ABSTRACT
Chloride concentration and freezing-point depression (=osmotic pressure) have been measured on samples of urine obtained from various parts of the crayfish antennal gland by micropuncture.
The chloride concentration of the urine is significantly below that of the blood in all parts of the antennal gland, but a marked drop is not seen until the distal tubule is reached.
The urine becomes progressively hypo-osmotic to the blood in the labyrinth, distal tubule and bladder. In the coelomosac, it is iso-osmotic; possible reasons for this are discussed.
Chloride concentration and osmotic pressure are lower in urine taken from the nephropore than in urine taken from the distal tubule near the place where it joins the bladder. This suggests that the bladder is in part responsible for the production of hypotonic urine.
The results of the present study are discussed in relation to the theory that filtration (in the limited sense discussed by Riegel & Kirschner, 1960) is the mechanism by which primary urine is formed in the crayfish antennal gland.
INTRODUCTION
The gross microscopical anatomy of the crayfish antennal gland has been well and repeatedly characterized (Marchal, 1892; Peters, 1925; Maluf, 1939). Examination of the anatomy of the organ has yielded little information elucidating its basic functional mechanism.
Since the work of Schlieper & Herrmann (1930) it has been known that the crayfish excretes a urine that is markedly hypotonic to the blood. However, the mechanism by which the animal does this is still obscure. Two conflicting theories have been advanced to explain the functional mechanism of the crayfish antennal gland.
Peters (1935) concluded that the primary urine is formed by filtration of blood into the most proximal portion (coelomosac) of the antennal gland, the filtrate being modified more distally in that organ. (The evidence for Peters’s conclusion will be reviewed in more detail in the Discussion.)
Maluf (1939,1941 a-c) made rather extensive studies of the anatomy and physiology of the crayfish antennal gland. From these studies he concluded that the antennal gland is a secretory kidney, secreting all normal urine components plus the renal test substance inulin.
Riegel & Kirschner (1960) re-examined the excretion of inulin and studied the excretion of glucose by the crayfish. They also critically examined Maluf’s evidence for secretion of inulin (and xylose). From their work, Riegel & Kirschner concluded that there is little evidence that the crayfish antennal gland forms primary urine by secretion. At the same time, they pointed out that there was little evidence to support the view that filtration under hydrostatic (arterial) pressure was responsible for primary urine formation, although it seemed likely that ‘filtration’ ‡ of some kind was involved.
Riegel (1961) has shown that hydration and low temperature have effects on excretion in crayfishes similar to their effects on excretion in animals that have filtration-reabsorption kidneys.
In the present work, freezing-point depression (= osmotic pressure) and chloride concentration have been measured on samples of urine taken by micropuncture from various parts of the crayfish antennal gland. This work was done in order to throw further light upon the mechanism of urine formation in the organ.
MATERIALS AND METHODS
Specimens of Austropotamobius pallipes pallipes (Lereboullet) were studied at Cambridge where the animals were kept in aquaria at room temperature (18–20° C.). Specimens of Orconectes virilis (Hagen) were studied at Queen’s University. They were maintained in temperature-controlled aquaria at 16° C.
Blood was collected from the sternal sinus with the aid of a c.c. syringe fitted with a 26- or 28-gauge needle. It was deposited under liquid paraffin in a siliconized watch-glass.
Urine samples were collected as follows : a crayfish was sacrificed and an antennal gland quickly removed, blotted on filter-paper and placed under liquid paraffin in a Petri dish. Under a stereoscopic microscope the various parts of the antennal gland were punctured with silica micropipettes which were moved into place with the aid of a micromanipulator. To aid visualization of their location in the antennal gland, the micropipettes were partially filled with liquid paraffin coloured with Sudan Blue. The micropipettes had orifices of 50–100 μ, and they were operated by mouth. This limited the lower size of their orifices, for if the orifices were less than about 50 μ, the contents could not be blown out. The urine samples were placed under liquid paraffin as described above for blood.
Samples were collected from all parts of the antennal gland except the proximal portion of the tubule. The small size and inaccessibility of that part made it impossible reliably to obtain samples from it.
Chloride was estimated by the second method of Ramsay, Brown & Croghan (1955), Freezing-point depressions (Δ) were estimated by the method of Ramsay & Brown (1955). Osmotic pressure was calculated as mm/1. NaCl from Δ using the relation: 0·605° C. ≅171 mm./l. NaCl.
Chloride concentrations and · were determined on three aliquots from each sample of blood and urine. The average of the three determinations was then taken.
RESULTS
Measurements were made on a total of thirteen crayfishes: five specimens of Austropotamobius (animals 1-5) and eight specimens of Orconectes (animals 6-13). No species differences were seen with regard to matters here investigated. Complete sets of determinations (i.e. both A and Cl of blood and of urine samples for all five parts of the antennal gland) were obtained on six crayfishes only (numbers 2, 5, 7, 9, 10, 12). All the data obtained are set out in Table 1 (chloride concentration) and Table 2 (osmotic pressure).
When the chloride concentrations of the urine from the various parts of the antennal gland are compared with the chloride concentration of the blood of the same animal it is found that in all parts of the gland the urine is significantly (P = < 0·02) hypotonic to the blood. The chloride concentration of the urine is seen to decline most sharply between the proximal and distal parts of the distal tubule and between the latter and the bladder.
The data for osmotic pressure have been treated in the same way and show that in all parts of the gland except the coelomosac the urine is significantly (P < 0·01) hypo-osmotic to the blood. In the coelomosac the urine can be either hypo-osmotic or hyperosmotic to the blood. The osmotic pressure of the urine declines most sharply between the distal portion of the distal tubule and the bladder.
DISCUSSION
The results of the chloride analyses differ from those of Peters (1935) only in that Peters found the urine in the coelomosac to be isotonic and the urine of the labyrinth to be hypertonic to the blood. Submission of Peter’s data to statistical analysis shows that the measured hypertonicity of urine chloride in the labyrinth is highly significant (P = < 0·01). No explanation is readily apparent for the discrepancy between the present work and that of Peters.
It is difficult to interpret the significance of the iso-osmoticity of the urine in the coelomosac, especially in view of the low chloride concentration there. Assuming that ‘filtration’ is responsible for primary urine formation, the high osmotic pressure (and low chloride concentration) might indicate the presence of large amounts of nonelectrolyte, possibly as a result of active secretion of non-electrolyte by the coelomosac cells. Alternatively, electrolytes may be reabsorbed there. Although the coelomosac does not exhibit the marked ‘secretory’ appearance of the labyrinth and distal tubule (Maluf, 1939, 1941 c;Riegel & Kirschner, 1960), it does show some manifestations of cellular activity. The cells of the coelomosac contain peculiar brownish inclusions which are surrounded by clear, vacuole-like spaces. This was also observed by Maluf (1939). Further, Maluf (1941b) found that the coelomosac accumulates a crystalline compound which he thought was CaCO3.
Kamemoto, Keister & Spalding (1962) have obtained experimental evidence which suggests that the urinary bladder takes part in elaboration of the hypotonic urine of the crayfish. They find that cholinesterase, a substance which is associated with sodium transport in many tissues (see their paper for a full discussion), is highly concentrated in the bladder epithelium. Further, 22Na injected into the bladder appears in the blood, and the appearance of 22Na in the blood can be partially blocked by eserine, a cholinesterase inhibitor.
It is to be noted, however,that Kamemoto et al. introduced 22Na into the urinary bladder by means of a small plastic catheter which was pushed through the nephropore opening into the bladder. As adequately demonstrated (and emphasized) by Maluf (1941b), the passage between the bladder and nephropore is S-shaped. Any object pushed through the nephropore might pass through the haemocoel before entering the bladder. The possibility does not seem to have been excluded that bladder urine might be contaminated by blood and vice versa.
In the present work the lower values of both chloride concentration and osmotic pressure in the bladder was compared with their values in the distal tubule appear to support the suggestion of Kamemoto et al. Even the lowest values for chloride concentration and osmotic pressure measured in the distal portion of the distal tubule (near the distal tubule-bladder junction) are very much higher than the corresponding values for the bladder.
The histological character of the bladder epithelium is not suggestive of secretory activity. This was also noted by Maluf (1939). Contrary to the reports of Maluf, however, the bladder appears to be well-supplied with blood vessels (at least in specimens of the two species with which the present work is concerned). Whether or not blood supply bears any relationship to the activity of the bladder remains to be established.
The results of the present study, considered in relation to the earlier studies of Riegel & Kirschner (1960) and of Riegel (1961) are compatible with the view that ‘filtration’ is the mechanism responsible for primary urine formation by the crayfish antennal gland.
It still cannot be said that ‘filtration’ in the crayfish antennal gland is brought about by arterial pressure. There is no direct evidence that arterial pressures in the region of the antennal gland are sufficiently high to effect filtration under hydrostatic pressure. Further, there is as yet no published evidence of a well-defined filtration site in the crayfish antennal gland.
Dr A. P. M. Lockwood (personal communication) has drawn attention to the fact that Picken’s (1939) measurements of haemocoelar pressures and colloid osmotic pressures of blood and urine in the crayfish do not, by themselves, provide conclusive evidence for filtration under hydrostatic pressure. The antennal gland is a non-rigid structure lying in the general haemocoel. Therefore it would be expected that the hydrostatic pressure of the haemocoel would be communicated to the fluid contents of the antennal gland and no difference of hydrostatic pressure would be available to effect filtration. In this situation, since the blood colloid osmotic pressure exceeds the urine colloid osmotic pressure, water would tend to be drawn from the urine to the blood. It is readily observed, however, that the blood actively circulates in the arteries and capillaries of the crayfish antennal gland. The arterial pressure must therefore exceed the haemocoelar pressure, and this implies that arterial pressure may possibly be sufficient to effect filtration in the crayfish antennal gland.
References
See Riegel & Kirschner (1960) for a full discussion of the use of this term.