ABSTRACT
The influence of water-loading and low-temperature stress on the physiology of the antennal gland was studied in specimens of the freshwater crayfish, Orconectes virilis.
The results of experiments on water-loading suggested that the crayfishes were able to excrete water injected into them, but they did so at the expense of an abnormal loss of salts (sodium). Heavy water-loads caused large and prolonged increases in inulin clearances and urine flow.
Low temperature had its primary effect in reducing urine flow and inulin clearance. Low temperature apparently had little effect on the rate of sodium excretion or intensity of water excretion.
The urine flow (at 16 ° C.) of specimens of O. virilis was determined to be 3 % of the body weight per 24 hr. This value agreed with the urine flow calculated from average inulin clearances and inulin U/B ratios obtained independently upon specimens of the same species.
The results presented in the present paper throw further light upon the function of the crayfish antennal gland. They agree fairly closely with results obtained in other animals where filtration is known to play a role in primary urine formation. However, because of limitations imposed, principally by the lack of morphological evidence for a filtration site in the crayfish kidney, it cannot be stated unequivocally that the crayfish antennal gland is a filtration kidney.
INTRODUCTION
The histological structures, which enabled morphologists to deduce the function of the vertebrate glomerular kidney before much of the physiological evidence was available (Smith, 1951), appear not to exist in the crayfish antennal gland. Despite this shortcoming, there exists physiological evidence which indicates that some form of ‘filtration’‡ is involved in primary urine formation in that organ. Peters (1935) was able to show that the antennal gland of the crayfish (Astacus astacus L.) was responsible for the markedly hypotonic urine of that animal; the chloride content of urine samples removed from various portions of the kidney decreased from proximal (coelomosac, urine c. blood-isotonic) to distal (bladder, urine hypotonic). Peters’s work did not provide unequivocal evidence that ‘filtration’ was responsible for primary urine formation, but it did show that that process was a strong possibility. Maluf (1941) studied the clearance of inulin, xylose and various dyes by the kidney of the crayfish Procambarus clarki (Girard). He concluded that his data showed the crayfish kidney to be a secretory kidney, since inulin was excreted by that organ in a manner which appeared to be dependent upon a transport mechanism rather than passive filtration. Further, Maluf decided that the crayfish antennal gland showed no threshold response to xylose (a substance which is only a stereotype for glucose according to Martin, 1957). Riegel & Kirschner (1960) have re-examined the role of the antennal gland in the excretion of inulin and glucose by the crayfishes Orconectes virilis (Hagen), Procambarus clarki (Girard) and Pacifastacus leniusculus (Dana). They found that glucose behaved like a threshold susbstance and that phlorizin caused a marked glucosuria. Their work did not rule out the possibility that the crayfish antennal gland is a secretory kidney, but it provided evidence supporting ‘filtration’ as a more likely mechanism of primary urine formation.
In the present paper are presented the results of studies which were designed to characterize the function of the crayfish antennal gland under conditions of waterloading and low-temperature stress. The experiments were undertaken in order to provide further insight into the physiology of that organ.
MATERIALS AND METHODS
Specimens of Orconectes virilis were used in all experiments outlined below. The specimens were maintained as described by Riegel & Kirschner (1960).
Three principal types of experiment were undertaken: (1) urine and sodium excretion of animals into which no injections were made (control experiment); (2) urine and sodium excretion and inulin clearance of crayfishes into which varying amounts of distilled water were injected (water-loading experiments); (3) urine and sodium excretion and inulin clearance of animals under the influence of low temperature (refrigeration experiments).
The procedures for the experiments and the methods of collecting and measuring blood and urine samples, measuring ion concentrations and weight, and making injections were generally the same as those described by Riegel & Kirschner (1960). Differences in procedures and methods between the present and previous work will be noted below as each of the three types of experiment is discussed in detail.
Control experiment
In this series of experiments ten crayfishes were weighed and their urinary bladders (on one side only) were evacuated. At intervals of 4, 8 and 12 hr., all of the urine that had accumulated in the bladder was collected, measured, and samples were saved for sodium analysis.
Water-loading experiments
The bladder on one side was evacuated and injections of either 0·05 ml. (9 animals) or 0·25 ml. (8 animals) of distilled water containing 50 μg. of 14C-inulin were then made. At 4, 8 and 12 hr. intervals after injection all the urine which had accumulated in the bladder was collected. Samples were saved for inulin and sodium analyses. Weight and blood samples were taken before and after the experiments to ascertain that no changes in either blood ions (Na, K and Ca) or weight occurred during the experiments.
Refrigeration experiments
This group of experiments was carried out in two parts. In part one, the bladders of eight crayfishes were evacuated (on one side), the inulin was injected (0·1 ml. of 0·1 % 14C-inulin), and the crayfishes were placed in the water-bath at 16° C. After 4−6 hr. at that temperature the bladders were again evacuated, the total quantity of urine was measured, and samples were saved for sodium and inulin analyses. The crayfishes were then refrigerated gradually (c. 2 hr.) to 10° C.; after 6−8 hr. at that temperature, the sample-taking procedure was repeated. The temperature was then lowered gradually to 4° C., and after 8 hr. at that temperature the bladders were evacuated, the total urine output was measured and samples were saved for inulin and sodium analyses. Six of the eight crayfishes were rewarmed to 16° C., the reverse of the foregoing procedure except that warming between 4° and 10° C. and between 10° and 16° C. was not gradual.
In part two of the experiments ten crayfishes were weighed and initial blood samples taken as follows : the ventral membrane between the carpus and merus of the cheliped (claw) was punctured with a fine needle. As soon as sufficient blood had oozed from the wound, a sample was drawn into a 10 μl. pipette and transferred to 2 ml. of deionized distilled water in a 3 ml. test-tube. The micropipette was rinsed in the resulting solution. The crayfishes were then refrigerated gradually (over c. 4 hr.) to 4° C. Twenty-four hours later the weighing and blood-collecting procedure was repeated.
By various control procedures, duplicating the actual experiments as closely as possible, it was found that the following average limits of error could be assigned to the various determinations. Blood and urine inulin could be determined within an average error of ±3·6%; blood and urine sodium, calcium and potassium could be determined within average errors of ±0·7%, ±2·0% and ±3·5%, respectively. The weight of the crayfishes could be determined within an average error of ± 0·9 %. The amount of error involved in the assumption that all the urine produced during a given period of time was collected from the nephropore could not be measured directly. However, that factor was checked as follows : urine was collected from the nephropores of several animals until no more could be obtained. The crayfishes were then sacrificed and the urinary bladders exposed. In all cases, the bladders were collapsed, indicating that all urine but a possible small residual amount had been removed.
RESULTS
Control experiment
The results of the control experiments are shown in Fig. 1. As can be seen, the average urine flow remained relatively constant between the 8 and 12 hr. sampling periods. The excretion of sodium tended to decrease between the two sampling periods.
The urine flow of ten crayfishes averaged 3·0% (s.D. = ±0·75; range = 1·6−4·0) of the body weight per 24 hr.
Water-loading experiments
The results of the water-loading experiments are shown in Fig. 1. The smaller of the two injections of distilled water (0·05 ml.) caused a short-term increase in urine flow, after which the rate of flow fell off to subnormal levels. Concomitant with the fall in urine flow was a decrease in the rate of sodium excretion and in the intensity of water excretion.* The inulin clearance did not change significantly during the 12 hr. period. The large injection of distilled water (0·25 ml.) caused the urine flow to remain relatively constant throughout the experimental period. At the same time the rate of sodium excretion increased enormously and there was a rather large increase in the inulin clearance. The intensity of water excretion decreased during the 12 hr. period, but not to the extent seen in the animals which had been injected with a smaller amount of distilled water.
Refrigeration experiments
The results of the refrigeration experiments are shown in Fig. 2. The general effect of low temperature on the crayfishes was seen in decreased urine flow and inulin clearance. There appeared to be a significant difference in the intensity of water excretion between refrigerated and non-refrigerated animals at 10° C., but there was none at 4° C. The rate of sodium excretion during the refrigeration phase of the experiments did not vary significantly from that of non-refrigerated crayfishes. During warming, the rate of sodium excretion of refrigerated animals far surpassed that of animals which had not been refrigerated.
Refrigeration to 4° C. over a 24 hr. period effected no changes either in weight or in blood ions (Na, K and Ca).
DISCUSSION
Normal urine production of Orconectes virilis
As shown in the results, the urine flow per 24 hr. for specimens of Orconectes virilis averaged 3 % of the body weight. Whether or not that value represents the normal urine flow is not known. The procedure used for collecting urine put a great deal of stress on the crayfishes. This was shown by the somewhat erratic urine flows in all parts of the present experiments. The erratic results were most pronounced in the control experiments, but they also manifested themselves in the water-loading and refrigeration experiments. One of the stress phenomena exhibited by crayfishes is a cessation or reduction in urine flow [see Riegel (1960) and Riegel & Kirschner (1960)].
The urine flow of Procambarus clarki has been determined by Maluf (1941) and Lienemann (1938) as approximately 5 % of the body weight per 24 hr. These investigators determined the urine flow by plugging the nephropores and either noting the change in weight (Maluf, 1941) or opening the nephropores after a period and aspirating the urine from them (Lienemann, 1938). Numerous determinations of the inulin clearance of specimens of P. clarki (unpublished) have been carried out by the writer.
The average inulin clearance amounted to 14·4% of the body weight per 24 hr. The usual inulin U/B ratio in specimens of that species varied between 2·5 and 3·0 (at 16 ° C.). Therefore, it appears that the urine flow calculated from inulin clearance and inulin U/B ratios agrees with the urine flow determined independently by Maluf and Lienemann. The average inulin clearance found in specimens of Or conectes virilis was 6·4 % of the body weight per 24 hr. Since the usual inulin U/B ratio of such specimens varied between 2·0 and 2·5, the independently obtained value of 3% of the body weight for urine flow per 24 hr. agrees with the urine flow calculated from average values of inulin clearance and inulin U/B ratios.
Water-loading
From Fig. 1 it may be seen that the injection of distilled water into crayfishes caused a pronounced diuresis, accompanied by an increased rate of sodium excretion. When a small water-load (0·05 ml.) was injected, the effect was short-termed. After the first 4 hr. of the experiment the inulin clearance remained relatively constant while the urine flow, the rate of sodium excretion and the intensity of water excretion decreased markedly. The above observations suggest that some physiological limit may have been exceeded during the first 4 hr. of the experiment. That is, it is possible that the crayfishes overcompensated for the water injected, perhaps by excreting too large a quantity of salt (sodium), in an effort to rid the body of the water. That is certainly suggested by the fact that the levels of sodium excretion were in excess of those in the control group. After 4 hr. the urine flow, the rate of sodium excretion and the intensity of water excretion decreased in the lightly water-loaded crayfishes. Since the inulin clearance remained steady, it is probable that the decreases in the aforementioned parameters were the result of compensatory adjustments under which possible deficits in salts (and water), incurred during the diuresis, were made up. In crayfishes into which the large quantity (0·25 ml.) of distilled water was injected, the results were similar in kind to those obtained with the lightly water-loaded crayfishes, but they differed in degree. The results suggested that the heavily water-loaded crayfishes were unable to excrete the water in the 12 hr. experimental period, despite the marked increase in the inulin clearance, and that in consequence it was necessary for them to excrete quantities of sodium much larger than normal. The overcompensation in urine flow, rate of sodium excretion and intensity of water excretion was not seen in the heavily water-loaded crayfishes, although those parameters did decrease after the first 4 hr. of the experiments.
In summary, crayfishes are able to excrete relatively large quantities of distilled water injected into them. However, in the process, it seems likely that they must suffer the loss of abnormal quantities of salt (sodium), which must be compensated for, after the body is rid of the excess water, by greatly increased reabsorption of salts (sodium) from the urine.
Burger (1957), in his study of excretion in the lobster, Homarus americanas, encountered animals which normally did not produce urine. He found that those anuric lobsters could be induced to produce erratic, transitory urine flows by injecting hyper- or hypotonic salt solutions into them. That fact seems puzzling when one considers that urine formation in the lobster could not be correlated with a rise in the arterial pressure (Burger, 1957). In view of this observation on the lobster caution must be exercised in making any statements about the causes of the increased urine flow in the crayfishes studied here. It is possible that the extra water, irrespective of the accompanying salt, is able to elicit the diuretic response.
Low temperature
The primary effects of low temperature on the excretory processes of the crayfishes appeared to be the results of a general interference with the metabolism of the experimental animals. Thus the inulin clearance decreased and there was a concomitant decrease in urine flow. The rate of sodium excretion appeared to vary little from that of non-refrigerated crayfishes. During rewarming, however, the rate of sodium excretion of the refrigerated crayfishes increased markedly. From the latter observation it seems possible that there was an accumulation of sodium during refrigeration.
Wikgren (1953) studied the effect of low temperature on the ion balance of the crayfish Potamobius ftuviatilis (which probably corresponds to the present-day species Astacus astacus’). Low temperature had the effect of causing a pronounced loss of ions from the crayfishes. Wikgren deduced that the loss was due to the inability of the ion-absorbing surfaces to keep pace with the diffusion of ions out of the crayfishes, rather than a many-fold increase in the concentration of ions in the urine. The results of the present experiments confirm Wikgren’s deduction and show, further, that if an ion loss occurred at low temperature, it was not accompanied by a decrease in the ion concentrations of the blood or by change in weight (i.e. water-loss or gain).
As far as can be ascertained, no research into the effect of low temperature on known secretory kidneys has been undertaken. However, a number of investigations have been made of the effect of low temperature on animals in which it is well established that filtration plays a role in urine formation. Potts (1954) studied the influence of low temperature on the clearance of inulin by the lamellibranch mollusc, Anodonta cygnea L. Low temperature markedly reduced the inulin clearance. In the lamprey, Petromyzon fluviatilis L., low temperature had effects remarkably similar to those seen in the crayfish. According to Wikgren (1953), the urine flow decreased considerably, there was an initial loss of salts from the surface, which reached a steadystate soon after, and the ion (chloride) concentration of the mine did not differ significantly from its value at room temperature. Jorgensen (1950) studied the effect of low temperature on osmoregulation in the frog, Rana escalenta L. Low temperature caused a reduction in urine flow and an accumulation of salt (followed by an increase in weight). Jorgensen concluded that the effects were due to the inability of the frog kidney to eliminate salt at low temperatures. If hypotonic (to the blood) saline was injected into the frogs, a diuresis and weight loss would result. However, injection of hypertonic saline caused a complete stoppage of urine flow and a weight increase.
General
The results of the experiments on water-loading and low-temperature effects on the crayfish antennal gland provide data which further characterize the physiology of that organ. Unfortunately, the data do not provide primary evidence as to the mechanism by which that organ forms the primary urine. The results of the water loading experiments were much as would be expected on the assumption that urine is formed by ‘filtration’. If the crayfish antennal gland were a secretory kidney, the injection of large amounts of water would not be expected to have such a pronounced effect on the urine flow and inulin clearance. As seen in the lightly water-loaded animals, the inulin clearance remained constant even when the urine flow dropped below normal. On the assumption that urine is formed by secretion, that observation might be rationalized to indicate that a maximal transport rate for inulin had been reached. However, the inulin clearance seen in the lightly water-loaded crayfishes apparently was not maximal, since that rate increased considerably in the heavily water-loaded crayfishes. Therefore, any attempt to explain the data obtained from the water-loading experiments on the basis of secretion as the mechanism of primary urine formation in crayfishes would seem highly dubious.
The response of the crayfish kidney to low temperature shows many similarities to responses shown by the kidney of a lamellibranch mollusc (Potts, 1954) and the mesonephric (glomerular) kidneys of vertebrates (Jorgensen, 1950; Wikgren, 1953). Nevertheless, however well the present results may agree with those seen in kidneys known to produce urine by filtration they fail to provide decisive evidence as to the method of primary urine formation in the antennal gland of the crayfish.
ACKNOWLEDGMENT
The writer wishes to express his gratitude to Dr Leonard B. Kirschner, of Washington State University, for permitting free use of his facilities during the period of this study. Thanks are also given to Dr A. P. M. Lockwood, of Cambridge University, and Dr P. A. Dehnel, of the University of British Columbia, for reading portions of the manuscript.
REFERENCES
The use of the term ‘filtration’ in this paper follows the restrictions outlined by Riegel & Kirschner (1960). This use of the term implies that urine is formed by the passive movement of fluid from which particles larger than an unknown critical size are restrained, but the cause of the fluid movement is not stated. Where the term is used without quotations, the usual sense of the word is meant—that is, movement of fluid through a porous membrane under the impetus of hydrostatic pressure.
No precedent for the use of the reciprocal of the inulin urine/blood ratio was found in the literature. Since the inulin U/B ratio is commonly used as an indicator of the intensity of water reabsorption, it was felt that the reciprocal of that value could be used to indicate the degree to which water is not reabsorbed, i.e. the intensity of water excretion.