a lOOO-fold range of weights. and a several lOO-fold range of weight specific Na’ influx rates, maximum Na’ uptake rate in fresh water crustaceans can be described by a single common equation, characterized by the 0.55 to the 056th power of body weight. Thts exponent appears to be significantly below the values from 0.66 to 1.0 (cf. e.g. Bertalanffy, 1957 and Weymouth et (I/.. 1944), which characterize the relation between body weight and metabolic rate for various freshwater crustaceans. It seems to us a useful working hypothesis to assume that some physiological function determined by the total surface area of a given individual may play a predominant role in determining the capacity of the sodium transport apparatus of freshwater crustaceans.

1

I.5

I-

Z I

0.5-

E 4

z

j H

O 0.!5-

-I -

-1.51 -2

0

-I log

I (Body

2

3

4

weight)

Fig. 5. Maximum Na+ influx rate as a function of body weight for three fresh water crustaceans (data from Shaw, 1959a.b; Shaw & Suttliffe. 1961) together with five Baikalian gammarids (see text for calculations).

Na+ transport in Lake Baikal gammarids A word may be in order with regard to the corrections applied to the results for shallow water gammarids in order to reduce them to a common temperature with the abyssal gammarids. The reason for choosing more than one reaction temperature in the first place was to minimize possible effects of thermal shock by testing the various animals at temperatures as close as possible to those prevailing in situ. A median temperature of 9°C seemed to us closer to the range of temperatures encountered by the shallow water species as a group than the 4°C proper for the abyssal forms, or the 56°C which may be presumed to prevail at depths between 50 and 1OOm in Lake Baikal. We are not aware of any published data in which the effect of temperature shifts upon Na+ flux rates in freshwater crustaceans has been determined directly. However, data have been published concerning the effects of temperature shifts on net sodium balance in several species, and indicate that increases in temperature are associated with increasingly positive net sodium balance values (cf. Lockwood, 1960). The magnitude of the reported effects, furthermore, is compatible with the assumption that this effect is exerted primarily upon the Na+ influx rates which in freshwater environments involve active transport. Reported activation energies for sodium transport in other biological systems range from 10,000 to 20,000 calories per mol (Caplan & Essig, 1977) corresponding to Q, 0 values between 2 and 3. A Qio near 2.0 also results from measurements of the temperature coefficient of activity of NaK ATPase isolated from fish gills (Moon, 1976). All things considered, we concluded that a temperature coefficient near the lower end of the range was more likely and, therefore, chose to make the revision shown in Fig. 2 on the basis of a Q LOof 2.0; using a larger Qlo would merely underscore the conclusion that Na+ influx rates at 1 atm and 4°C for abyssal gammarids are not smaller than those for shallow water gammarids. Clearly, a more precise definition of temperature effects upon this transport mechanism will require direct experiments in which the effect of temperature on at least two of the three parameters, sodium balance, sodium influx and sodium efflux, is measured simultaneously. From the point of view of the application of these results to the present discussion, it should be noted that the hypothesis utilized here, e.g. the assumption that influx rate alone is affected by changes in the reaction temperature, would have the effect of minimizing differences between deep and shallow water forms. As illustrated by Fig. 4, if efflux, as well as influx, rates were assumed to be temperature sensitive, the resulting temperature corrected points representing the relation between sodium influx and sodium efflux in shallow water forms would correspond to even more positive values of Na+ balance than shown by the double arrowheads. Such doubly correct values would then, of necessity, be even more remote from the values shown for abyssal gammarids, all of which fall above the line 4 = l, i.e. in the sector corresponding to negative Na balance in Fig. 5. The degree of sodium depletion implicit in the negative sodium balance of abyssal gammarids during the first day after capture is by no means negligible: if one assumes that these animals achieve a new sodium balance as soon as 12 hr after capture, integration

125

under the curves describing the time course of sodium loss leads to estimates of 420% loss of total exchangeable sodium (cf. Robertson, 1961, for relation between total and exchangeable Na in crustaceans) in large and small abyssal gammarids, respectively. Such losses may be compatible with survival, but no data are available as yet to describe the manner in which they would be reflected in changes of the fluid and electrolyte make-up of these animals. The magnitude of the effect, however, is such that it might well become a significant component in the biochemical events responsible for the inability of crustaceans retrieved from depths twice as great as those prevailing in Lake Baikal to survive under a 1 atm regime (cf. MacDonald, 1975). The negative sodium balance observed in the deep water gammarids at 1 atm could reflect any or all of the following: pecularities of the sodium uptake mechanism, of renal excretion or of percutaneous sodium losses. Data obtained in freshwater gammarids and in other freshwater crustaceans (cf. Lockwood, 1961 and 1967) suggest that renal sodium losses can hardly account for more than 2-3% of the total sodium loss in small gammarids and perhaps as much as 15-20x in larger forms. While there are no data concerning the effect of pressure on urine flow in crustaceans, there are observations to indicate that stress, due to manipulation of the animals, may result in reduced urine flow or anuria (Riegel & Kirschner, 1960) and, hence, tend to decrease net Na+ efflux. Althogether, we think it extremely unlikely that changes in renal function can account for the excessively high values of sodium efflux in the abyssal gammarids. The data illustrated in Fig. 2 suggest that, under our conditions, sodium uptake rates at 4°C are virtually the same for abyssal gammarids as for shallow water gammarids of corresponding weight. Together with the observed negative sodium balance, this implies that sodium efflux rates at 1 atm must be substantially larger in the abyssal than in the shallow water gammarids with respect to both, a common body weight, and a common sodium influx, a conclusion borne out by our analysis of Fig. 3 (see Results). It is self-evident that, in their normal habitats, both abyssal and shallow water gammarids must be in neutral or slightly positive sodium balance during most of their life span. With regard to the shallow water forms, the data incorporated in Figs 2 and 5 suggest that environmental temperature by itself may suffice to account for the positive sodium balances observed in our experiments, and that correction to temperatures prevailing in the natural habitat of the animals would return these animals very nearly to neutral sodium balance. In the case of the abyssal gammarids. the experimental observations on ParapaNasea lagowski and Odontogammarus margaritaceus suggest that returning the animals to their habitat pressure entails a very substantial increase in the rate of sodium uptake. As illustrated in Fig. 4, the amount of this increase by itself would appear to be sufficient to return the animals to normal sodium balance, assuming such pressures produced no additional modifications of sodium efflux rates. Since the conflicting data in the literature dealing with pressure effects on membrane permeability (cf. e.g. Pequeux, 1972; Goldinger

et cd.. 1978) do not permit any inference regarding the effect of 1OOatm on aggregate cutaneous Na’ permeability in crustaceans, the importance of this factor can only be assessed on the basis of direct experimentation. The increase of Na’ influx rates under the influence of pressures of the order of 10Oatm may well prove to be a specific characteristic of animals residing at depths of 1OOOm or more and, hence, adapted to such pressures. While we have obtained no data concerning the effect of elevated hydrostatic pressures upon sodium transport rates in shallow water Baikatian gammarids, we do have such results of pilot studies for North American fresh water, shallow water gammarids (Crtrngo~z,w sp.). These indicate that. in contrast to the abyssal forms, in these animals pressures of 80 100 atm result in a measurable decrease of the rate of sodium uptake. Concerning the nature of the pressure-induced effects upon the sodium transport mechanism and, in particular. of the difference in the response pattern associated with adaptation to shallow or to abyssal regimes. one can only speculate at the present time. The effect might reflect pressure-induced changes in intracellular ionic make-up. perhaps secondary to pr~ssLlre-induced changes in dissociation constants of weak electrolytes, but it is di%icult to see how such an effect could be made the basis of the observed adaptive differences. It seems more likely that the different responses reflect changes in the structure of the energy coupled transport system itself and. more particularly, perhaps in the transport ATPases which it seems reasonable to surmize as the molecular basis for the observed Na’ transport against a thousandfold concentration gradient (Spencer c’t ul.. 1979). This hypothesis is made more appealing by the observations of Moon (1976). who compared NaK ATPase isolated from the gills of shallow water and deep-sea fish. His data reveal that while in shallow water forms pressures of 169 atm and 338 atm reduced the activity of the NaK ATPase. the corresponding system isolated from the gills of deep-sea fish showed an increase in activity under the same pressure conditions. The magnitude of the increases and the decreases in the two systems (259, increase or XY’,, decrease at 169atm) are of an order quite compatible with the changes in the overall Na’ transport fLlnct~ons observed in our deep water and shallow water crustacean preparations, respectively. We fully recognize that to transfer conclusions from a vertebrate to our invertebrate systems might be hazardous. At the very least, however. we feel that the striking parallelism observed lends plausibiiity to the more general conclusion that we are inclined to draw from our own data. To recapitulate the conclusions we draw from the present study: Evidence has been obtained to demonstrate that abyssai gammarids from Lake Baikai, when exposed to 1 atm pressure at habitat temperature, go into negative sodium balance. while shallow water gammarids remain in neutral or positive balance under the same conditions. The data suggest that the rate of sodium uptake observed in abyssal forms under these test conditions follows the same relation to body weight as that for shallow water gammarids and, by implication, indicates that sodium

and. hence. permcabllit~ of ab>ssal gammarlds to sodium ion at I atm is higher than for shallow water gammarids. Direct evidence shows that at habitat pressure (i.e. about 1OOatm) the rate of sodium uptake of abyssal gammarids increases over the levels obt~~il~ing 1 atm. while data to he presented in detail elsewhere indicate that similar prcsaures applied to shallow water gammarids product an actual decrease in the rate of sodium uptake. The pressure-induced increase m sodium uptake rate appears sufficienti) large to return the negative sodium balance observed in these animals at I atm to a neutral sodium balance at habitat pressure. ArgLi~tlents have been advanced to suggest that the difference in transport function indicated b> these results reflects adaptation to a high pressure regime at a molecular keel. The data here presented provide the first r\,idence known to us of a specific physiological regulatory mechanism rnvolved in adaptation to high pressure regimeb. and suggest at least one basis for the stress experienced b! deep water animals when retricced to and maintained at surface pressure. efllux

.~tc,krto~~~lrdy~~rlu,lts The authors M.I$~ to acknowledge the encouragement and help received throughout the conduct of this research from Professor G. 1. Galazii. Director of the Institute OF Limnolog). Siberian Division, USSR Academy of Sciences. The authors htrthcr gratefully acknowledge the generous assistance of Professor R. K. Salayev. Director of the Institute of Plant Physiology and Biochemistry. Siberian Divlston. l’SSR Academy of Sciences. who authorized use of the facihtics of the Isotope Cabinet of that Institute in connection aith the work here reported. Our sincere thanks are due to Professor K. K. Votyntzev. of the Institute of Limnoloy\. Siberian Division, USSR Academy of Sciences. for graoiuusly making iC possible to obtain these analyses. This work was supported in part hq grants from The National Geographic Society and from The Grifiis Foundation REFERENCES

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