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Volume regulation in Nereis diversicolor—I. the steady state
Comp.Biochm. Physiol.,1974, Vol. 47A, pp.1199 to 1214. Pwgamon Press. Printed in Great Britain
VOLUME REGULATION IN NEREIS DIVERSICOLOR-I. THE STEADY STATE C. R. FLETCHER Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England (Received 3 May 1973)
Abstract-l.
The steady-state parameters of volume regulation in a population of Nereis diversicolor have been examined. 2. The water permeability changes as a function of the salinity of acclimatization even when expressed in a form independent of the animals’ water content, and is highest in full strength sea water. 3. Drinking does not contribute significantly to water movements. 4. Water permeability measured by osmotic flow is substantially larger than that determined by movement of labelled water molecules, and the difference is not explained by isotope effects. 5. The rate of urine production is highest in animals acclimatized to 2% sea water, but is substantial even in animals acclimatized to full strength sea water. 6. Hydrostatic pressure is not the primary cause of fluid flow through the nephridia.
INTRODUCTION
NEREIS DIVERSICOLOR is an estuarine polychaete
which is subject to both low and variable salinities, and may be most abundant where the salinity is most variable (Smith, 1956). It has been used in investigations on osmotic and ionic regulation as it is a good osmoregulator at low salinities but osmoconforms in higher salinities (Smith, 1955; Hohendorf, 1963). However, it has been shown that these animals lose their ability to control their volume in the absence of external calcium ions (Beadle, 1937; Ellis, 1937) and these authors’ explanation that this results from an increased water permeability has not yet been substantiated. It has also been suggested that adaptation to low salinities is in part accomplished by a reduction in water permeability (Jorgenson & Dales, 1957), but their evidence was criticized by Potts & Parry (1964) on the basis that they had failed adequately to control the temperatures, had not determined the concentrations of the body fluids and had assumed that the rates of urine production did not However, Smith (1970a) has shown that the change in their experiments. permeability measured by the influx of labelled water molecules does change, when expressed as a rate constant. This work ignores the fact that water permeabilities measured by tracer and osmotic flow methods seldom agree (e.g. Rudy, 1967; Motais et al., 1969). Also it has been shown that the water content of these animals is higher when adapted to lower salinities (Oglesby, 1970), and 1199
1200
C. R. FLETCHER
this will result in a reduced rate constant even if the permeability of the presumably unchanged surface area is unaltered. N. diversicolor’s normal environment is one in which the salinity may fluctuate substantially during the tidal cycle as well as seasonally, and with the exception of early work (Beadle, 1937; Ellis, 1937), which showed that these animals have substantial abilities to control their volumes in these conditions, little work has been published on events occurring in response to a salinity change, and this has been largely concerned with ionic fluxes, and has raised more difficulties than it has illuminated the mechanisms of adaptation (Oglesby, 1972). Experiments have been undertaken to determine whether the osmotic water permeability is reduced in response to a reduction in salinity, to determine whether lack of calcium does result in an increase in water permeability and to determine the mechanisms by which the animal adapts to a new steady state after an experimental change of salinity. These last two items require a detailed understanding of the water balance in the steady-state (acclimatized) animal. It has been suggested that animals from different locations may differ considerably (Ellis, 1937), and although this was not supported by Smith (1955) it was thought desirable to determine the parameters of water balance for acclimatized animals for the population used in the subsequent studies rather than relying on published data. This paper reports the results of experiments on the water balance of acclimatized animals and on the existence of true changes in their osmotic water permeability; subsequent papers will record the results of experiments on the effect of calcium and on the mechanisms of adaptation to a changed salinity.
MATERIALS
AND
METHODS
Specimens of Nereis dive&color weighing between 0.25 and 0.8 g were collected from the upper reaches of Bridlington harbour on the East Coast of England on a number of occasions between April 1970 and October 1972 and were maintained in the laboratory in 20% sea water at 12 f 1°C until required. They were adapted gradually to other salinities as already described (Fletcher, 1970a). Each animal spent at least 6 days in the final acclimatization salinity, the medium being changed twice. All experiments were conducted at 12 + 0.5”C. Sea water was obtained from the beach at Bridlington or from the Wellcome Marine Laboratory at Robin Hoods Bay. Dilutions were prepared with distilled water, and when concentrations greater than full strength sea water were required they were prepared by dissolving the appropriate quantities of salts in sea water, using the values of Hale (1965). The chlorosity of all solutions was checked with an Aminco amperometric chloride titrator. Samples of coelomic fluid for osmotic pressure measurements were taken in clean melting point capillary tubes which had been drawn to a fine point at one end which were inserted through the dorsal body wall adjacent to the blood vessel. The capillaries were then sealed at the remote end and the cellular material sedimented in a microhaematocrit centrifuge. The capillaries were broken just above the cell/fluid interface and the supernatant used immediately for osmotic pressure determinations, Osmotic pressure measurements were made using a differential method based on the difference in vapour pressure and hence equilibrium temperature of two drops of fluid hanging on hermetically sealed matched bead thermistors in a temperature-controlled
VOLUME REGULATION
IN NEREIS DIVERSICOLOR--I
1201
chamber saturated with water vapour, the differences in temperature being determined by differences in resistance of the thermistors connected in a transformer bridge circuit. The measurements were reproducible to f 3 mosmolal SD. and were independent of the nature of the solute. The water content of animals and organic dry weight were determined by weighing the animals, drying them under vacuum over silica gel, re-weighing and then ashing them in a muffle furnace at 600°C and weighing them again. All weighings were to f 0.1 mg. Diffusional permeabilities were measured with tritiated water from the Radiochemical Centre, Amersham, England, which was incorporated in the appropriate experimental solution to 0.05 &i/ml. The animals were placed in 50 ml of labelled medium for 10 min during which time the medium was stirred by a stream of air bubbles from a fine pipette, and then removed, rinsed for about 5 set in a nylon teastrainer with unlabelled medium from a wash bottle and blotted dry. They were stored individually in polythene capped vials at - 20°C. The body water was sublimed off under high vacuum (< 10e3 torr) and collected on a cold finger containing liquid nitrogen, and subsequently allowed to melt in a dry atmosphere and collected in a clean tube. Aliquots (0.1 ml f 1 per cent) were taken for assay of specific activity by liquid scintillation counting using 5 ml of toluene-based scintillator containing 67 ml/l of the blending agent NE 520 (Nuclear Enterprises Ltd.). Samples of loading media were similarly handled to provide standards and since the counting mixture was of constant composition quenching corrections were unnecessary. The counting efficiency was about 40 per cent in a Beckman 1650 counter. Tests were conducted to ensure that there was no significant isotopic fractionation in the sublimation of water from the animals by partly drying one group of loaded animals, and then subsequently removing the residual water. The mean ratio of the specific activities of the two fractions was 1.02 with an SE. of 0.012. Tests were also conducted to show that all the water in the animals exchanged as one well-mixed pool by loading sixty animals for 4 hr in 20% sea water, when they were close to isotopic equilibrium, and then allowing them to efflux into a large volume of 20% sea water for times from 0 to 100 min before removing them for determination of the specific activity of their body water. The results are shown in Fig. 1, and demonstrate that the whole body water exchanges as though it were in one well-mixed compartment, i.e. internal exchanges are much more rapid than exchanges with the environment. Thus we may use a relatively simple formula to determine the rate constant for isotopic water exchange, K: 1 A0 K = ;loge(&A) where A0 is the specific activity of the medium and A is the specific activity of the animal’s body water after a time t in the medium. In order to investigate the significance of isotope effects one experiment was performed in which deuterium was used as well as tritium as a tracer for water molecules, and the medium contained 10% DzO as well as tritiated water. This medium was allowed to stand at room temperature for a week before use to allow isotopic equilibrium to be established. Pure water was extracted as already described and deuterium concentration measured in a density gradient column of xylene and carbon tetrachloride with suitable standards. Osmotic permeabilities were deduced from measurements of weight changes occurring after relatively small changes of external salinity. It was found that after such a change the animal initially starts to swell or shrink, but after an hour or two the rate of change of weight becomes very much reduced below that which would be expected of a perfect osmometer, or indeed of an animal lacking any direct means of controlling its volume (Fig. 2). A theoretical analysis of such an animal is given in the following paper on adaptation to a reduction in salinity. Indeed departures from such theoretical predictions are
1202
C. R. FLETCHER
Efflux
time,
min
FIG. 1. Loss of Iabelled water from animals. The animals were loaded for 4 hr and then placed in unlabelled medium for the time shown before the specific activity of their body water was determined. Each point is the mean of six observations + S.D.
Time,
hr
FIG. 2. Volume changes of animals acclimatized to 50% sea water after transfer to 70, 50 or 30% sea water. The theoretical behaviour of animals which lacked means of volume control are shown by broken lines. In this and subsequent figures variation about a mean is shown by a vertical line of + 1 SD. and a central block of f 1 S.E.
VOLUME REGULATION
1203
fN AWZ.?ZS LtIVEASICOLOR-1
apparent as little as 1 hr, but not 30 min after changes of salinity, implying that mechanisms of volume control are being brought into effect after a delay of 30 min or more after the change of salinity. Further evidence of this is presented in the same paper. Thus we may reasonably assume that weight changes during the first 30 min after such salinity changes are caused by osmotic water movements, and enable us to determine the osmotic water permeabilities of the animals. ISince the animals are not normally iso-osmotic in any salinity the formula describing the behaviour of the weight of an animal after transfer but before urine flow or total internal solutes change is not very simple, and derivation is given in the later paper. The formula is -1 p = t(C,+C,i-Co)
a0 Co1 If+c,+CO’-CO)‘op”
f(C, -t-Co’ - Co) ( l-
ao(C0 -
C,)
II’
where P is the permeability in kg H,O]kg animal per hr per unit osmolal concentration difference, Co is the osmolality of the acclimatization medium, Co1 is the osmolality of the animal’s body fluids in the acclimatization medium, z. is the water content of the animals, kg/kg animal when acclimatized to Co, Cr is the osmolality of the medium to which they are transferred and f is the fractional increase in weight t hr after transfer, relative to controls, Thus by substituting observed data for f as a function of t using determined values of Co, C,, Col-Co, and z. we may determine P subject to the above assumptions, and a decrease of the value of P as t increases indicates where the assumptions cease to be valid, and demonstrate the operation of some form of volume regulation. Since the results are based on relatively small changes in weight a reliable and accurate way of weighing wet animals with a minimum of stress was needed. The water containing the animal was poured through a nylon tea strainer, collecting the animal. The underside of the strainer was blotted on absorbent paper and the worm emptied onto a filter paper and rapidly weighed to 0.1 mg. The worm was returned to its water in a clean beaker and the filter paper reweighed. This was found to give weights reproducible to kO.2 mg; the animals were out of water about 20 set and were not touched at all. The relative constancy of the mean weight of control worms showed that this procedure did not disturb their volume unduIy. In order to ascertain whether drinking formed a significant part of the water flows the rate of a?!5 sulphate uptake was determined as a measure of drinking, after the method of Potts et al (1967). Yl labelled sodium sulphate was added to acclimatization media to a concentration of 0.2 &i/ml, and animals removed after times between 0.5 and 8 hr. The worms were washed externally by three 5-min periods in unlabelled media with rinsing between each and then blotted dry and dried under vacuum over silica gel. The labelled sulphate was released by the oxygen flask combustion technique of Dobbs (1963) and dissolved in 10 ml of his basic scintillator, of which a 9-ml aliquot was counted. Standards of 0.05 ml of the labelled media were dried on absorbent paper and subsequently handled like the animals. Quenching corrections were made by the external standard channels ratio method. Measurements of the hydrostatic pressure difference across the body walls of these animals were made using a capacitance pressure transducer in a bridge circuit driving a Beckman pen recorder. The pressure cannula, containing sea water diluted to approximate to the animals’ body fluids, was made of fine polythene tubing with a small cigar-shaped glass tip which was inserted through the dorsal body wall into the coelomic cavity. Elasticity of the body wall held the cannula in place without visible leakage. The animal was allowed to swim or crawl freely in a small glass dish. The response of the system was limited by the recorder to about 1 sec. This piece of work was performed at the University of Birmingham using animals obtained from Christchurch harbour, Hants.
C. R. FLETCHER
1204
RESULTS
Osmotic concentrations of the body jluids The osmotic concentration of cell-free coelomic fluid samples were determined from seventeen or more animals acclimatized to each of a range of salinities Measurements were made relative between full strength and 0.5% sea water. to the acclimatization media, and these were determined relative to distilled water. The results are shown in Fig. 3, and it is observed that the worms were significantly hyperosmotic to the media in all salinities, the difference being largest in worms acclimatized to 2 and 1% sea water.
Acclimot~sot~on
FIG.
3.
medium,
mosmolal
Osmotic concentration difference between the coelomic acclimatized animals and the external medium.
fluids of
Water content and ash free dry weight This was measured using groups of nineteen or twenty animals acclimatized to a similar range of salinities, and the results are given in Table 1. The water content is seen to vary markedly in the higher salinities, and if the ash-free dry weight is assumed to be constant we may deduce the changes in weight of individuals when they are acclimatized to a different salinity. Osmotic permeability This was measured using animals acclimatized to 100, 50, 30 and water. The first group consisted of sixty animals, twenty of which were individually in 1OOo/o sea water, twenty of which were transferred to water and twenty of which were transferred to 120% sea water, and their as fractions of the original weight determined after t, 4, 1 and 2$ h.
10% sea retained 80% sea weights Animals
VOLUME
TABLE l-WATERCONTRNT
REGULATION
1205
IN NEREIS DIVERSICOLOR--I
OF ANIMALSASAFUNCTIONOFACCLIMATIZATION
Sea water (%)
Chlorosity of medium (mm/l.)
100 50 20 10 5 2 1
533 255 105 52.1 27.0 11.0 5.63
Water content (g/kg of animal)
794.2 + 3.2 840.1+ 2.5 865.3 + 2.3 868-S + 2.1 878.3 + 2.3 879.3 f 4.4 887.3 z!z2.6
SALINITY
Ash free dry weight (g/kg of animal)
178.5 144.9 125.1 123.6 114.0 112-l 104.8
(20) (20) (20) (20) (19) (19) (19)
+ 2.9 ?r:2.5 + 2.4 + 2-l * 2.2 * 4.4 + 2.6
(20) (20) (20) (20) (19) (19) (19)
Means + S.E. are given with number of observations in parentheses.
from the other salinities were similarly treated except that the transfers used for animals acclimatized to lOo/o sea water were to 0 and 20% sea water. A typical set of experimental results is given in Table 2, and the permeabilities calculated from these data in Table 3. The overall mean of PO, from the &hr and TABLE 2-WEIGHT OF ANIMALS ACCLIMATISED TO FULL STRENGTH
As A FRACTION OF THE ORIGINAL WEIGHT OF ANIMALS SEA WATER AFTER TRANSFER TO 80 OR 120% SEA WATER
Time after transfer (hi-)
Sea water Osmotic concentration of experimental media (%) (osmolal) *
*
1
2*
80
O-806
1.0227 +0*0015 (20)
1.0412 *o-o019 (20)
1*0705 & O-0028 (20)
1.1052 rf:0.0039 (20)
100
l-008
l-0014 f 00IlO (20)
1 a0004 * 0~0017 (20)
0.9997 f O-0016 (20)
O-9965 + 0.0033 (20)
120
1.222
0.9779 zb0.0018 (19)
0.9561 + 0.0043 (20)
O-9270 f o-0043 (20)
0.8878 + 0.0047 (20)
Means -I SE.
are given with number of observations in parentheses.
+hr measurements is taken as the permeability. Since the &hr and &hr measurements relate to the same animals they can be regarded as independent only insofar as the variability reflects experimental errors, not individual variability of the animals. Since the standard error of the weight changes rises almost linearly with time and is similar for controls and both groups of experimental animals it appears that the primary source of variability arises from the fact that
C. R. FLETCHER
1206 TABLE
~-VALUES
FOR
WATER
PERMEABILITY
CALCULATED
FROM
DATA
IN TABLE
2,
in kg H,O/kg animal per hr per 1 osmolal concentration difference Time (hr) +
2f
0.469 + 0.035 0.484 i: 0.061 0.477 t 0.035
0,462 + 0,028 0.459 & O-043 0,460 -1:0.026
0.384 + 0,031 0.413 + 0*057 0,398 ? 0.032
‘s 0.456 t 0,041 0.473 i 0.044 0.464 + 0.030
Swelling
Shrinking Mean
-1
Means + S.E. are given. Since twenty animals gave the swelling data and nineteen or twenty animals the shrinking data, the final value used in subsequent calculations was O-470 _+0.030 (IV = 39).
at any time an individual’s weight may be rising or falling in the absence of any change in external salinity. Thus the &- and &hr figures are taken as not independent but the swelling and shrinking data are independent. The standard error is a real measurement of the reliability of the mean but not an adequate measurement of individual variability. TABLE
~-WATER
PERMRABILITIRS MEASURGD BY OSMOTIC TO VARIOUS SALINITIES
FLOW
IN ANIMALS
ACCLIMATIZED
Acclimatization media Sea water (%)
Chlorosity (mm/l.)
p0lI
100 50 30 10
533 274 161 54‘4
0.470 + 0.030 0.280 + 0.022 0.161+ 0.016 0,116 t 0.023
p0, 2.63 1.93 1.23 0.94
+_0.17 z!z0.16 + 0.12 If:o-19
iv
39 37 40 38
P, in kg H,O/kg animal per hr per unit osmolal concentration difference. Posl in kg HzO/kg ash free dry weight of animal per hr per unit osmolal concentration difference. Means 4 S.E. are given. N is the number of experimental animals from which each figure is derived.
The pooled results for PO, for each acclimation salinity with standard errors are given in Table 4 and Fig. 4, and it is apparent that PO, does decline markedly as the salinity falls, but it may be noticed that PO, is here defined relative to the weight of the animal which is itself variable. Consequently the permeabilities have also been calculated relative to the ash-free dry weight using the data in Table 1. These values, given as POsl are also recorded in Table 4, and it is seen that the permeability is indeed reduced in the lower salinities.
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DIVERSICOLOR-I
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Diflusional permeability This was measured in several salinities to determine whether it was similar to the permeability measured by osmotic flow. Since preliminary results showed that it was not, experiments were undertaken to see whether the discrepancy could be accounted for by isotope effects by comparing permeabilities measured simultaneously with heavy water and tritiated water, and also to determine whether the isotopic flow varied significantly as a function of the osmotic concentration as diffusional permeabilities should be measured when there is no nett flow. Thus rate constants were determined for SHHO and for DHO influx from 50% sea water into animals acclimatized to the same salinity, and for sHH0 influx from salinities between 0 and 40% sea water into animals TABLE
S-RATE
CONSTANTS
FOR THE
EXCHANGE OF ISOTOPICALLY PERMBABILITIES
Acclimatization salinity (% sea water)
K hr-l
Pd
100 50 50 20 20
5.79 * 0.25 5*90+0.15 6.21 + O-17 3.43 f 0.10 3.63 + 0.04
0.131 -I-0.006 0.127 + 0.003 O-133 + 0.004 0.071 f 0.002 0.076 + O-001
LABBLLED
WATER
AND
Method and No. of animals 0.734 f. 0.036 O-876 + O-026 0.918 + O-032 O-568 f 0.019 0.607 f. 0.014
aH *H D 3H 3H
influx influx influx influx efflux
(N = (N = (IV = (IV = (N =
12) 20) 20) 10) 60)
Units: Pa kg HaO/kg animal per hr per unit osmolal concentration difference. Pdl kg H,O/kg ash free dry weight of animal per hr per unit osmolal concentration difference. Means + S.E. are given. TABLE
~-EFFECT
OF OSMOTIC
GRADIENTS ON THE RATE CONSTANTS EXCHANGE
OF TRITIATED WATER
Acclimatization medium chlorosity
Test medium chlorosity,
(mm/l.)
(mm/l.)
529
774 642 528 418 320
5.27 + O-18 (12) 5.65 Z!T 0.09 (12) 5.79 + 0.25 (12) 5-72 zb0.22 (12) 6.16 I!Z0.08 (12)
108
0.06 52.7 106 158 205
3.96 3.43 3.43 3.46 3.75
Khr-'
f f f f +
O-10 (10) o-11 (10) o-10 (10) 0.10 (10) 0.14 (10)
Means &-SE. are given with number of observations in parentheses.
C. R. FLETCHER
1208
acclimatized to 20% sea water, and for 3HH0 influx from salinities between 60 and 140% sea water into animals acclimatized to 100% sea water. The results are given in Tables 5 and 6. Also values for Pd, the diffusional permeabilities have been calculated from the values of K in Table 5, and are also given in Table 5, using the relationship
where Pd is defined in the same units as PO,, x0 is the water content of the animal and No is the number of moles in 1 kg of water = 55.5. The approximate sign is used since this will only be exactly true if the mole fraction of water outside is unity, but the effect of this will be less than 2 per cent. The values of Pa are also shown in Fig. 4. It can be shown that all values of Pd are very much smaller
0
I
I 20
I
40 Salinity,
I
I
60 80 % sea water
I
100 S
FIG. 4. Water permeabilities of animals acclimatized to different salinities, measured by osmotic flow (P,,, open symbols) or by labelled water fluxes (Pd closed symbols) + SE. Permeabilities expressed in kg Hz0 per kg animal per hr per 1 osmolal concentration difference.
than PO,, and the two appear to vary relatively independently. Like PO,, the diffusional permeability may also be expressed in terms independent of the water content of the animal, Pd’, and these are also given in Table 5. It may also be seen from Table 6 that the diffusional influx of 3HH0 in animals acclimatized to 20% sea water does not vary regularly with the external salinity (correlation coefficient probability > 0*05), but in the animals acclimatized to lOOo/o sea water the isotopic flux is significantly dependent on the external salinity (correlation coefficient probability < O-01).
VOLUME
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1209
The mean of the ratio of K for SHHO to K for aHHO was O-951 + O-006 S.E. (N = 20). Thus there is a significant isotope effect. If we assume that the isotope effect is a linear function of mass of the water molecule we may judge that the isotopic fluxes measured with sHH0 are about 10 per cent less than the flux rates for H,O. This ignores the fact that in this experiment a significant proportion of the sH fluxes were carried by 3H2H0 molecules, and a significant proportion of the deuterium fluxes were carried by 2H20 molecules, A detailed analysis suggests that these two factors cancel to make the original deduction correct to within 0.7 per cent. Drinking rate
The rates of uptake of 35S sulphate were determined as already described. No checks were made to ensure that the isotope was confined to the gut as it is difficult to dissect out without rupture, but the rates were low and establish an upper limit to the rate of drinking. After only 30 min all animals contained some tracer and the quantity rose linearly thereafter suggesting it had not started to leave the anus within 8 hr. A regression analysis of the quite variable data gave slopes which are given in Table 7, and intercepts significantly different to zero. TABLE
~---RATE OF UPTAKE OF TS SULPHATE (DRINKING RATE) Salinity (% sea water) 100
10 1
Rate of uptake (d/kg
per hr)
0.94 z!z0.07 (16) 0.48 k 0.03 (16) 1.60 + 0.07 (16)
Means f SE. are given with number of animals in parentheses.
The latter may have been caused by the habit of these animals of everting their proboscis in response to handling and possibly taking in some medium when it is retracted, or could be caused by an exchange phenomenon with the mucus or cuticle.
This was normally recorded over a period of about 10 min, and was relatively steady with small fluctuations when the animal was crawling or quiescent, but rose dramatically when the animal displayed the startle response of longitudinal contraction or commenced to swim. Since the behaviour resulting in these high values is seldom displayed by undisturbed animals they were ignored in taking an average pressure for each animal, which were then used to calculate the means shown in Fig. 5 : each point is the mean from ten to twelve animals. It is observed that whilst all the values are small they are significantly larger in the less swollen animals acclimatized to higher salinities.
1210
C.
I
FLETCHER
I
I
I
I
IO
30
too
300
I
0 a
R.
Acclimatisotion
medium chloride concentration,
I+
1000 mM
FIG. 5. Hydrostatic pressure differences across the body walls of animals acclimatized to a range of salinities.
Rate of urine flow From the information given in Fig. 3 and Table 4 we can cafculate the osmotic influx in a range of salinities, and if drinking is neglected this must equal the rate of urine flow. Since the uptake of sulphate was not shown to occur by drinking this may be true, and even if all the sulphate uptake occurs by drinking the latter will not contribute significantly to the urine flow. No values for PO, have been obtained in salinities below 10% sea water and it has been assumed to remain constant in lower salinities. The rates of urine flow calculated on these assumptions are shown in Fig. 6 with all the uncertainties shown as S.E.‘s.
0
I
o-3
i
I
I
I
I
3
IO
30
100 ’
Acclimatisation
FIG. 6.
medium,
% seawater
Calculated mean rates of urine production of animals acclimatized to a range of salinities f S.E. of estimate.
VOLUME REGULATION
IN NEREIS DIVERSICOLOR--I
1211
DISCUSSION
The differences in osmotic concentration between the animal and its environment were uniformly about 30 mosmolal higher than those reported by Hohendorf (1963) as freezing point depressions for animals from Kiel. However, the data of Smith (1955) show differences of up to 70 mM in coelomic fluid chloride levels of animals from different locations. He concludes that these differences were not significant since the higher values were from the survivors of experimental groups which had suffered high mortalities in the laboratory, but the data still suggest that distinct physiological races or environmental factors may be significant. The methods used for determining the osmotic permeabilities are related to those used by Jorgenson & Dales (1957), and the transfers used were of similar magnitude except in worms acclimatized to 10% sea water. However, in this paper it has been shown that whilst mechanisms of volume control are operating an hour or more after such changes in external salinity there is no evidence of their operation over shorter times. Furthermore, it will be shown in a subsequent paper that the rate of inulin clearance from the animals is scarcely altered in the first hour after a reduction of external salinity but rises considerably thereafter. Thus whilst this technique is subject to large random errors due to the inconstancy of the weight of individuals it seems that the means should not be subject to significant systematic errors. The osmotic water permeability is larger in worms acclimatized to higher salinities, and this is a true effect since it is still apparent when expressed in terms independent of the water content of the animals. This confirms the conclusions of Smith (1970a) based on measurements of diffusional permeabilities and not corrected for changes in the water content of the animals with salinity. The measurements of diffusional permeability reported here are one-third to one-half of the values of Smith (1970a) who was working with rather smaller animals at temperatures 6-12°C higher. Since the Qi,,‘s for diffusional water fluxes through epithelia are about 2 (Isaia, 1972) these data are not inconsistent. However, the data presented here clearly show that the permeability measured by osmotic flow is several times that measured with isotopic tracers, and the data also indicate no close correlation between the two (Fig. 4) since P,, rises rapidly at high salinities where Pd is effectively constant. The rates of urine flow calculated by Smith are thus likely to be substantial underestimates for his animals This may have been even more true at high at his experimental temperatures. salinities where he assumes the relatively low osmotic concentration difference of 10 mosmolal after Hohnedorf (1963). If his animals had been similar to the animals used in this study it would have been three times higher. Indeed one of the most interesting features of this study is the surprisingly high rate of urine flow in animals acclimatized to high salinities. The differences between osmotic and diffusional measurements of permeability can be accounted for by isotope effects to a small extent. This is in marked contrast to the conclusions of Smith (1970b) relating to crustacea. However,
1212
C. R. FLETCHER
his deuterated and tritiated water fluxes were not determined on the same individuals or at the same time so the large differences he reports may have been due to differences between the animals or experimental conditions. As already observed there is no significant direct correlation between diffusional influx and the external salinity in animals acclimatized to 20% sea water, but there is in animals acclimatized to 100% sea water. Variations in the diffusional influx may be expected on a number of grounds. Firstly the mole fraction of water outside the animal decreases as the salinity is raised, but this would only be expected to affect the diffusional influx by O-8 and 1.6 per cent respectively in these experiments, which would not be significant. If the water movements were occurring by bulk flow through pores this would be expected to increase or decrease the isotopic flow. With the permeabilities determined here this effect might be expected to result in an overall change in the tracer influx of about 2 per cent at the lower salinity, which would not be significant, and 5 per cent at the higher salinity, where the observed change is 17 per cent. Also if the diffusional permeability were limited by unstirred layers external to the membrane the presence of an osmotic flow in either direction might be expected to cause convective stirring which would increase the tracer influx by reducing the effective thickness of the unstirred layers (Everitt, Redwood & Haydon,1969). Since the animals acclimatized to each salinity are approximately iso-osmotic in 40 and 103 o/o sea water respectively it can be seen that there is no evidence for this process in either group as correlation coefficients between the modulus of the osmotic pressure differences across the body walls and the diffusional rate constants have probabilities of > 0.05 and > 0.5. The final possible cause of the effect observed in animals acclimatized to sea water is that these are rapid changes in the limiting membrane, possibly swelling in the lower salinities and conversely, and thus affecting the thickness of unstirred layers within the membrane. There is thus no adequate evidence to decide whether the differences between PO, and Pd are due to unstirred layers within the membrane or pores through it, or low blood flow adjacent to the permeable areas of the body wall. The rates of sulphate uptake suggest that drinking is not an important source of urinary water in these animals, despite the lack of evidence that all or most of the sulphate enters the animal by drinking. However, this seems probable since the rate is highest in low salinities where the ionic permeability might be expected to be lowest and where there is a substantial electrical potential which might be expected to reduce the sulphate influx by a factor of about 10 (Fletcher, 1970b). The substantial reduction in osmotic water permeability in low salinities is of obvious physiological significance in minimizing the energy requirements of ionic regulation. The function of a high permeability in high salinities is less obvious, but it could be speculated that a high permeability in high salinities allowing a high rate of urine production allows the animal to reduce its internal salt content rapidly in response to an external reduction in salinity and thus approach its new acclimatized state rapidly. Also a high water permeability in an acclimatized animal will allow a high rate of urinary salt loss. Such a
VOLUMEREGULATION IN NEREIS
DIVERSICOLORI
1213
mechanism cannot make the animal hypo-osmotic but it can reduce the internal salt concentrations to below those in the media, as has frequently been observed for N. diversicolor in high salinities. It is interesting to observe that similar adaptive permeability changes have been observed in another estuarine animal with similar patterns of osmotic and ionic regulation, Gummarus duebeni (Lockwood et al., 1973). Detailed measurements of the nephridial canal of N. diversicolor have not been published, but measurements are available for the nephridia of the closely related N. Zimnicoh (Jones, 1967). If we take the average overall length she records and subdivide it according to the frequency-diameter histogram she gives for animals from a low salinity environment we may calculate the fluid flow for a given hydrostatic pressure. Assuming the nephridial fluid to be no more viscous than water, and taking a hydrostatic pressure of 3 cm of water as observed for animals acclimatized to low salinities we can calculate a flow rate of 15 nl/hr. An animal with 120 such nephridia weighing 0.4 g would thus have a total flow rate due to hydrostatic pressure of 4.5 ml/kg per hr. This is much less than the values deduced for such animals. Whilst the estimate is critically dependent on the few small sections where the lumen is down to 2-3 pm diameter, it ignores the viscous drag on the cilia with which most of the nephridial canal is endowed (Goodrich, 1893). We may thus presume that ciliary beating is the main force moving fluid through the nephridia. Beadle (1937), in an ingenious experiment, showed that urine flow was dependent on the volume of the animal rather than the salt concentration of the coelomic fluid, and assumed that this was due to a higher hydrostatic pressure driving a higher flow rate through the nephridia. The data presented here suggest that this cannot be true, especially since the hydrostatic pressure is lower in animals whose urine flow is higher, and demonstrates that there must be some form of feedback between body volume and the nephridial cilia. REFERENCES BEADLEL. C. (1937) Adaptation to changes of salinity in the polychaetes-I. Control of body volume and body fluid concentration in Nereis diversicolor. J. exp. Biol. 14, 56-70. DOBBSH. E. (1963) Oxygen flask method for the assay of tritium-carbon-14- and sulphur35-labelled compounds. An&t. Chem. 35, 783-786. ELLIS W. G. (1937) Water and electrolyte exchanges of Nereis diversicolor (Miiller). J. exp. Biol. 14, 340-350. EVERITT C. T., REDWOODW. R. & HAYDOND. A. (1969) Problems of boundary layers in the exchange diffusion of water across bimolecular lipid membranes. r. theor. Biol. 22, 20-32. FLETCHER C. R. (1970a) The metabolism of iodine by a polychaete. Comp. Biochem. Physiol. 35, 105-123. FLETCHER C. R. (1970b) The regulation of calcium and magnesium in the brackish water polychaete, Nereis diversicolor 0. F. M. J. exp. Biol. 53, 425443. GOODRICH E. S. (1893) On a new organ in the Lycoridea, and on the nephridium in Nereis diversicolor, 0. F. Miill. Ql. J. microsc. Sci. 34, 387-402. HALE L.J. (1965) Biological Laboratory Data, 2nd edn. Methuen, London.
1214
C. R. FLETCHER
HOHENDORFK. (1963). Der Einfluss der Temperatur auf de Salzgehaltstoteranz und Osmoregulation von Nereis diversicolor 0. F. Muell. Kieler Meeresforch. 19, 196-218. ISAIAJ. (1972) Comparative effects of temperature on the sodium and water permeabilities of the gills of a stenohaline fresh water fish (Carassius auratus) and a stenohaline marine fish (Serranus scriba, Serranus cabrilla). J. exp. Biol. 57, 359-366. JONES M. L. (1967) On the morphology of the nephridia of Nereis Zimnicola Johnson. Biol. Bull., mar. biol. Lab., Woods Hole 132, 362-380. JORGENSENC. B. & DALES R. P. (1957) The regulation of volume and osmotic regulation in some Nereid polychaetes. Physiol. camp. Oecol. 4, 357-374. LOCKWOODA. P. M., INMANC. B. E. & COURTNAYT. H. (1973) The influence of environmental salinity on the water fluxes of the amphipod crustacean Gammarus duebeni. J. exp. Biol. 58, 137-148. MOTAISR., ISAIAJ., RANKINJ. C. & MAETZ J. (1969) Adaptive changes of the water permeability of the teleostean gill epithelium in relation to external salinity. J. exp. Biol. 51, 529-546. OGLESBY L. C. (1970) Studies on the salt and water balance of Nereis diversicolor-I. Steady-state parameters. Comp. Biochem. Physiol. 36, 449-466. OGLESBY L. C. (1972) Studies on the salt and water balance of Nereis diversicolor-II. Components of total sodium efflux. Comp. Biochem. Physiol. 41A, 765-790. POTTS W. T. W., FOSTER M.A., RUDY P. P. & PARRY HOWELLS G. (1967) Sodium and water balance in the cichlid teleost Tilapia mossambica. J. exp. Biol. 47, 461-470. POTTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Pergamon Press, Oxford. RUDY P. P., JR. (1967) Water permeability of selected decapod crustacea. Camp. Biochem. Physiol. 22, 581-589. SMITH R. I. (1955) Comparison of the level of chloride regulation by Nereis diversicolor in different parts of its geographical range. Biol. Bull. mar. biol. Lab., Woods Hole 109, 453-474. SMITH R. I. (1956) The ecology of the Tamar estuary-VII. Observations on the interstitial salinity of intertidal muds in the estuarine habitat of Nereis diversicolor. J. mar. Biol. Ass. U.K. 35, 81-104. SMITH R. I. (1970a) Chloride regulation at low salinities by Nereis diversicolor (Annelida, polychaeta)-II. Water fluxes and apparent permeability to water. -7. exp. BioE. 53, 93-100. SMITH R. I. (1970b) The apparent water permeability of Carcinus maenas (Crustacea, Brachyura, Portunidae) as a function of salinity. Biol. Bull. mar. biol. Lab., Woods Hole 139, 351-362. Key Word Index-Nereis diversicolor ; polychaete ; estuarine ; brackish water ; euryhaline ; volume regulation; osmotic regulation; water permeability; osmotic flow; urine flow; isotope effect.
VOLUME REGULATION IN NEREIS DIVERSICOLOR-I. THE STEADY STATE C. R. FLETCHER Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England (Received 3 May 1973)
Abstract-l.
The steady-state parameters of volume regulation in a population of Nereis diversicolor have been examined. 2. The water permeability changes as a function of the salinity of acclimatization even when expressed in a form independent of the animals’ water content, and is highest in full strength sea water. 3. Drinking does not contribute significantly to water movements. 4. Water permeability measured by osmotic flow is substantially larger than that determined by movement of labelled water molecules, and the difference is not explained by isotope effects. 5. The rate of urine production is highest in animals acclimatized to 2% sea water, but is substantial even in animals acclimatized to full strength sea water. 6. Hydrostatic pressure is not the primary cause of fluid flow through the nephridia.
INTRODUCTION
NEREIS DIVERSICOLOR is an estuarine polychaete
which is subject to both low and variable salinities, and may be most abundant where the salinity is most variable (Smith, 1956). It has been used in investigations on osmotic and ionic regulation as it is a good osmoregulator at low salinities but osmoconforms in higher salinities (Smith, 1955; Hohendorf, 1963). However, it has been shown that these animals lose their ability to control their volume in the absence of external calcium ions (Beadle, 1937; Ellis, 1937) and these authors’ explanation that this results from an increased water permeability has not yet been substantiated. It has also been suggested that adaptation to low salinities is in part accomplished by a reduction in water permeability (Jorgenson & Dales, 1957), but their evidence was criticized by Potts & Parry (1964) on the basis that they had failed adequately to control the temperatures, had not determined the concentrations of the body fluids and had assumed that the rates of urine production did not However, Smith (1970a) has shown that the change in their experiments. permeability measured by the influx of labelled water molecules does change, when expressed as a rate constant. This work ignores the fact that water permeabilities measured by tracer and osmotic flow methods seldom agree (e.g. Rudy, 1967; Motais et al., 1969). Also it has been shown that the water content of these animals is higher when adapted to lower salinities (Oglesby, 1970), and 1199
1200
C. R. FLETCHER
this will result in a reduced rate constant even if the permeability of the presumably unchanged surface area is unaltered. N. diversicolor’s normal environment is one in which the salinity may fluctuate substantially during the tidal cycle as well as seasonally, and with the exception of early work (Beadle, 1937; Ellis, 1937), which showed that these animals have substantial abilities to control their volumes in these conditions, little work has been published on events occurring in response to a salinity change, and this has been largely concerned with ionic fluxes, and has raised more difficulties than it has illuminated the mechanisms of adaptation (Oglesby, 1972). Experiments have been undertaken to determine whether the osmotic water permeability is reduced in response to a reduction in salinity, to determine whether lack of calcium does result in an increase in water permeability and to determine the mechanisms by which the animal adapts to a new steady state after an experimental change of salinity. These last two items require a detailed understanding of the water balance in the steady-state (acclimatized) animal. It has been suggested that animals from different locations may differ considerably (Ellis, 1937), and although this was not supported by Smith (1955) it was thought desirable to determine the parameters of water balance for acclimatized animals for the population used in the subsequent studies rather than relying on published data. This paper reports the results of experiments on the water balance of acclimatized animals and on the existence of true changes in their osmotic water permeability; subsequent papers will record the results of experiments on the effect of calcium and on the mechanisms of adaptation to a changed salinity.
MATERIALS
AND
METHODS
Specimens of Nereis dive&color weighing between 0.25 and 0.8 g were collected from the upper reaches of Bridlington harbour on the East Coast of England on a number of occasions between April 1970 and October 1972 and were maintained in the laboratory in 20% sea water at 12 f 1°C until required. They were adapted gradually to other salinities as already described (Fletcher, 1970a). Each animal spent at least 6 days in the final acclimatization salinity, the medium being changed twice. All experiments were conducted at 12 + 0.5”C. Sea water was obtained from the beach at Bridlington or from the Wellcome Marine Laboratory at Robin Hoods Bay. Dilutions were prepared with distilled water, and when concentrations greater than full strength sea water were required they were prepared by dissolving the appropriate quantities of salts in sea water, using the values of Hale (1965). The chlorosity of all solutions was checked with an Aminco amperometric chloride titrator. Samples of coelomic fluid for osmotic pressure measurements were taken in clean melting point capillary tubes which had been drawn to a fine point at one end which were inserted through the dorsal body wall adjacent to the blood vessel. The capillaries were then sealed at the remote end and the cellular material sedimented in a microhaematocrit centrifuge. The capillaries were broken just above the cell/fluid interface and the supernatant used immediately for osmotic pressure determinations, Osmotic pressure measurements were made using a differential method based on the difference in vapour pressure and hence equilibrium temperature of two drops of fluid hanging on hermetically sealed matched bead thermistors in a temperature-controlled
VOLUME REGULATION
IN NEREIS DIVERSICOLOR--I
1201
chamber saturated with water vapour, the differences in temperature being determined by differences in resistance of the thermistors connected in a transformer bridge circuit. The measurements were reproducible to f 3 mosmolal SD. and were independent of the nature of the solute. The water content of animals and organic dry weight were determined by weighing the animals, drying them under vacuum over silica gel, re-weighing and then ashing them in a muffle furnace at 600°C and weighing them again. All weighings were to f 0.1 mg. Diffusional permeabilities were measured with tritiated water from the Radiochemical Centre, Amersham, England, which was incorporated in the appropriate experimental solution to 0.05 &i/ml. The animals were placed in 50 ml of labelled medium for 10 min during which time the medium was stirred by a stream of air bubbles from a fine pipette, and then removed, rinsed for about 5 set in a nylon teastrainer with unlabelled medium from a wash bottle and blotted dry. They were stored individually in polythene capped vials at - 20°C. The body water was sublimed off under high vacuum (< 10e3 torr) and collected on a cold finger containing liquid nitrogen, and subsequently allowed to melt in a dry atmosphere and collected in a clean tube. Aliquots (0.1 ml f 1 per cent) were taken for assay of specific activity by liquid scintillation counting using 5 ml of toluene-based scintillator containing 67 ml/l of the blending agent NE 520 (Nuclear Enterprises Ltd.). Samples of loading media were similarly handled to provide standards and since the counting mixture was of constant composition quenching corrections were unnecessary. The counting efficiency was about 40 per cent in a Beckman 1650 counter. Tests were conducted to ensure that there was no significant isotopic fractionation in the sublimation of water from the animals by partly drying one group of loaded animals, and then subsequently removing the residual water. The mean ratio of the specific activities of the two fractions was 1.02 with an SE. of 0.012. Tests were also conducted to show that all the water in the animals exchanged as one well-mixed pool by loading sixty animals for 4 hr in 20% sea water, when they were close to isotopic equilibrium, and then allowing them to efflux into a large volume of 20% sea water for times from 0 to 100 min before removing them for determination of the specific activity of their body water. The results are shown in Fig. 1, and demonstrate that the whole body water exchanges as though it were in one well-mixed compartment, i.e. internal exchanges are much more rapid than exchanges with the environment. Thus we may use a relatively simple formula to determine the rate constant for isotopic water exchange, K: 1 A0 K = ;loge(&A) where A0 is the specific activity of the medium and A is the specific activity of the animal’s body water after a time t in the medium. In order to investigate the significance of isotope effects one experiment was performed in which deuterium was used as well as tritium as a tracer for water molecules, and the medium contained 10% DzO as well as tritiated water. This medium was allowed to stand at room temperature for a week before use to allow isotopic equilibrium to be established. Pure water was extracted as already described and deuterium concentration measured in a density gradient column of xylene and carbon tetrachloride with suitable standards. Osmotic permeabilities were deduced from measurements of weight changes occurring after relatively small changes of external salinity. It was found that after such a change the animal initially starts to swell or shrink, but after an hour or two the rate of change of weight becomes very much reduced below that which would be expected of a perfect osmometer, or indeed of an animal lacking any direct means of controlling its volume (Fig. 2). A theoretical analysis of such an animal is given in the following paper on adaptation to a reduction in salinity. Indeed departures from such theoretical predictions are
1202
C. R. FLETCHER
Efflux
time,
min
FIG. 1. Loss of Iabelled water from animals. The animals were loaded for 4 hr and then placed in unlabelled medium for the time shown before the specific activity of their body water was determined. Each point is the mean of six observations + S.D.
Time,
hr
FIG. 2. Volume changes of animals acclimatized to 50% sea water after transfer to 70, 50 or 30% sea water. The theoretical behaviour of animals which lacked means of volume control are shown by broken lines. In this and subsequent figures variation about a mean is shown by a vertical line of + 1 SD. and a central block of f 1 S.E.
VOLUME REGULATION
1203
fN AWZ.?ZS LtIVEASICOLOR-1
apparent as little as 1 hr, but not 30 min after changes of salinity, implying that mechanisms of volume control are being brought into effect after a delay of 30 min or more after the change of salinity. Further evidence of this is presented in the same paper. Thus we may reasonably assume that weight changes during the first 30 min after such salinity changes are caused by osmotic water movements, and enable us to determine the osmotic water permeabilities of the animals. ISince the animals are not normally iso-osmotic in any salinity the formula describing the behaviour of the weight of an animal after transfer but before urine flow or total internal solutes change is not very simple, and derivation is given in the later paper. The formula is -1 p = t(C,+C,i-Co)
a0 Co1 If+c,+CO’-CO)‘op”
f(C, -t-Co’ - Co) ( l-
ao(C0 -
C,)
II’
where P is the permeability in kg H,O]kg animal per hr per unit osmolal concentration difference, Co is the osmolality of the acclimatization medium, Co1 is the osmolality of the animal’s body fluids in the acclimatization medium, z. is the water content of the animals, kg/kg animal when acclimatized to Co, Cr is the osmolality of the medium to which they are transferred and f is the fractional increase in weight t hr after transfer, relative to controls, Thus by substituting observed data for f as a function of t using determined values of Co, C,, Col-Co, and z. we may determine P subject to the above assumptions, and a decrease of the value of P as t increases indicates where the assumptions cease to be valid, and demonstrate the operation of some form of volume regulation. Since the results are based on relatively small changes in weight a reliable and accurate way of weighing wet animals with a minimum of stress was needed. The water containing the animal was poured through a nylon tea strainer, collecting the animal. The underside of the strainer was blotted on absorbent paper and the worm emptied onto a filter paper and rapidly weighed to 0.1 mg. The worm was returned to its water in a clean beaker and the filter paper reweighed. This was found to give weights reproducible to kO.2 mg; the animals were out of water about 20 set and were not touched at all. The relative constancy of the mean weight of control worms showed that this procedure did not disturb their volume unduIy. In order to ascertain whether drinking formed a significant part of the water flows the rate of a?!5 sulphate uptake was determined as a measure of drinking, after the method of Potts et al (1967). Yl labelled sodium sulphate was added to acclimatization media to a concentration of 0.2 &i/ml, and animals removed after times between 0.5 and 8 hr. The worms were washed externally by three 5-min periods in unlabelled media with rinsing between each and then blotted dry and dried under vacuum over silica gel. The labelled sulphate was released by the oxygen flask combustion technique of Dobbs (1963) and dissolved in 10 ml of his basic scintillator, of which a 9-ml aliquot was counted. Standards of 0.05 ml of the labelled media were dried on absorbent paper and subsequently handled like the animals. Quenching corrections were made by the external standard channels ratio method. Measurements of the hydrostatic pressure difference across the body walls of these animals were made using a capacitance pressure transducer in a bridge circuit driving a Beckman pen recorder. The pressure cannula, containing sea water diluted to approximate to the animals’ body fluids, was made of fine polythene tubing with a small cigar-shaped glass tip which was inserted through the dorsal body wall into the coelomic cavity. Elasticity of the body wall held the cannula in place without visible leakage. The animal was allowed to swim or crawl freely in a small glass dish. The response of the system was limited by the recorder to about 1 sec. This piece of work was performed at the University of Birmingham using animals obtained from Christchurch harbour, Hants.
C. R. FLETCHER
1204
RESULTS
Osmotic concentrations of the body jluids The osmotic concentration of cell-free coelomic fluid samples were determined from seventeen or more animals acclimatized to each of a range of salinities Measurements were made relative between full strength and 0.5% sea water. to the acclimatization media, and these were determined relative to distilled water. The results are shown in Fig. 3, and it is observed that the worms were significantly hyperosmotic to the media in all salinities, the difference being largest in worms acclimatized to 2 and 1% sea water.
Acclimot~sot~on
FIG.
3.
medium,
mosmolal
Osmotic concentration difference between the coelomic acclimatized animals and the external medium.
fluids of
Water content and ash free dry weight This was measured using groups of nineteen or twenty animals acclimatized to a similar range of salinities, and the results are given in Table 1. The water content is seen to vary markedly in the higher salinities, and if the ash-free dry weight is assumed to be constant we may deduce the changes in weight of individuals when they are acclimatized to a different salinity. Osmotic permeability This was measured using animals acclimatized to 100, 50, 30 and water. The first group consisted of sixty animals, twenty of which were individually in 1OOo/o sea water, twenty of which were transferred to water and twenty of which were transferred to 120% sea water, and their as fractions of the original weight determined after t, 4, 1 and 2$ h.
10% sea retained 80% sea weights Animals
VOLUME
TABLE l-WATERCONTRNT
REGULATION
1205
IN NEREIS DIVERSICOLOR--I
OF ANIMALSASAFUNCTIONOFACCLIMATIZATION
Sea water (%)
Chlorosity of medium (mm/l.)
100 50 20 10 5 2 1
533 255 105 52.1 27.0 11.0 5.63
Water content (g/kg of animal)
794.2 + 3.2 840.1+ 2.5 865.3 + 2.3 868-S + 2.1 878.3 + 2.3 879.3 f 4.4 887.3 z!z2.6
SALINITY
Ash free dry weight (g/kg of animal)
178.5 144.9 125.1 123.6 114.0 112-l 104.8
(20) (20) (20) (20) (19) (19) (19)
+ 2.9 ?r:2.5 + 2.4 + 2-l * 2.2 * 4.4 + 2.6
(20) (20) (20) (20) (19) (19) (19)
Means + S.E. are given with number of observations in parentheses.
from the other salinities were similarly treated except that the transfers used for animals acclimatized to lOo/o sea water were to 0 and 20% sea water. A typical set of experimental results is given in Table 2, and the permeabilities calculated from these data in Table 3. The overall mean of PO, from the &hr and TABLE 2-WEIGHT OF ANIMALS ACCLIMATISED TO FULL STRENGTH
As A FRACTION OF THE ORIGINAL WEIGHT OF ANIMALS SEA WATER AFTER TRANSFER TO 80 OR 120% SEA WATER
Time after transfer (hi-)
Sea water Osmotic concentration of experimental media (%) (osmolal) *
*
1
2*
80
O-806
1.0227 +0*0015 (20)
1.0412 *o-o019 (20)
1*0705 & O-0028 (20)
1.1052 rf:0.0039 (20)
100
l-008
l-0014 f 00IlO (20)
1 a0004 * 0~0017 (20)
0.9997 f O-0016 (20)
O-9965 + 0.0033 (20)
120
1.222
0.9779 zb0.0018 (19)
0.9561 + 0.0043 (20)
O-9270 f o-0043 (20)
0.8878 + 0.0047 (20)
Means -I SE.
are given with number of observations in parentheses.
+hr measurements is taken as the permeability. Since the &hr and &hr measurements relate to the same animals they can be regarded as independent only insofar as the variability reflects experimental errors, not individual variability of the animals. Since the standard error of the weight changes rises almost linearly with time and is similar for controls and both groups of experimental animals it appears that the primary source of variability arises from the fact that
C. R. FLETCHER
1206 TABLE
~-VALUES
FOR
WATER
PERMEABILITY
CALCULATED
FROM
DATA
IN TABLE
2,
in kg H,O/kg animal per hr per 1 osmolal concentration difference Time (hr) +
2f
0.469 + 0.035 0.484 i: 0.061 0.477 t 0.035
0,462 + 0,028 0.459 & O-043 0,460 -1:0.026
0.384 + 0,031 0.413 + 0*057 0,398 ? 0.032
‘s 0.456 t 0,041 0.473 i 0.044 0.464 + 0.030
Swelling
Shrinking Mean
-1
Means + S.E. are given. Since twenty animals gave the swelling data and nineteen or twenty animals the shrinking data, the final value used in subsequent calculations was O-470 _+0.030 (IV = 39).
at any time an individual’s weight may be rising or falling in the absence of any change in external salinity. Thus the &- and &hr figures are taken as not independent but the swelling and shrinking data are independent. The standard error is a real measurement of the reliability of the mean but not an adequate measurement of individual variability. TABLE
~-WATER
PERMRABILITIRS MEASURGD BY OSMOTIC TO VARIOUS SALINITIES
FLOW
IN ANIMALS
ACCLIMATIZED
Acclimatization media Sea water (%)
Chlorosity (mm/l.)
p0lI
100 50 30 10
533 274 161 54‘4
0.470 + 0.030 0.280 + 0.022 0.161+ 0.016 0,116 t 0.023
p0, 2.63 1.93 1.23 0.94
+_0.17 z!z0.16 + 0.12 If:o-19
iv
39 37 40 38
P, in kg H,O/kg animal per hr per unit osmolal concentration difference. Posl in kg HzO/kg ash free dry weight of animal per hr per unit osmolal concentration difference. Means 4 S.E. are given. N is the number of experimental animals from which each figure is derived.
The pooled results for PO, for each acclimation salinity with standard errors are given in Table 4 and Fig. 4, and it is apparent that PO, does decline markedly as the salinity falls, but it may be noticed that PO, is here defined relative to the weight of the animal which is itself variable. Consequently the permeabilities have also been calculated relative to the ash-free dry weight using the data in Table 1. These values, given as POsl are also recorded in Table 4, and it is seen that the permeability is indeed reduced in the lower salinities.
VOLUME
REGULATION
IN NEREIS
DIVERSICOLOR-I
1207
Diflusional permeability This was measured in several salinities to determine whether it was similar to the permeability measured by osmotic flow. Since preliminary results showed that it was not, experiments were undertaken to see whether the discrepancy could be accounted for by isotope effects by comparing permeabilities measured simultaneously with heavy water and tritiated water, and also to determine whether the isotopic flow varied significantly as a function of the osmotic concentration as diffusional permeabilities should be measured when there is no nett flow. Thus rate constants were determined for SHHO and for DHO influx from 50% sea water into animals acclimatized to the same salinity, and for sHH0 influx from salinities between 0 and 40% sea water into animals TABLE
S-RATE
CONSTANTS
FOR THE
EXCHANGE OF ISOTOPICALLY PERMBABILITIES
Acclimatization salinity (% sea water)
K hr-l
Pd
100 50 50 20 20
5.79 * 0.25 5*90+0.15 6.21 + O-17 3.43 f 0.10 3.63 + 0.04
0.131 -I-0.006 0.127 + 0.003 O-133 + 0.004 0.071 f 0.002 0.076 + O-001
LABBLLED
WATER
AND
Method and No. of animals 0.734 f. 0.036 O-876 + O-026 0.918 + O-032 O-568 f 0.019 0.607 f. 0.014
aH *H D 3H 3H
influx influx influx influx efflux
(N = (N = (IV = (IV = (N =
12) 20) 20) 10) 60)
Units: Pa kg HaO/kg animal per hr per unit osmolal concentration difference. Pdl kg H,O/kg ash free dry weight of animal per hr per unit osmolal concentration difference. Means + S.E. are given. TABLE
~-EFFECT
OF OSMOTIC
GRADIENTS ON THE RATE CONSTANTS EXCHANGE
OF TRITIATED WATER
Acclimatization medium chlorosity
Test medium chlorosity,
(mm/l.)
(mm/l.)
529
774 642 528 418 320
5.27 + O-18 (12) 5.65 Z!T 0.09 (12) 5.79 + 0.25 (12) 5-72 zb0.22 (12) 6.16 I!Z0.08 (12)
108
0.06 52.7 106 158 205
3.96 3.43 3.43 3.46 3.75
Khr-'
f f f f +
O-10 (10) o-11 (10) o-10 (10) 0.10 (10) 0.14 (10)
Means &-SE. are given with number of observations in parentheses.
C. R. FLETCHER
1208
acclimatized to 20% sea water, and for 3HH0 influx from salinities between 60 and 140% sea water into animals acclimatized to 100% sea water. The results are given in Tables 5 and 6. Also values for Pd, the diffusional permeabilities have been calculated from the values of K in Table 5, and are also given in Table 5, using the relationship
where Pd is defined in the same units as PO,, x0 is the water content of the animal and No is the number of moles in 1 kg of water = 55.5. The approximate sign is used since this will only be exactly true if the mole fraction of water outside is unity, but the effect of this will be less than 2 per cent. The values of Pa are also shown in Fig. 4. It can be shown that all values of Pd are very much smaller
0
I
I 20
I
40 Salinity,
I
I
60 80 % sea water
I
100 S
FIG. 4. Water permeabilities of animals acclimatized to different salinities, measured by osmotic flow (P,,, open symbols) or by labelled water fluxes (Pd closed symbols) + SE. Permeabilities expressed in kg Hz0 per kg animal per hr per 1 osmolal concentration difference.
than PO,, and the two appear to vary relatively independently. Like PO,, the diffusional permeability may also be expressed in terms independent of the water content of the animal, Pd’, and these are also given in Table 5. It may also be seen from Table 6 that the diffusional influx of 3HH0 in animals acclimatized to 20% sea water does not vary regularly with the external salinity (correlation coefficient probability > 0*05), but in the animals acclimatized to lOOo/o sea water the isotopic flux is significantly dependent on the external salinity (correlation coefficient probability < O-01).
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The mean of the ratio of K for SHHO to K for aHHO was O-951 + O-006 S.E. (N = 20). Thus there is a significant isotope effect. If we assume that the isotope effect is a linear function of mass of the water molecule we may judge that the isotopic fluxes measured with sHH0 are about 10 per cent less than the flux rates for H,O. This ignores the fact that in this experiment a significant proportion of the sH fluxes were carried by 3H2H0 molecules, and a significant proportion of the deuterium fluxes were carried by 2H20 molecules, A detailed analysis suggests that these two factors cancel to make the original deduction correct to within 0.7 per cent. Drinking rate
The rates of uptake of 35S sulphate were determined as already described. No checks were made to ensure that the isotope was confined to the gut as it is difficult to dissect out without rupture, but the rates were low and establish an upper limit to the rate of drinking. After only 30 min all animals contained some tracer and the quantity rose linearly thereafter suggesting it had not started to leave the anus within 8 hr. A regression analysis of the quite variable data gave slopes which are given in Table 7, and intercepts significantly different to zero. TABLE
~---RATE OF UPTAKE OF TS SULPHATE (DRINKING RATE) Salinity (% sea water) 100
10 1
Rate of uptake (d/kg
per hr)
0.94 z!z0.07 (16) 0.48 k 0.03 (16) 1.60 + 0.07 (16)
Means f SE. are given with number of animals in parentheses.
The latter may have been caused by the habit of these animals of everting their proboscis in response to handling and possibly taking in some medium when it is retracted, or could be caused by an exchange phenomenon with the mucus or cuticle.
This was normally recorded over a period of about 10 min, and was relatively steady with small fluctuations when the animal was crawling or quiescent, but rose dramatically when the animal displayed the startle response of longitudinal contraction or commenced to swim. Since the behaviour resulting in these high values is seldom displayed by undisturbed animals they were ignored in taking an average pressure for each animal, which were then used to calculate the means shown in Fig. 5 : each point is the mean from ten to twelve animals. It is observed that whilst all the values are small they are significantly larger in the less swollen animals acclimatized to higher salinities.
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C.
I
FLETCHER
I
I
I
I
IO
30
too
300
I
0 a
R.
Acclimatisotion
medium chloride concentration,
I+
1000 mM
FIG. 5. Hydrostatic pressure differences across the body walls of animals acclimatized to a range of salinities.
Rate of urine flow From the information given in Fig. 3 and Table 4 we can cafculate the osmotic influx in a range of salinities, and if drinking is neglected this must equal the rate of urine flow. Since the uptake of sulphate was not shown to occur by drinking this may be true, and even if all the sulphate uptake occurs by drinking the latter will not contribute significantly to the urine flow. No values for PO, have been obtained in salinities below 10% sea water and it has been assumed to remain constant in lower salinities. The rates of urine flow calculated on these assumptions are shown in Fig. 6 with all the uncertainties shown as S.E.‘s.
0
I
o-3
i
I
I
I
I
3
IO
30
100 ’
Acclimatisation
FIG. 6.
medium,
% seawater
Calculated mean rates of urine production of animals acclimatized to a range of salinities f S.E. of estimate.
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DISCUSSION
The differences in osmotic concentration between the animal and its environment were uniformly about 30 mosmolal higher than those reported by Hohendorf (1963) as freezing point depressions for animals from Kiel. However, the data of Smith (1955) show differences of up to 70 mM in coelomic fluid chloride levels of animals from different locations. He concludes that these differences were not significant since the higher values were from the survivors of experimental groups which had suffered high mortalities in the laboratory, but the data still suggest that distinct physiological races or environmental factors may be significant. The methods used for determining the osmotic permeabilities are related to those used by Jorgenson & Dales (1957), and the transfers used were of similar magnitude except in worms acclimatized to 10% sea water. However, in this paper it has been shown that whilst mechanisms of volume control are operating an hour or more after such changes in external salinity there is no evidence of their operation over shorter times. Furthermore, it will be shown in a subsequent paper that the rate of inulin clearance from the animals is scarcely altered in the first hour after a reduction of external salinity but rises considerably thereafter. Thus whilst this technique is subject to large random errors due to the inconstancy of the weight of individuals it seems that the means should not be subject to significant systematic errors. The osmotic water permeability is larger in worms acclimatized to higher salinities, and this is a true effect since it is still apparent when expressed in terms independent of the water content of the animals. This confirms the conclusions of Smith (1970a) based on measurements of diffusional permeabilities and not corrected for changes in the water content of the animals with salinity. The measurements of diffusional permeability reported here are one-third to one-half of the values of Smith (1970a) who was working with rather smaller animals at temperatures 6-12°C higher. Since the Qi,,‘s for diffusional water fluxes through epithelia are about 2 (Isaia, 1972) these data are not inconsistent. However, the data presented here clearly show that the permeability measured by osmotic flow is several times that measured with isotopic tracers, and the data also indicate no close correlation between the two (Fig. 4) since P,, rises rapidly at high salinities where Pd is effectively constant. The rates of urine flow calculated by Smith are thus likely to be substantial underestimates for his animals This may have been even more true at high at his experimental temperatures. salinities where he assumes the relatively low osmotic concentration difference of 10 mosmolal after Hohnedorf (1963). If his animals had been similar to the animals used in this study it would have been three times higher. Indeed one of the most interesting features of this study is the surprisingly high rate of urine flow in animals acclimatized to high salinities. The differences between osmotic and diffusional measurements of permeability can be accounted for by isotope effects to a small extent. This is in marked contrast to the conclusions of Smith (1970b) relating to crustacea. However,
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C. R. FLETCHER
his deuterated and tritiated water fluxes were not determined on the same individuals or at the same time so the large differences he reports may have been due to differences between the animals or experimental conditions. As already observed there is no significant direct correlation between diffusional influx and the external salinity in animals acclimatized to 20% sea water, but there is in animals acclimatized to 100% sea water. Variations in the diffusional influx may be expected on a number of grounds. Firstly the mole fraction of water outside the animal decreases as the salinity is raised, but this would only be expected to affect the diffusional influx by O-8 and 1.6 per cent respectively in these experiments, which would not be significant. If the water movements were occurring by bulk flow through pores this would be expected to increase or decrease the isotopic flow. With the permeabilities determined here this effect might be expected to result in an overall change in the tracer influx of about 2 per cent at the lower salinity, which would not be significant, and 5 per cent at the higher salinity, where the observed change is 17 per cent. Also if the diffusional permeability were limited by unstirred layers external to the membrane the presence of an osmotic flow in either direction might be expected to cause convective stirring which would increase the tracer influx by reducing the effective thickness of the unstirred layers (Everitt, Redwood & Haydon,1969). Since the animals acclimatized to each salinity are approximately iso-osmotic in 40 and 103 o/o sea water respectively it can be seen that there is no evidence for this process in either group as correlation coefficients between the modulus of the osmotic pressure differences across the body walls and the diffusional rate constants have probabilities of > 0.05 and > 0.5. The final possible cause of the effect observed in animals acclimatized to sea water is that these are rapid changes in the limiting membrane, possibly swelling in the lower salinities and conversely, and thus affecting the thickness of unstirred layers within the membrane. There is thus no adequate evidence to decide whether the differences between PO, and Pd are due to unstirred layers within the membrane or pores through it, or low blood flow adjacent to the permeable areas of the body wall. The rates of sulphate uptake suggest that drinking is not an important source of urinary water in these animals, despite the lack of evidence that all or most of the sulphate enters the animal by drinking. However, this seems probable since the rate is highest in low salinities where the ionic permeability might be expected to be lowest and where there is a substantial electrical potential which might be expected to reduce the sulphate influx by a factor of about 10 (Fletcher, 1970b). The substantial reduction in osmotic water permeability in low salinities is of obvious physiological significance in minimizing the energy requirements of ionic regulation. The function of a high permeability in high salinities is less obvious, but it could be speculated that a high permeability in high salinities allowing a high rate of urine production allows the animal to reduce its internal salt content rapidly in response to an external reduction in salinity and thus approach its new acclimatized state rapidly. Also a high water permeability in an acclimatized animal will allow a high rate of urinary salt loss. Such a
VOLUMEREGULATION IN NEREIS
DIVERSICOLORI
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mechanism cannot make the animal hypo-osmotic but it can reduce the internal salt concentrations to below those in the media, as has frequently been observed for N. diversicolor in high salinities. It is interesting to observe that similar adaptive permeability changes have been observed in another estuarine animal with similar patterns of osmotic and ionic regulation, Gummarus duebeni (Lockwood et al., 1973). Detailed measurements of the nephridial canal of N. diversicolor have not been published, but measurements are available for the nephridia of the closely related N. Zimnicoh (Jones, 1967). If we take the average overall length she records and subdivide it according to the frequency-diameter histogram she gives for animals from a low salinity environment we may calculate the fluid flow for a given hydrostatic pressure. Assuming the nephridial fluid to be no more viscous than water, and taking a hydrostatic pressure of 3 cm of water as observed for animals acclimatized to low salinities we can calculate a flow rate of 15 nl/hr. An animal with 120 such nephridia weighing 0.4 g would thus have a total flow rate due to hydrostatic pressure of 4.5 ml/kg per hr. This is much less than the values deduced for such animals. Whilst the estimate is critically dependent on the few small sections where the lumen is down to 2-3 pm diameter, it ignores the viscous drag on the cilia with which most of the nephridial canal is endowed (Goodrich, 1893). We may thus presume that ciliary beating is the main force moving fluid through the nephridia. Beadle (1937), in an ingenious experiment, showed that urine flow was dependent on the volume of the animal rather than the salt concentration of the coelomic fluid, and assumed that this was due to a higher hydrostatic pressure driving a higher flow rate through the nephridia. The data presented here suggest that this cannot be true, especially since the hydrostatic pressure is lower in animals whose urine flow is higher, and demonstrates that there must be some form of feedback between body volume and the nephridial cilia. REFERENCES BEADLEL. C. (1937) Adaptation to changes of salinity in the polychaetes-I. Control of body volume and body fluid concentration in Nereis diversicolor. J. exp. Biol. 14, 56-70. DOBBSH. E. (1963) Oxygen flask method for the assay of tritium-carbon-14- and sulphur35-labelled compounds. An&t. Chem. 35, 783-786. ELLIS W. G. (1937) Water and electrolyte exchanges of Nereis diversicolor (Miiller). J. exp. Biol. 14, 340-350. EVERITT C. T., REDWOODW. R. & HAYDOND. A. (1969) Problems of boundary layers in the exchange diffusion of water across bimolecular lipid membranes. r. theor. Biol. 22, 20-32. FLETCHER C. R. (1970a) The metabolism of iodine by a polychaete. Comp. Biochem. Physiol. 35, 105-123. FLETCHER C. R. (1970b) The regulation of calcium and magnesium in the brackish water polychaete, Nereis diversicolor 0. F. M. J. exp. Biol. 53, 425443. GOODRICH E. S. (1893) On a new organ in the Lycoridea, and on the nephridium in Nereis diversicolor, 0. F. Miill. Ql. J. microsc. Sci. 34, 387-402. HALE L.J. (1965) Biological Laboratory Data, 2nd edn. Methuen, London.
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HOHENDORFK. (1963). Der Einfluss der Temperatur auf de Salzgehaltstoteranz und Osmoregulation von Nereis diversicolor 0. F. Muell. Kieler Meeresforch. 19, 196-218. ISAIAJ. (1972) Comparative effects of temperature on the sodium and water permeabilities of the gills of a stenohaline fresh water fish (Carassius auratus) and a stenohaline marine fish (Serranus scriba, Serranus cabrilla). J. exp. Biol. 57, 359-366. JONES M. L. (1967) On the morphology of the nephridia of Nereis Zimnicola Johnson. Biol. Bull., mar. biol. Lab., Woods Hole 132, 362-380. JORGENSENC. B. & DALES R. P. (1957) The regulation of volume and osmotic regulation in some Nereid polychaetes. Physiol. camp. Oecol. 4, 357-374. LOCKWOODA. P. M., INMANC. B. E. & COURTNAYT. H. (1973) The influence of environmental salinity on the water fluxes of the amphipod crustacean Gammarus duebeni. J. exp. Biol. 58, 137-148. MOTAISR., ISAIAJ., RANKINJ. C. & MAETZ J. (1969) Adaptive changes of the water permeability of the teleostean gill epithelium in relation to external salinity. J. exp. Biol. 51, 529-546. OGLESBY L. C. (1970) Studies on the salt and water balance of Nereis diversicolor-I. Steady-state parameters. Comp. Biochem. Physiol. 36, 449-466. OGLESBY L. C. (1972) Studies on the salt and water balance of Nereis diversicolor-II. Components of total sodium efflux. Comp. Biochem. Physiol. 41A, 765-790. POTTS W. T. W., FOSTER M.A., RUDY P. P. & PARRY HOWELLS G. (1967) Sodium and water balance in the cichlid teleost Tilapia mossambica. J. exp. Biol. 47, 461-470. POTTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Pergamon Press, Oxford. RUDY P. P., JR. (1967) Water permeability of selected decapod crustacea. Camp. Biochem. Physiol. 22, 581-589. SMITH R. I. (1955) Comparison of the level of chloride regulation by Nereis diversicolor in different parts of its geographical range. Biol. Bull. mar. biol. Lab., Woods Hole 109, 453-474. SMITH R. I. (1956) The ecology of the Tamar estuary-VII. Observations on the interstitial salinity of intertidal muds in the estuarine habitat of Nereis diversicolor. J. mar. Biol. Ass. U.K. 35, 81-104. SMITH R. I. (1970a) Chloride regulation at low salinities by Nereis diversicolor (Annelida, polychaeta)-II. Water fluxes and apparent permeability to water. -7. exp. BioE. 53, 93-100. SMITH R. I. (1970b) The apparent water permeability of Carcinus maenas (Crustacea, Brachyura, Portunidae) as a function of salinity. Biol. Bull. mar. biol. Lab., Woods Hole 139, 351-362. Key Word Index-Nereis diversicolor ; polychaete ; estuarine ; brackish water ; euryhaline ; volume regulation; osmotic regulation; water permeability; osmotic flow; urine flow; isotope effect.