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Kidney function in the North American crayfish Pacifastacus leniusculus (Dana) stepwise acclimated to dilutions of sea water
Comp. Biochem. Physiol., 1970, Vol. 35, pp. 427 to 437. Pergamon Press. Printed in Great Britain
K I D N E Y F U N C T I O N IN T H E N O R T H AMERICAN CRAYFISH P A C I F A S T A C U S L E N I U S C U L U S (DANA) STEPWISE ACCLIMATED TO D I L U T I O N S OF SEA WATER* A U S T I N W. PRITCHARD and DAVID E. KERLEY Department of Zoology, Oregon State University, Corvallis, Oregon 97331
(Received 13 ffanuary 1970) Abstract--1. The Na +, CI- and osmotic concentration was determined in urine from crayfish, Pacifastacus leniusculus (Dana), stepwise acclimated to 20, 40 and 70% sea water (100% seawater = 31~oo). The osmotic concentration of the urine increased in the higher salinities but never equilibrated with the blood. 2. Urine-blood (U/B) ratios for Na +, C1- and osmotic concentration were less than 1 at all salinities. The U/B ratio for inulin was greater than 1 and did not change with salinity. 3. Urine production rate, as measured by weighing the crayfishafter plugging the nephropore, decreased slightly in 60 and 70% sea water. 4. Inulin clearance rates, measured by the rate of appearance of inulin-14C, were greatly reduced in the higher salinity. INTRODUCTION IN THEIRnormal fresh-water environment, crayfish excrete an extremely hyposmotic urine. Pressures available for formation of "primary urine" are low, but the evidence available favors the operation of a filtrafion-reabsorption kidney in crayfish, with the site of filtration in the coelomosac (Riegel & Kirschner, 1960; Kirschner & Wagner, 1965). It is well known that sodium and chloride are efficiently reabsorbed by the antennal gland, mostly in the distal portion of the nephridial canal and in the bladder (Peters, 1935; Riegel, 1963; Kirschner, 1967; Riegel, 1968). Blood osmotic regulation of crayfish stressed to a range of salinities has been investigated in a number of studies (Hermann, 1931; Bogncki, 1934; Lienemann, 1938; Bryan, 1960c; Kerley & Pritchard, 1967). The role of the antennal gland (kidney) in regulating water and salt balance in salinifies greater than fresh water has received less attention. Bryan (1960c) studied sodium elimination in the urine of NaCl-loaded Astacus fluviatilis. After equilibration in 300 mM NaC1 urine sodium concentration was almost isoionic to blood. Kerley (1962) measured osmotic concentrations in urine of the western North American crayfish, Pacifastacus leniusculus, acclimated to various salinitles, and found that * This investigation was supported by a Grant (GB-1114) from the National Science Foundation and by a Grant from Eastern Oregon College Research Fund, Eastern Oregon College, LaGrande, Oregon. 427
428
AUSTIN W. PRITCHARD AND DAVID E. KERLEY
t h e o s m o t i c c o n c e n t r a t i o n of the u r i n e did n o t a p p r o a c h that of the blood, even in t h e h i g h e r salinities. U r i n e samples, moreover, were very difficult to o b t a i n at the h i g h e r salinities, s u g g e s t i n g a decrease i n t h e flow of u r i n e u n d e r these c o n d i t i o n s . I n this p a p e r we p r e s e n t data o n u r i n e o s m o t i c a n d ionic c o n c e n t r a t i o n s , u r i n e p r o d u c t i o n a n d i n u l i n clearance rate f r o m crayfish (Pacifastacus [eniusculus) stepwise a c c l i m a t e d to 20, 40 a n d 7 0 % sea water. T h e p u r p o s e of the s t u d y is to gain a n u n d e r s t a n d i n g of t h e role of the k i d n e y in osmotic r e g u l a t i o n in salinity-stressed crayfish. M A T E R I A L S AND M E T H O D S Crayfish were captured in minnow traps from a pond near Corvallis. They were maintained in dechlorinated tap water for at least 1 week before an experiment and fed daily. Only intermolt males were used. All experiments were conducted at 15°C.
Experimental protocol Crayfish were left 48 hr in fresh water, at which time ten animals were sampled. The remaining animals were transferred to 20% sea water, and after 48 hr another group of ten animals was sampled. The procedure was repeated for 40 and 70% sea water. One hundred per cent sea water was established as 31%° (970 mOsm/1.). Blood and urine sampling Blood samples were obtained from the ventral body sinus by puncturing the membrane of the coxal segment on the fourth or fifth walking leg with a capillary tube, and letting the blood flow into a test-tube. The blood was allowed to clot and the clotted blood was then frozen until analyses were performed. As the frozen clot thawed it was broken up with a small glass stirring rod and filtered through an 8/~ Millipore paper. The resulting "serum" was used for analyses. Urine samples were obtained by gently teasing the antennal gland pore with Pasteur pipets drawn out and slightly hooked at the ends. Urine and blood sodium were determined with a Coleman flame photometer. Chloride was analyzed with a Cotlove chloridometer. Osmotic concentrations were determined with a Mechrolab vapour pressure osmometer. Procedure for estimating inulin clearance rate T h e traditional procedure for determining clearance necessitates knowing urine concentration, plasma concentration and urine production rate. The arrangement of the antennal gland in crayfish makes it very difficult to obtain a direct measure of urine flow. In most cases it has been estimated by blocking the nephropore and following the weight change of the animal, a procedure which places the animal in a rather abnormal state (see Results). In the present study, inulin clearance is estimated by an indirect method which obviates the necessity of determining urine volume flow. The method is described by Parsons & Alvarado (1968), and in essence involves following the rate of appearance of labelled inulin into a small, measured quantity of medium containing a single animal. Riegel & Kirschner (1960) obtained a fairly constant inulin urine-blood (U]B) ratio in Pacifastacus over a wide range of blood inulin concentrations, and concluded that inulin is neither reabsorbed nor secreted by the antennal gland tubules. If this is so, and assuming that primary urine in crayfish is formed by filtration, then the inulin clearance rate serves as an estimate of filtration rate. It is calculated from the following equation : UV C R = - -p , (1) where CR is the inulin clearance rate, U is the concentration of inulin/ml of urine, V is the volume in ml of urine produced]unit of time and P is the concentration of inulin/ml of blood.
KIDNEY FUNCTION I N THE NORTH AMERICAN CRAYFISH
429
If one assumes that the cpm of radioactive inulin in a sample are proportional to the quantity of inulin, then one can substitute as in equation (2): CR = (cpm/ml) of urine x (ml/hour) of urine (cpm/ml) of blood ' (2) Cancelling the ml's in equation (2), equation (3) is obtained: CR = (cpm/hour) appearing in bath due to urination (cpm/ml) of blood
(3)
T h e numerator is obtained by constructing a time-concentration curve from samples taken from the bath. In order to obtain samples with enough radioactivity, a small test chamber had to be used. Riegel & Kirschner (1960) have shown that handling causes the kidney to cease functioning for various periods of time. Since the method is based on the assumption that the crayfish was a more-or-less continuous urinator (Kamemoto & Ono, 1968), the following precautions were taken. The animals were placed in the test chamber, 5½ x 3½ in. plastic dishes, in 100 ml of the appropriate sea-water dilutions, 36 hr before the start of the experiment. Twelve hr before the start of the experiment the crayfish were injected with 0"2 ml of Ringer's containing approximately 3/~c of inulin-llC. To prevent leakage during the experiment, the syringe hole was glued shut with Lock-Tite cement. One hr before the start of the experiment the animals were gently transferred to clean containers of the same size, containing 100 ml of fresh sea-water dilution. One-ml samples of the bath were then taken at 2-hr intervals for 8 hr. T h e radioactivity was determined by a Nuclear-Chicago liquid scintillation counter, Model No. 6819. One-ml samples of the bath were mixed with 10 ml of liquid scintillation medium described by Patterson & Green (1965) containing toluene as a primary solvent and T r i t o n - X 100 as the secondary solvent or emulsifying agent in a ratio of 2 : 1. T h e primary solute of fluor was 2,5-diphenuloxazole (PPO) and the secondary solute was 1,4-bis-2(5-phenuloxazolyl)-benzene (POPOP). Patterson & Green (1965) showed that the efficiency of this medium remained essentially the same even though the water in the sample or the quantity of radioactivity varied. To test the effects of sea water on this system, inulin-14C was mixed with various sea-water dilutions and counted over an 800-rain period. Moreover, some of the stock inulin-14C labeled sea water was stored for 2 weeks, and then prepared and counted to see if storage at room temperature would affect it. Activity of the emulsion was the same under all conditions. Blood and urine samples were taken at the end of the experiment and analyzed as described in foregoing sections. Blood and urine radioactivity was measured using the liquid scintillation method. RESULTS
Analysis of ionic and osmotic concentrations of urine O s m o t i c a n d ionic c o n c e n t r a t i o n of t h e u r i n e of crayfish s t e p w i s e a c c l i m a t e d to i n c r e a s i n g salinities w e r e a n a l y z e d in t w o e x p e r i m e n t s . O s m o t i c a n d s o d i u m c o n c e n t r a t i o n s w e r e d e t e r m i n e d in t h e first e x p e r i m e n t , a n d c h l o r i d e v a l u e s in t h e s e c o n d . C o n c u r r e n t b l o o d s a m p l e s w e r e t a k e n a n d a n a l y z e d for t h e s a m e p a r a m e t e r s . T h e results are p r e s e n t e d in T a b l e 1. L o w c o n c e n t r a t i o n s o f s o d i u m a n d c h l o r i d e a p p e a r in t h e u r i n e in salinities t h r o u g h 4 0 % . I n 70% sea w a t e r t h e r e was a m a r k e d i n c r e a s e in t h e e x c r e t i o n o f b o t h ions. T h e o s m o t i c c o n c e n t r a t i o n of u r i n e r e m a i n e d u n c h a n g e d in 20~/o sea water, c o m p a r e d t o f r e s h - w a t e r c o n t r o l a n i m a l s . I n t h e 400/o stress, o s m o t i c c o n c e n t r a t i o n d o u b l e s a n d d o u b l e s again in 70~/o. S i n c e s o d i u m a n d c h l o r i d e c o n c e n t r a t i o n s in t h e u r i n e d o n o t i n c r e a s e in
23 (8) 8 (10) 10 (10) 7 (9)
n
14"18+2'3 (6.8 _+2.5) 1 5 " 5 +-2"9 (8"8 + 1"5) 2 4 " 5 _+7"2 (9"1 _+1-7) 65"4 _+8'6 (55.0 _+8.6)
Urine
n 25 (10) 14 (10) 12 (10) 21 (10)
Sodium
200+4-4 (224_+6"2) 230_+3"5 (224_+2"7) 212+8"2 (227 + 5"7) 246 + 8"7 (251 _+4.0)
Blood
9
10
10
10
n
83'0 + 32-4
5'7_+ 1"7
5"2+- 1"7
3'8+_ 1"4
Urine
10
10
10
10
n
Chloride
298.0-+ 5"8
241"0+-4"9
229'0+4"1
t96'9+-3"8
Blood
48'1_+12'9
20"0+- 2.1
19'7_+ 3-2
Urine
18 102.8_+ 19"7
13
14
25
n
22
14
10
26
n
266 + 4'9
228_+ 3-4
223+-12'4
207_+ 3-9
Blood
Osmotic concentration
C H L O R I D E AND O S M O T I C C O N C E N T R A T I O N S OF U R I N E F R O M C R A Y F I S H S T E P W I S E A C C L I M A T E D TO SEA "WATER D I L U T I O N S
Sodium and osmotic concentrations from the first (1966) experiment; chloride values from the second (1968) experiment. Osmotic concentrations in mlVI/NaC1 per 1.; sodium concentrations in mNI/1. Mean values with standard errors are given. n = number of individual animals sampled. 100°/o sea water = 30"67~o. T h e numbers in parentheses are sodium values from the second experiment. Blood values from concurrently sampled animals are shown.
70% sea water
40%seawater
20%seawater
Freshwater
Medium
TABLE 1--SoDIUM~
7,
~J
>
x
f~
431
K I D N E Y F U N C T I O N I N THE N O R T H AMERICAN CRAYFISH
40%, the rise in urine osmotic concentration reflects increased excretion of an unmeasured osmotic constituent of the urine, possibly potassium. Riegel (1968) reports potassium concentrations of 10-30 raM/1, in the definitive urine of .4stacus
fluviatilis. The U/B ratios of the various parameters are presented in Table 2. Ratios for sodium and chloride remain essentially unchanged in fresh water, 20 and 40% sea water. Chloride appears to be reabsorbed more effectively than sodium, as indicated by the lower U/B ratios for the former. In 70% sea water U/B ratios for all parameters increase, but are still considerably less than one. Moreover, the U/B ratios for sodium and chloride are the same in this stress. Included in Table 2 are U/B ratios for inulin, obtained from the study of inulin clearance rates, to be described later. The ratios are greater than 1.0, indicating reabsorption of water. TABLE
2--U/B RATIOS FOR Medium
Fresh water 20% sea water 40% sea water 70% sea water
CRAYFISH STEPWISE ACCLIMATED TO SEA-WATER D I L U T I O N S
Sodium
Chloride
Osmotic
Inulin
0"07 (0.03) 0"068 (0.04) 0"115 (0.04) 0"266 (0.219)
0'016
0"095
2"11
0'023
0"089
2"18
0"024
0"210
2"30
0"278
0"380
2"13
Numbers in parentheses are sodium U/B ratios calculated from a 1966 experiment.
Estimate of urine production rate Estimates of urine production in crayfish stressed to increasing salinities were accomplished by weighing the animal, plugging the nephropore with a small piece of filter paper soaked in Lock-Tire tissue cement, and then reweighing the animal several hours after it was replaced in the medium. Care was taken to dry the animals in a standardized manner before weighing. The increase in weight represents the amount of water that would normally be removed by the kidney as urine. Bryan (1960a) found that the values for urine production rate (expressed as percentage weight gained per 24 hr) obtained by weighing the animals 8 hr after plugging the nephropore were higher than those obtained if he weighed the crayfish 24 hr after plugging. This is not surprising since the crayfish kidney is a filtration system composed of a number of connected anatomical parts, without intervening valves (Maluf, 1939, 1941); any back pressure caused by an excess of urine in the bladder could reduce the rate of filtration. Turgor pressure in the animal could, moreover, result in a decrease in the osmotic flow of water into the animal. We have confirmed Bryan's (1960a) findings in some preliminary tests in
AUSTIN W . PRITCHARD AND DAVID E. KERLEY
432
which crayfish were weighed at 8 hr, and again at 24 hr. In many cases the total weight gained after 24 hr was actually less than that gained after 8 hr. For this reason we decided to base our estimates of urine production rate (in 8-hr measurements. T h e accuracy of the weight-gain procedure has been discussed recently by K a m e m o t o & Ono (1968). T h e s e authors constructed a special crayfish holder and managed to attach small tubes to the nephropores so that urine production could be measured directly. W h e n the data were calculated as percentage body weight gain, an average value of 4.27 per cent gain/24 hr was obtained for the crayfish Procambarus clarkii. T w o crayfish in their study gained 12:96 and 9.6 per cent body wt. in 24 hr. W e fitted two Pacifastacus with catheter tubes in a manner similar to that described by K a m e m o t o & Ono, and obtained the urine production rates shown in Table 3. Although higher than the average value of Kamemoto & Ono, the rates are within the range of values for fresh-water animals in Table 4. TABLE 3--URINE
PRODUCTION OF TWO CRAYFISH OVER A
Crayfish 1 Crayfish 2
2-day PERIOD
First 24 hr
Second 24 hr
8"0 6'2
7-1 7.8
Both crayfish had a tube glued over nephropore papillae. Data presented as percentage weight gained (urine wt.+crayfish wt.)/24 hr, so that it may be compared to Table 4. T A B L E t]
E S T I M A T I O N OF URINE PRODUCTION IN CRAYFISH STEPWISE ACCLIMATED TO SEAWATER DILUTIONS
Medium
n
Urine production
Average initial crayfish weight
Fresh water 20% sea water 40% sea water 70% sea water
10 11 8 13
6"18 + 1-2 3-84 +_0-7 2-53 + 1"2 3"21 + 0'4
37-97 36"40 37"30 37'24
Data expressed as percentage weight gained in 24 hr in animals whose nephropores were plugged. Mean values with standard errors are given, n = number of individual animals sampled. Table 4 summarizes the results of the measurements of urine production rate, estimated by the weight-gain method, of crayfish stressed to increasing salinities. T h e results for fresh-water animals are similar to those obtained on other crayfish, e.g. 6 per cent weight gain/24 hr for Astacusfluviatilis (Bryan, 1960c) and a 7 per cent weight gain/24 hr for Procambarus clarkii (Kamemoto et al., 1966). A marked
433
KIDNEY FUNCTION I N THE NORTH AMERICAN CRAYFISH
decrease in urine production rate occurs in 20% sea water. This would be expected because the osmotic gradient between blood and medium is reduced. There is some further reduction in 40% sea water. In 70% sea water the osmotic gradient has been reversed, yet these animals still seem to be gaining weight; at a rate, in fact, slightly greater than in 40%, but still considerably less than in fresh water. One explanation would be that the animals are drinking and reabsorbing salts and water in the gut, in a manner reminiscent of marine teleosts. To test this a number of crayfish were acclimated to 70% sea water for 0, 3, 7, 8 and 11 days. Radioactive inulin was then added to the medium and animals were sampled 4 hr later; in one case 24 hr later. The animals were autopsied and the amount of radioactivity in the intestinal fluid was the same as background. This agrees with studies of Bryan (1960b) on Astacusfluviatilis, using radioactive inulin and various dyes. It appears questionable, therefore, that the weight-gain method estimates the true urine production in the higher salinities.
Inulin clearance rates Inulin clearance rates obtained by analyzing the appearance of inulin-t4C in the bath are summarized in Table 5. Two assumptions are made in assessing the validity of the method. First, it is assumed that negligible quantities of inulin leave the animal by extrarenal routes. To test this, five crayfish were injected with inulin-t4C and their nephropores plugged with tissue cement. When the bath was analyzed, little, if any, activity appeared during the first 13-hr period (Table 5). Two crayfish (Nos. 6 and 7, Table 5) showed some leakage at the end of 29 hr. T A B L E 5 - - A P P E A R A N C E OF I N U L I N - 1 4 C I N THE BATH AFTER NEPHROPORE P L U G G I N G
Crayfish No. Time after injection (hr) 1 5 8 13 17 20 29
6
7
8
9
10
225 41 50 125 . . 1282
170 7 145 541
16 19 23 21 . . 48
16 16 21 17
13 24 20 21
18
19
. .
. . 1834
. .
AnimaLs were injected with approximately 3/~c of inulin and the nephropores were plugged. Data reported in cpm/ml of 100 ml of bath. This could have been due to a leaking plug. A second assumption is that the crayfish urinates more or less continuously over the period of time of sampling, since the slope of the curve constructed from appearance of inulin-14C in the bath is used to calculate the inulin clearance. Kamemoto & Ono (1968) indicated that Procambarus clarkii urinated intermittently, but fairly regularly. The crayfish
434
AUSTIN W . PRITCHABD AND DAVID E. KERLEY
catheterized in the preliminary test described earlier appeared to urinate continuously. To determine whether inulin would appear in the water at a constant
rate or whether it would appear in irregular "surges", five freshwater control animals were injected with inulin and sampled over a 29-hr period (Table 6). TABLE 6--APPEARANCE OF RADIOACTIVITY IN THE BATH AFTER INJECTION OF INULIN-14C
Crayfish No. Time after injection (hr) 1 5 8 13 17 20 29
1
2
3
4
5
759 661 1241 1968 2409 2257 1852
833 303 4032 2415 4115 2695 3460
44 23 1519 2717 3236 3086 2392
466 1628 2127 4273 4716 4444 2967
30 1222 1506 1631 1919 1196 1675
Normal animals injected with about 3/zc of inulin. One-ml samples were taken from the bath and are reported as cpm/ml of 100 ml bath.
Animals 1, 2 and 3 appear not to urinate for the first 5 hr. These animals may be reflecting the effects of handling. Riegel & Kirschner (1960), for example, noted that Cambarus would not produce urine for some time after being handled. Although the data in T a b l e 6 suggest an intermittent pattern of urination, there is a steady increase of radio-inulin in the bath up to 17 hr, with the exception of an inexplicable decrease in animal No. 2 at 13 hr. After 20 hr there may be some bacterial decay of inulin. T h e inulin clearance rates obtained f r o m crayfish stepwise acclimated to increasing salinities are s u m m a r i z e d in T a b l e 7. T h e r e is a striking reduction in the a m o u n t of inulin being cleared by the 40 and 70% stressed animals compared to fresh-water and 2 0 % animals. I t appears as if the kidney almost completely shuts down in the isosmotic and hyperosmotic media. T h e discrepancy in the effects of salinity stress on inulin clearance rates (Table 7) and urine production rates estimated by weight gain (Table 4) will be discussed later. TABLE 7 - - I N U L I N CLEARANCE RATES FOR SALINITY-STRESSED CRAYFISH
Medium Fresh water 20% sea water 40% sea water 70% sea water
n 9 8 8 10
Inulin clearance 2.45 2"66 0.008 0.033
+0-47 _+0"70 _+0"004 _+0"024
Mean weight 38"27 39"13 40-16 45"54
Rates expressed as ml/kg per hr. Mean values with standard errors are given. n = number of individual animals sampled.
K I D N E Y F U N C T I O N IN THE N O R T H AMERICAN CRAYFISH
435
DISCUSSION The crayfish kidney is a primary site of water loss and salt conservation. The normal fresh-water crayfish produces large quantities of dilute urine. The primary urine is apparently produced by filtration and the secondary or definitive urine by reabsorption of salts and water by the various parts of the kidney (Martin, 1957; Riegel & Kirschner, 1960; Kamemoto, 1961; Kamemoto et al., 1962; Riegel, 1963, 1965, 1966a, b, 1968; Kirschner & Wagner, 1965). If the kidney of a crayfish subjected to a hyperosmotic stress were to continue to produce urine at the same rate as in fresh water, the animals would presumably become dehydrated. In an earlier paper (Kerley & Pritchard, 1967), we reported no change in tissue water content or blood volume in crayfish stepwise acclimated to 70% sea water, a hyperosmotic medium. The possibility that water is obtained through the gut by drinking has been tested by Maluf (1940), Bryan (1960a) and by the present authors, using dyes and radio-inulin. In no case has clear evidence been presented that crayfish drink the medium. If Pacifastacus does not take in water by drinking and if the body fluids are isosmotic or hyposmotic to the medium then the total output of the kidney must be reduced. This is supported by the inulin clearance data (Table 7) and the urine production rate data (Table 4), both of which show reduction in kidney function with increasing salinity. Moreover, we have observed that it is extremely difficult to obtain urine samples from animals stressed to 70% sea water. The inulin clearance information, obtained by the method used in this study, does not directly tell us whether the filtration rate is reduced in the higher salinities. The inulin U/B ratios, however, do not change with salinity (Table 2), and this indicates that equal proportions of water are absorped by the kidney tubule in all salinities tested. This in turn leads us to suggest that the main response to the higher salinities (40 and 70% sea water) is a great reduction in the formation of primary urine by the antennal gland of
Pacifastacus. The urine production rates show a different trend with increasing salinity than the inulin clearance values (Tables 4 and 7). Specifically, there is proportionally a much greater reduction in inulin clearance rates in 40 and 70% stresses than in urine production rates. At this time we can offer no explanation for the discrepancy outside of the unlikely one that water is secreted into the tubules. As mentioned previously, the weight-gain method may not accurately reflect the true urine production rate in the higher salinities. When Pacifastacus leniusculus is stepwise acclimated to increasing salinities the osmotic and ionic concentrations of the urine remain low in fresh water, 20 and 40% sea water (Table 1). The U/B ratios (Table 2) are very low in these stresses, reflecting the reabsorption of sodium and chloride from the tubules (Peters, 1935; Schmidt-Nielsen & Laws, 1963 ; Riegel, 1968). Even in the hyperosmotic medium of 70% sea water, the ratios are all less than 0.05. These findings contrast to those of Bryan (1960c) who found sodium U/B ratios of 1.0 in Astacus fluviatilis immersed from 5 to 6 days in 300 m M NaC1. Although our animals were brought to the highest test salinity in stepwise fashion, it is possible that a steady state had not
436
AUSTIN W . PRITCHARD AND DAVID E. KERLEY
been reached at the time of sampling. Even if this were the case, however, it is not likely that a further small change in blood concentration would alter dramatically the excretion of salts by the kidney. T h e relatively low U/B ionic and osmotic concentration ratios, together with the greatly reduced urine output, would indicate that Pacifastacus stressed to hyperosmotic media does not eliminate large quantities of salt via the kidney. Examination of extra-renal sources of salt loss, for example gills, under conditions of hyperosmotic stress should prove interesting.
REFERENCES BOGDCKI M. (1934) Recherches sur la regulation de la composition min~rale da sung chez l'6crevisse (Astacus fluviatilis L.). Archs int. Physiol. 38, 172-179. BRYAN G. W. (1960a) Sodium regulation in the cryafish Astacusfluviatilis--I. The normal animal. J. exp. Biol. 37, 83-99. BRYANG. W. (1960b) Sodium regulation in the crayfish Astacusfluviatilis-- II. Experiments with sodium-depleted animals. J. exp. Biol. 37, 100-112. BRYANG. W. (1960c) Sodium regulation in the crayfish Astacusfluviatilis--I I I. Experiments with NaCl-loaded animals. J. exp. Biol. 37, 113-128. HERMANNF. (1931) l~ber den Wasserhaushalt des Flusskrebses (Potamobius astacus Leach). Z. vergl. Physiol. 14, 479-524. KAMEMOTO F. I. (1961) The effects of eserine on sodium regulation in crayfish. Comp. Biochem. Physiol. 3, 297-303. KAMEMOTO F. I., KATO K. N. & TUCKERL. E. (1966). Neurosecretion and salt and water balance in the Annelida and Crustacea. Am. Zoologist 6, 213-219. KAMEMOTO F. I., KEISTER S. M. & SPALDINGA. E. (1962) Cholinesterase activities and sodium movement in the crayfish kidney. Comp. Biochem. Physiol. 7, 81-87. KAMEMOTOF. I. • ONO J. (1968) Urine flow determinations by continuous collection in the crayfish Procambarus clarkii. Comp. Biochem. Physiol. 27, 851-857. KERLEYD. E. (1962) Osmoregulation in two geographically isolated populations of crayfish. Master's thesis, Corvallis, Oregon State University. KERLEY n . E. & PRITCHARDA. W. (1967) Osmotic regulation in the crayfish, Pacifastacus leniusculus, stepwise acclimated to dilutions of sea water. Comp. Biochem. Physiol. 20, 101-113. KIRSCHNER L. B. (1967) Comparative physiology: invertebrate excretory organs. A. Rev. Physiol. 29, 169-196. KIRSCHNER L. B. & WAGNERS. (1965) The site and permeability of the filtration locus in crayfish antennal gland. 37. exp. Biol. 43, 385-395. LIENEMANN L. J. (1938) The green gland as a mechanism for osmotic regulation in the crayfish Cambarus clarkii Girard. 37. cell. eomp. Physiol. 11, 149-161. MALUF N. S. R. (1939) On the anatomy of the kidney of the crayfish and on the absorption of chloride from fresh water by this animal. Zool. 3tb. 59, 515-534. MALUF N. S. R. (1940) The uptake of inorganic electrolytes by the crayfish. J. gen. Physiol. 24, 151-167. MALUF N. S. R. (1941) Micturition in the crayfish and further observations on the anatomy of the mephron of this animal. Biol. Bull. 81, 134-148. MARTIN A. W. (1957) Recent advances in knowledge of invertebrate renal function. In Recent Advances in Invertebrate Physiology (Edited by SCHEERBRADLEYT.), pp. 247-276. Eugene, University of Oregon Publications. MARTINA. W. (1958) Comparative physiology (excretion). A. Rev. Physiol. 20, 225-242.
K I D N E Y F U N C T I O N I N THE N O R T H AMERICAN CRAYFISH
437
PARSONSR. H. & ALVARADOR. H. (1968) Effect of temperature on water and ion balance in larval Ambystoma gracile. Comp. Biochem. Physiol. 24, 61-72. PATTERSONM. S. & GREENR. C. (1965) Measurement of low energy/~-emitters in aqueous solutions by liquid scintillation counting of emulsions. Analyt. Chem. 37, 854-857. PETERS H. (1935) ~ber den Einfluss des Salzgehaltes im Aussenmedium auf den Ban und die Funktion der Exkretionsorgane dekapoder Crustaceen (nach Untersuchungen an Potamobius fluviatilis and Homaris vulgaris). Z. Morph. Okol. 30, 355-381. RIECEr. J. A. (1963) Micropuncture studies of chloride concentration and osmotic pressure in the crayfish antennal gland. ~t. exp. Biol. 40, 457--492. RmOEL J. A. (1965) Micropuncture studies of the concentration of sodium, potassium and inulin in the crayfish antennal gland, ft. exp. Biol. 42, 379-384. RIEOEr. J. A. (1966a) Analysis of formed bodies in urine removed from crayfish antennal gland by micropuncture, ft. exp. Biol. 44, 389-395. RmGEL J. A. (1966b) Micropuncture studies of formed-bodies secreted by the excretory organs of the crayfish, frog and stick insects..7, exp. Biol. 48, 587-596. RmCEL J. A. (1968) Analysis of the distribution of sodium, potassium, and osmotic pressure in the urine of the crayfish, ft. exp. Biol. 48, 587-596. RIEGELJ. A. & KIRSCHNERL. B. (1960) The excretion of inulin and glucose by the crayfish antennal gland. Biol. Bull. 118, 296-307. SCHMIDT-NmLsEN B. & LAWS D. F. (1963) Invertebrate mechanisms for diluting and concentrating the urine. A. Rev. Physiol. 25, 631-658.
Key Word Index--Acclimation; kidney function in crayfish; Pascifastacus peniusculus; osmoregulation in crayfish.
K I D N E Y F U N C T I O N IN T H E N O R T H AMERICAN CRAYFISH P A C I F A S T A C U S L E N I U S C U L U S (DANA) STEPWISE ACCLIMATED TO D I L U T I O N S OF SEA WATER* A U S T I N W. PRITCHARD and DAVID E. KERLEY Department of Zoology, Oregon State University, Corvallis, Oregon 97331
(Received 13 ffanuary 1970) Abstract--1. The Na +, CI- and osmotic concentration was determined in urine from crayfish, Pacifastacus leniusculus (Dana), stepwise acclimated to 20, 40 and 70% sea water (100% seawater = 31~oo). The osmotic concentration of the urine increased in the higher salinities but never equilibrated with the blood. 2. Urine-blood (U/B) ratios for Na +, C1- and osmotic concentration were less than 1 at all salinities. The U/B ratio for inulin was greater than 1 and did not change with salinity. 3. Urine production rate, as measured by weighing the crayfishafter plugging the nephropore, decreased slightly in 60 and 70% sea water. 4. Inulin clearance rates, measured by the rate of appearance of inulin-14C, were greatly reduced in the higher salinity. INTRODUCTION IN THEIRnormal fresh-water environment, crayfish excrete an extremely hyposmotic urine. Pressures available for formation of "primary urine" are low, but the evidence available favors the operation of a filtrafion-reabsorption kidney in crayfish, with the site of filtration in the coelomosac (Riegel & Kirschner, 1960; Kirschner & Wagner, 1965). It is well known that sodium and chloride are efficiently reabsorbed by the antennal gland, mostly in the distal portion of the nephridial canal and in the bladder (Peters, 1935; Riegel, 1963; Kirschner, 1967; Riegel, 1968). Blood osmotic regulation of crayfish stressed to a range of salinities has been investigated in a number of studies (Hermann, 1931; Bogncki, 1934; Lienemann, 1938; Bryan, 1960c; Kerley & Pritchard, 1967). The role of the antennal gland (kidney) in regulating water and salt balance in salinifies greater than fresh water has received less attention. Bryan (1960c) studied sodium elimination in the urine of NaCl-loaded Astacus fluviatilis. After equilibration in 300 mM NaC1 urine sodium concentration was almost isoionic to blood. Kerley (1962) measured osmotic concentrations in urine of the western North American crayfish, Pacifastacus leniusculus, acclimated to various salinitles, and found that * This investigation was supported by a Grant (GB-1114) from the National Science Foundation and by a Grant from Eastern Oregon College Research Fund, Eastern Oregon College, LaGrande, Oregon. 427
428
AUSTIN W. PRITCHARD AND DAVID E. KERLEY
t h e o s m o t i c c o n c e n t r a t i o n of the u r i n e did n o t a p p r o a c h that of the blood, even in t h e h i g h e r salinities. U r i n e samples, moreover, were very difficult to o b t a i n at the h i g h e r salinities, s u g g e s t i n g a decrease i n t h e flow of u r i n e u n d e r these c o n d i t i o n s . I n this p a p e r we p r e s e n t data o n u r i n e o s m o t i c a n d ionic c o n c e n t r a t i o n s , u r i n e p r o d u c t i o n a n d i n u l i n clearance rate f r o m crayfish (Pacifastacus [eniusculus) stepwise a c c l i m a t e d to 20, 40 a n d 7 0 % sea water. T h e p u r p o s e of the s t u d y is to gain a n u n d e r s t a n d i n g of t h e role of the k i d n e y in osmotic r e g u l a t i o n in salinity-stressed crayfish. M A T E R I A L S AND M E T H O D S Crayfish were captured in minnow traps from a pond near Corvallis. They were maintained in dechlorinated tap water for at least 1 week before an experiment and fed daily. Only intermolt males were used. All experiments were conducted at 15°C.
Experimental protocol Crayfish were left 48 hr in fresh water, at which time ten animals were sampled. The remaining animals were transferred to 20% sea water, and after 48 hr another group of ten animals was sampled. The procedure was repeated for 40 and 70% sea water. One hundred per cent sea water was established as 31%° (970 mOsm/1.). Blood and urine sampling Blood samples were obtained from the ventral body sinus by puncturing the membrane of the coxal segment on the fourth or fifth walking leg with a capillary tube, and letting the blood flow into a test-tube. The blood was allowed to clot and the clotted blood was then frozen until analyses were performed. As the frozen clot thawed it was broken up with a small glass stirring rod and filtered through an 8/~ Millipore paper. The resulting "serum" was used for analyses. Urine samples were obtained by gently teasing the antennal gland pore with Pasteur pipets drawn out and slightly hooked at the ends. Urine and blood sodium were determined with a Coleman flame photometer. Chloride was analyzed with a Cotlove chloridometer. Osmotic concentrations were determined with a Mechrolab vapour pressure osmometer. Procedure for estimating inulin clearance rate T h e traditional procedure for determining clearance necessitates knowing urine concentration, plasma concentration and urine production rate. The arrangement of the antennal gland in crayfish makes it very difficult to obtain a direct measure of urine flow. In most cases it has been estimated by blocking the nephropore and following the weight change of the animal, a procedure which places the animal in a rather abnormal state (see Results). In the present study, inulin clearance is estimated by an indirect method which obviates the necessity of determining urine volume flow. The method is described by Parsons & Alvarado (1968), and in essence involves following the rate of appearance of labelled inulin into a small, measured quantity of medium containing a single animal. Riegel & Kirschner (1960) obtained a fairly constant inulin urine-blood (U]B) ratio in Pacifastacus over a wide range of blood inulin concentrations, and concluded that inulin is neither reabsorbed nor secreted by the antennal gland tubules. If this is so, and assuming that primary urine in crayfish is formed by filtration, then the inulin clearance rate serves as an estimate of filtration rate. It is calculated from the following equation : UV C R = - -p , (1) where CR is the inulin clearance rate, U is the concentration of inulin/ml of urine, V is the volume in ml of urine produced]unit of time and P is the concentration of inulin/ml of blood.
KIDNEY FUNCTION I N THE NORTH AMERICAN CRAYFISH
429
If one assumes that the cpm of radioactive inulin in a sample are proportional to the quantity of inulin, then one can substitute as in equation (2): CR = (cpm/ml) of urine x (ml/hour) of urine (cpm/ml) of blood ' (2) Cancelling the ml's in equation (2), equation (3) is obtained: CR = (cpm/hour) appearing in bath due to urination (cpm/ml) of blood
(3)
T h e numerator is obtained by constructing a time-concentration curve from samples taken from the bath. In order to obtain samples with enough radioactivity, a small test chamber had to be used. Riegel & Kirschner (1960) have shown that handling causes the kidney to cease functioning for various periods of time. Since the method is based on the assumption that the crayfish was a more-or-less continuous urinator (Kamemoto & Ono, 1968), the following precautions were taken. The animals were placed in the test chamber, 5½ x 3½ in. plastic dishes, in 100 ml of the appropriate sea-water dilutions, 36 hr before the start of the experiment. Twelve hr before the start of the experiment the crayfish were injected with 0"2 ml of Ringer's containing approximately 3/~c of inulin-llC. To prevent leakage during the experiment, the syringe hole was glued shut with Lock-Tite cement. One hr before the start of the experiment the animals were gently transferred to clean containers of the same size, containing 100 ml of fresh sea-water dilution. One-ml samples of the bath were then taken at 2-hr intervals for 8 hr. T h e radioactivity was determined by a Nuclear-Chicago liquid scintillation counter, Model No. 6819. One-ml samples of the bath were mixed with 10 ml of liquid scintillation medium described by Patterson & Green (1965) containing toluene as a primary solvent and T r i t o n - X 100 as the secondary solvent or emulsifying agent in a ratio of 2 : 1. T h e primary solute of fluor was 2,5-diphenuloxazole (PPO) and the secondary solute was 1,4-bis-2(5-phenuloxazolyl)-benzene (POPOP). Patterson & Green (1965) showed that the efficiency of this medium remained essentially the same even though the water in the sample or the quantity of radioactivity varied. To test the effects of sea water on this system, inulin-14C was mixed with various sea-water dilutions and counted over an 800-rain period. Moreover, some of the stock inulin-14C labeled sea water was stored for 2 weeks, and then prepared and counted to see if storage at room temperature would affect it. Activity of the emulsion was the same under all conditions. Blood and urine samples were taken at the end of the experiment and analyzed as described in foregoing sections. Blood and urine radioactivity was measured using the liquid scintillation method. RESULTS
Analysis of ionic and osmotic concentrations of urine O s m o t i c a n d ionic c o n c e n t r a t i o n of t h e u r i n e of crayfish s t e p w i s e a c c l i m a t e d to i n c r e a s i n g salinities w e r e a n a l y z e d in t w o e x p e r i m e n t s . O s m o t i c a n d s o d i u m c o n c e n t r a t i o n s w e r e d e t e r m i n e d in t h e first e x p e r i m e n t , a n d c h l o r i d e v a l u e s in t h e s e c o n d . C o n c u r r e n t b l o o d s a m p l e s w e r e t a k e n a n d a n a l y z e d for t h e s a m e p a r a m e t e r s . T h e results are p r e s e n t e d in T a b l e 1. L o w c o n c e n t r a t i o n s o f s o d i u m a n d c h l o r i d e a p p e a r in t h e u r i n e in salinities t h r o u g h 4 0 % . I n 70% sea w a t e r t h e r e was a m a r k e d i n c r e a s e in t h e e x c r e t i o n o f b o t h ions. T h e o s m o t i c c o n c e n t r a t i o n of u r i n e r e m a i n e d u n c h a n g e d in 20~/o sea water, c o m p a r e d t o f r e s h - w a t e r c o n t r o l a n i m a l s . I n t h e 400/o stress, o s m o t i c c o n c e n t r a t i o n d o u b l e s a n d d o u b l e s again in 70~/o. S i n c e s o d i u m a n d c h l o r i d e c o n c e n t r a t i o n s in t h e u r i n e d o n o t i n c r e a s e in
23 (8) 8 (10) 10 (10) 7 (9)
n
14"18+2'3 (6.8 _+2.5) 1 5 " 5 +-2"9 (8"8 + 1"5) 2 4 " 5 _+7"2 (9"1 _+1-7) 65"4 _+8'6 (55.0 _+8.6)
Urine
n 25 (10) 14 (10) 12 (10) 21 (10)
Sodium
200+4-4 (224_+6"2) 230_+3"5 (224_+2"7) 212+8"2 (227 + 5"7) 246 + 8"7 (251 _+4.0)
Blood
9
10
10
10
n
83'0 + 32-4
5'7_+ 1"7
5"2+- 1"7
3'8+_ 1"4
Urine
10
10
10
10
n
Chloride
298.0-+ 5"8
241"0+-4"9
229'0+4"1
t96'9+-3"8
Blood
48'1_+12'9
20"0+- 2.1
19'7_+ 3-2
Urine
18 102.8_+ 19"7
13
14
25
n
22
14
10
26
n
266 + 4'9
228_+ 3-4
223+-12'4
207_+ 3-9
Blood
Osmotic concentration
C H L O R I D E AND O S M O T I C C O N C E N T R A T I O N S OF U R I N E F R O M C R A Y F I S H S T E P W I S E A C C L I M A T E D TO SEA "WATER D I L U T I O N S
Sodium and osmotic concentrations from the first (1966) experiment; chloride values from the second (1968) experiment. Osmotic concentrations in mlVI/NaC1 per 1.; sodium concentrations in mNI/1. Mean values with standard errors are given. n = number of individual animals sampled. 100°/o sea water = 30"67~o. T h e numbers in parentheses are sodium values from the second experiment. Blood values from concurrently sampled animals are shown.
70% sea water
40%seawater
20%seawater
Freshwater
Medium
TABLE 1--SoDIUM~
7,
~J
>
x
f~
431
K I D N E Y F U N C T I O N I N THE N O R T H AMERICAN CRAYFISH
40%, the rise in urine osmotic concentration reflects increased excretion of an unmeasured osmotic constituent of the urine, possibly potassium. Riegel (1968) reports potassium concentrations of 10-30 raM/1, in the definitive urine of .4stacus
fluviatilis. The U/B ratios of the various parameters are presented in Table 2. Ratios for sodium and chloride remain essentially unchanged in fresh water, 20 and 40% sea water. Chloride appears to be reabsorbed more effectively than sodium, as indicated by the lower U/B ratios for the former. In 70% sea water U/B ratios for all parameters increase, but are still considerably less than one. Moreover, the U/B ratios for sodium and chloride are the same in this stress. Included in Table 2 are U/B ratios for inulin, obtained from the study of inulin clearance rates, to be described later. The ratios are greater than 1.0, indicating reabsorption of water. TABLE
2--U/B RATIOS FOR Medium
Fresh water 20% sea water 40% sea water 70% sea water
CRAYFISH STEPWISE ACCLIMATED TO SEA-WATER D I L U T I O N S
Sodium
Chloride
Osmotic
Inulin
0"07 (0.03) 0"068 (0.04) 0"115 (0.04) 0"266 (0.219)
0'016
0"095
2"11
0'023
0"089
2"18
0"024
0"210
2"30
0"278
0"380
2"13
Numbers in parentheses are sodium U/B ratios calculated from a 1966 experiment.
Estimate of urine production rate Estimates of urine production in crayfish stressed to increasing salinities were accomplished by weighing the animal, plugging the nephropore with a small piece of filter paper soaked in Lock-Tire tissue cement, and then reweighing the animal several hours after it was replaced in the medium. Care was taken to dry the animals in a standardized manner before weighing. The increase in weight represents the amount of water that would normally be removed by the kidney as urine. Bryan (1960a) found that the values for urine production rate (expressed as percentage weight gained per 24 hr) obtained by weighing the animals 8 hr after plugging the nephropore were higher than those obtained if he weighed the crayfish 24 hr after plugging. This is not surprising since the crayfish kidney is a filtration system composed of a number of connected anatomical parts, without intervening valves (Maluf, 1939, 1941); any back pressure caused by an excess of urine in the bladder could reduce the rate of filtration. Turgor pressure in the animal could, moreover, result in a decrease in the osmotic flow of water into the animal. We have confirmed Bryan's (1960a) findings in some preliminary tests in
AUSTIN W . PRITCHARD AND DAVID E. KERLEY
432
which crayfish were weighed at 8 hr, and again at 24 hr. In many cases the total weight gained after 24 hr was actually less than that gained after 8 hr. For this reason we decided to base our estimates of urine production rate (in 8-hr measurements. T h e accuracy of the weight-gain procedure has been discussed recently by K a m e m o t o & Ono (1968). T h e s e authors constructed a special crayfish holder and managed to attach small tubes to the nephropores so that urine production could be measured directly. W h e n the data were calculated as percentage body weight gain, an average value of 4.27 per cent gain/24 hr was obtained for the crayfish Procambarus clarkii. T w o crayfish in their study gained 12:96 and 9.6 per cent body wt. in 24 hr. W e fitted two Pacifastacus with catheter tubes in a manner similar to that described by K a m e m o t o & Ono, and obtained the urine production rates shown in Table 3. Although higher than the average value of Kamemoto & Ono, the rates are within the range of values for fresh-water animals in Table 4. TABLE 3--URINE
PRODUCTION OF TWO CRAYFISH OVER A
Crayfish 1 Crayfish 2
2-day PERIOD
First 24 hr
Second 24 hr
8"0 6'2
7-1 7.8
Both crayfish had a tube glued over nephropore papillae. Data presented as percentage weight gained (urine wt.+crayfish wt.)/24 hr, so that it may be compared to Table 4. T A B L E t]
E S T I M A T I O N OF URINE PRODUCTION IN CRAYFISH STEPWISE ACCLIMATED TO SEAWATER DILUTIONS
Medium
n
Urine production
Average initial crayfish weight
Fresh water 20% sea water 40% sea water 70% sea water
10 11 8 13
6"18 + 1-2 3-84 +_0-7 2-53 + 1"2 3"21 + 0'4
37-97 36"40 37"30 37'24
Data expressed as percentage weight gained in 24 hr in animals whose nephropores were plugged. Mean values with standard errors are given, n = number of individual animals sampled. Table 4 summarizes the results of the measurements of urine production rate, estimated by the weight-gain method, of crayfish stressed to increasing salinities. T h e results for fresh-water animals are similar to those obtained on other crayfish, e.g. 6 per cent weight gain/24 hr for Astacusfluviatilis (Bryan, 1960c) and a 7 per cent weight gain/24 hr for Procambarus clarkii (Kamemoto et al., 1966). A marked
433
KIDNEY FUNCTION I N THE NORTH AMERICAN CRAYFISH
decrease in urine production rate occurs in 20% sea water. This would be expected because the osmotic gradient between blood and medium is reduced. There is some further reduction in 40% sea water. In 70% sea water the osmotic gradient has been reversed, yet these animals still seem to be gaining weight; at a rate, in fact, slightly greater than in 40%, but still considerably less than in fresh water. One explanation would be that the animals are drinking and reabsorbing salts and water in the gut, in a manner reminiscent of marine teleosts. To test this a number of crayfish were acclimated to 70% sea water for 0, 3, 7, 8 and 11 days. Radioactive inulin was then added to the medium and animals were sampled 4 hr later; in one case 24 hr later. The animals were autopsied and the amount of radioactivity in the intestinal fluid was the same as background. This agrees with studies of Bryan (1960b) on Astacusfluviatilis, using radioactive inulin and various dyes. It appears questionable, therefore, that the weight-gain method estimates the true urine production in the higher salinities.
Inulin clearance rates Inulin clearance rates obtained by analyzing the appearance of inulin-t4C in the bath are summarized in Table 5. Two assumptions are made in assessing the validity of the method. First, it is assumed that negligible quantities of inulin leave the animal by extrarenal routes. To test this, five crayfish were injected with inulin-t4C and their nephropores plugged with tissue cement. When the bath was analyzed, little, if any, activity appeared during the first 13-hr period (Table 5). Two crayfish (Nos. 6 and 7, Table 5) showed some leakage at the end of 29 hr. T A B L E 5 - - A P P E A R A N C E OF I N U L I N - 1 4 C I N THE BATH AFTER NEPHROPORE P L U G G I N G
Crayfish No. Time after injection (hr) 1 5 8 13 17 20 29
6
7
8
9
10
225 41 50 125 . . 1282
170 7 145 541
16 19 23 21 . . 48
16 16 21 17
13 24 20 21
18
19
. .
. . 1834
. .
AnimaLs were injected with approximately 3/~c of inulin and the nephropores were plugged. Data reported in cpm/ml of 100 ml of bath. This could have been due to a leaking plug. A second assumption is that the crayfish urinates more or less continuously over the period of time of sampling, since the slope of the curve constructed from appearance of inulin-14C in the bath is used to calculate the inulin clearance. Kamemoto & Ono (1968) indicated that Procambarus clarkii urinated intermittently, but fairly regularly. The crayfish
434
AUSTIN W . PRITCHABD AND DAVID E. KERLEY
catheterized in the preliminary test described earlier appeared to urinate continuously. To determine whether inulin would appear in the water at a constant
rate or whether it would appear in irregular "surges", five freshwater control animals were injected with inulin and sampled over a 29-hr period (Table 6). TABLE 6--APPEARANCE OF RADIOACTIVITY IN THE BATH AFTER INJECTION OF INULIN-14C
Crayfish No. Time after injection (hr) 1 5 8 13 17 20 29
1
2
3
4
5
759 661 1241 1968 2409 2257 1852
833 303 4032 2415 4115 2695 3460
44 23 1519 2717 3236 3086 2392
466 1628 2127 4273 4716 4444 2967
30 1222 1506 1631 1919 1196 1675
Normal animals injected with about 3/zc of inulin. One-ml samples were taken from the bath and are reported as cpm/ml of 100 ml bath.
Animals 1, 2 and 3 appear not to urinate for the first 5 hr. These animals may be reflecting the effects of handling. Riegel & Kirschner (1960), for example, noted that Cambarus would not produce urine for some time after being handled. Although the data in T a b l e 6 suggest an intermittent pattern of urination, there is a steady increase of radio-inulin in the bath up to 17 hr, with the exception of an inexplicable decrease in animal No. 2 at 13 hr. After 20 hr there may be some bacterial decay of inulin. T h e inulin clearance rates obtained f r o m crayfish stepwise acclimated to increasing salinities are s u m m a r i z e d in T a b l e 7. T h e r e is a striking reduction in the a m o u n t of inulin being cleared by the 40 and 70% stressed animals compared to fresh-water and 2 0 % animals. I t appears as if the kidney almost completely shuts down in the isosmotic and hyperosmotic media. T h e discrepancy in the effects of salinity stress on inulin clearance rates (Table 7) and urine production rates estimated by weight gain (Table 4) will be discussed later. TABLE 7 - - I N U L I N CLEARANCE RATES FOR SALINITY-STRESSED CRAYFISH
Medium Fresh water 20% sea water 40% sea water 70% sea water
n 9 8 8 10
Inulin clearance 2.45 2"66 0.008 0.033
+0-47 _+0"70 _+0"004 _+0"024
Mean weight 38"27 39"13 40-16 45"54
Rates expressed as ml/kg per hr. Mean values with standard errors are given. n = number of individual animals sampled.
K I D N E Y F U N C T I O N IN THE N O R T H AMERICAN CRAYFISH
435
DISCUSSION The crayfish kidney is a primary site of water loss and salt conservation. The normal fresh-water crayfish produces large quantities of dilute urine. The primary urine is apparently produced by filtration and the secondary or definitive urine by reabsorption of salts and water by the various parts of the kidney (Martin, 1957; Riegel & Kirschner, 1960; Kamemoto, 1961; Kamemoto et al., 1962; Riegel, 1963, 1965, 1966a, b, 1968; Kirschner & Wagner, 1965). If the kidney of a crayfish subjected to a hyperosmotic stress were to continue to produce urine at the same rate as in fresh water, the animals would presumably become dehydrated. In an earlier paper (Kerley & Pritchard, 1967), we reported no change in tissue water content or blood volume in crayfish stepwise acclimated to 70% sea water, a hyperosmotic medium. The possibility that water is obtained through the gut by drinking has been tested by Maluf (1940), Bryan (1960a) and by the present authors, using dyes and radio-inulin. In no case has clear evidence been presented that crayfish drink the medium. If Pacifastacus does not take in water by drinking and if the body fluids are isosmotic or hyposmotic to the medium then the total output of the kidney must be reduced. This is supported by the inulin clearance data (Table 7) and the urine production rate data (Table 4), both of which show reduction in kidney function with increasing salinity. Moreover, we have observed that it is extremely difficult to obtain urine samples from animals stressed to 70% sea water. The inulin clearance information, obtained by the method used in this study, does not directly tell us whether the filtration rate is reduced in the higher salinities. The inulin U/B ratios, however, do not change with salinity (Table 2), and this indicates that equal proportions of water are absorped by the kidney tubule in all salinities tested. This in turn leads us to suggest that the main response to the higher salinities (40 and 70% sea water) is a great reduction in the formation of primary urine by the antennal gland of
Pacifastacus. The urine production rates show a different trend with increasing salinity than the inulin clearance values (Tables 4 and 7). Specifically, there is proportionally a much greater reduction in inulin clearance rates in 40 and 70% stresses than in urine production rates. At this time we can offer no explanation for the discrepancy outside of the unlikely one that water is secreted into the tubules. As mentioned previously, the weight-gain method may not accurately reflect the true urine production rate in the higher salinities. When Pacifastacus leniusculus is stepwise acclimated to increasing salinities the osmotic and ionic concentrations of the urine remain low in fresh water, 20 and 40% sea water (Table 1). The U/B ratios (Table 2) are very low in these stresses, reflecting the reabsorption of sodium and chloride from the tubules (Peters, 1935; Schmidt-Nielsen & Laws, 1963 ; Riegel, 1968). Even in the hyperosmotic medium of 70% sea water, the ratios are all less than 0.05. These findings contrast to those of Bryan (1960c) who found sodium U/B ratios of 1.0 in Astacus fluviatilis immersed from 5 to 6 days in 300 m M NaC1. Although our animals were brought to the highest test salinity in stepwise fashion, it is possible that a steady state had not
436
AUSTIN W . PRITCHARD AND DAVID E. KERLEY
been reached at the time of sampling. Even if this were the case, however, it is not likely that a further small change in blood concentration would alter dramatically the excretion of salts by the kidney. T h e relatively low U/B ionic and osmotic concentration ratios, together with the greatly reduced urine output, would indicate that Pacifastacus stressed to hyperosmotic media does not eliminate large quantities of salt via the kidney. Examination of extra-renal sources of salt loss, for example gills, under conditions of hyperosmotic stress should prove interesting.
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Key Word Index--Acclimation; kidney function in crayfish; Pascifastacus peniusculus; osmoregulation in crayfish.