The Physiology of the Antennal Gland of Carcinus Maenas (L.) III. Glucose Reabsorption

Numerous analyses have established that both in marine and in freshwater Crustacea the antennal glands are important in determining the ionic composition of body fluids (Robertson, 1957; Parry, 1960; Lockwood, 1962). Other aspects of antennal gland function usually associated with the physiology of excretory organs (reabsorption of metabolites, elimination of nitrogenous compounds, secretion of some materials) have received much less attention.

Study of carbohydrate reabsorption in Crustacea has been confined to investigations on one marine lobster and several species of freshwater crayfish. Forster & Zia-Walroth (1941) were unable to show reabsorption of reducing sugars from the urine of Homarus and concluded that in this animal cellular reabsorption was not a factor in urine formation. Other experiments have shown that sugars are reabsorbed by the antennal gland of Homarus and that there is an apparent blood threshold concentration above which glycosuria occurs (Burger, 1957). Reabsorption of glucose from the urine and a similar threshold have also been shown in freshwater crayfish (Riegel & Kirschner, 1960). Information presented by Maluf (1941) purports to show that xylose is secreted by the antennal gland of Cambarus clarkii, though criticism has been made of the experimental techniques used (Martin, 1957) and the interpretation of the results of this work (Riegel & Kirschner, 1960). Evidence concerning the reabsorption of metabolites by the antennal glands of Crustacea is therefore limited and inconclusive.

During investigations of the mechanism of primary urine formation (Binns, 1969 a), urine/blood (U/B) ratios of 1 were found for both inulin and sorbitol. Passive movement of a protein-free filtrate of the blood into the antennal gland, indicated by these U/B ratios, would result in the loss of essential metabolites if these were not subsequently reabsorbed from the urine. A study of metabolite reabsorption by the antennal gland of Carcinus has not previously been made. This paper describes some features of the mechanism of glucose reabsorption in this animal.

Details of general experimental procedure and methods of sampling of body fluids have been described elsewhere (Binns, 1969a).

Total reducing substances (T.R.S.) in blood and urine were determined by the method of Park & Johnson (1949). Proteins in blood were precipitated using 10% trichloracetic acid. To ensure that reducing substances detected in body fluids were not due to contamination, micropipettes were rinsed several times before being used and all other glassware was washed thoroughly in de-ionized water. Also, portions of urine samples were treated with the protein precipitant to make certain that there had been no contamination of urine with blood during sampling.

Concentrations of glucose in blood and urine were estimated using ‘Glucostat’ enzymic reagent, after the method of Saifer & Gerstenfeld (1958).

Both of the above are colorimetric methods, and concentrations of T.R.S. and glucose were determined using an H 810 Biochem Absorptiometer with the appropriate filters. The limits of error of the analyses for T.R.S. were ±4% and for glucose ±2%.

For the estimation of ‘normal’ concentrations of T.R.S. and glucose, blood and urine samples were taken as soon as possible after animals were collected. The delay between capture and sampling was never more than 3 hr. Before experiments involving the injection of glucose into crabs the animals were starved for several days to bring blood glucose to a fasting level.

To ascertain whether glucose is, in fact, reabsorbed from the urine, ‘normal’ blood and urine concentrations of T.R.S. and glucose were estimated in freshly captured crabs (Table 1). These results indicate that glucose is conserved by the antennal gland, since it has been shown previously that there is no movement of water into the urine as it passes through the antennal gland (Binns, 1969a). However, reabsorption was often incomplete; eleven of the sixteen urine samples analysed contained detectable amounts of glucose.

Concentrations of T.R.S. in urine were high relative to blood levels. Several U/B ratios of around 1 were recorded, and in two cases ratios of 2·54 and 2·46 were found. Qualitative tests showed that carbohydrates and glucose may be absent from urine samples even when relatively high levels of T.R.S. were recorded. The nature of the reducing substances other than glucose is not known.

Glucose reabsorption having been established, the phenomenon was investigated experimentally.

Taking the effective blood volume to be 19% of the body weight (Binns, 1969b), a calculated amount of a 20 g. % aqueous solution of glucose was injected into each crab to give a blood concentration of 300 mg. %. At intervals after the injection, blood and urine samples were taken from successive groups of three crabs. Glycosuria produced by frequent handling has been observed in crayfish (Riegel, 1960) and it is possible that Carcinus may be similarly affected. For this reason, only one sample of blood and one of urine were taken from each crab. A composite picture of the changes in glucose concentration is shown in Fig. 1a. Blood glucose was rapidly reduced to a mean value of 24·7 mg. % in 7 hr. Glucose was not detected in the urine until 2·5 hr. after the injection and as the experiment continued the urine again became glucose free.

A similar experiment was carried out with animals in 50% sea water, to determine the effect of a high rate of urine production on the changes in the concentration of glucose in blood and urine (Fig. 1b). Under these conditions the fall in blood glucose concentration, though marked, was not as rapid as when animals were in 100% sea water. All urine samples collected contained glucose, relatively large amounts being present in samples taken soon after injection. Glucose in bladder urine was presumably lost as urine was voided into the external medium.

To check that glucose concentrations in blood and urine are not normally raised when Carcinus is in dilute media, unfed animals were placed directly into 50% sea water and body fluid samples were taken over a period of several days. Two experiments were carried out, the reactions of the animals being distinctly different in the two groups. In the first group, animals were placed individually into small tanks containing 500 ml. of non-aerated medium. Blood glucose concentration increased in these crabs, all of which died some time after the last blood samples were taken (Fig. 2a). The second group of crabs was kept individually in containers holding 1 l. of continuously aerated medium. Blood glucose concentrations in these animals did not increase. In fact, there was a tendency for blood glucose to fall, presumably due to a metabolism of glucose by the animals during the experimental period. All these animals survived and were apparently in good condition (Fig. 2b).

Urine was collected from both these groups of crabs in 50% sea water, and glucose was detected in only one of forty-two samples taken. Glucose was never detected in the urine of any of the crabs which eventually died. This was surprising since the animals were obviously distressed by the non-aerated medium and under such conditions one might expect to detect some failure of metabolic functions. However, urine was glucose-free even at the highest blood concentration recorded of 67·5 mg. %, which was more than thirteen times the initial blood concentration in that particular animal.

Glucose appears in the urine only briefly when the blood concentration is raised to 300 mg. % by injection, and the urine may be glucose-free in starved animals. This indicates that there is a marked reabsorption of glucose by the antennal gland and that there may be a threshold value, below which glucose is largely reabsorbed. At the higher rate of urine production in 50% sea water the postulated threshold value seems to be lowered. Nevertheless, reabsorption is still complete in this medium at a blood concentration of 67·5 mg. glucose %, therefore any threshold must at least exceed this figure.

For an investigation of glucose thresholds it was considered impracticable to maintain any required blood glucose concentration by continuous perfusion. It has been shown above that in animals in 100% sea water glucose appears in the urine 2·5 hr. after blood glucose is suddenly increased. In the following experiments glucose was injected into crabs living in 100 and 50% sea water to give a range of initial blood concentrations of 50−400 mg. % and 25−400 mg. % respectively. If the initial blood concentration exceeded a threshold concentration, some glucose would pass through the antennal gland into the bladder. If not, the urine collected would be glucose free. The results of analyses of urine samples from crabs in both media collected 2·5 hr after injection are shown in Fig. 3.

Consider first the animals in 100% sea water. Glucose was absent from urine samples collected from eight animals which had an initial blood glucose concentration calculated to be 50 mg. %. Only one of ten urine samples from crabs with 100 mg. % in the blood initially contained glucose. At a blood concentration of 150 mg. % reabsorption became incomplete and above this level urine glucose concentrations increased as the initial blood concentration was raised.

In 50% sea water, urine was glucose-free when the initial blood glucose level was 25 mg. %. One of five urine samples collected from animals with 50 mg. % in the blood had a measurable amount of glucose. Above this level urine glucose tended to increase as the initial blood glucose concentration was raised.

In normal sea water at a blood glucose concentration of 150 mg. % the threshold is exceeded, reabsorption is not complete and glycosuria occurs. In 50% sea water the threshold is lowered and lies between 50 and 100 mg. glucose %, i.e. approximately half the threshold value in normal sea water. Controls showed that the reduction in threshold is not reversed when Carcinus becomes fully adapted to the dilute medium.

As in the vertebrate nephron, the mechanism for the reabsorption of glucose by the antennal gland shows a characteristic blood threshold level. The analogy was tested further by determining the effect of phloridzin on glucose reabsorption. Animals were injected with concentrated glucose solution so that the initial blood concentration in all of them was 300 mg. %. A concentratrated solution of phloridzin was then injected and the animals were replaced in 100% sea water. Changes in glucose concentrations in blood and urine compared with those observed in animals not treated with phlorid-zin, but having the same initial blood glucose concentration, are shown in Fig. 4.

All urine samples from crabs treated with phloridzin contained glucose, and concentrations tended to increase as the experiment was continued. After 6·5 hr. the mean urine glucose concentration was 47·4 mg. % and the blood concentration 64·0 mg. %. U/B ratios of 1 were found in two of the five animals sampled in the latter part of the experiment and in one of these the urine glucose rose to 113·3 mg. %. This contrasts markedly with the slight transient increase in urine glucose observed in the animals not treated with phloridzin.

The mean blood concentration of T.R.S. found in Carcinus is within the range of values for ‘blood sugars’ determined by other workers using similar methods (see Florkin (1960) for details). The use of such non-specific methods for determining blood sugars has been criticized (Wyatt, 1961) on the grounds that they tend to give spuriously high values, due to the presence of other reducing substances in body fluids. Comparison of T.R.S. and glucose concentration in both blood and urine of Carcinus substantiates this objection. The normal blood concentration of T.R.S. was almost three times the glucose concentration, and urine samples contained relatively large concentrations of T.R.S. though it was shown both qualitatively and quantitatively that glucose is present in much smaller amounts. Similarly, in the blood of Callinectes sapidus glucose represents only 20−25% of the concentration of T.R.S. (Dean & Vernberg, 1965). Values for ‘reducing sugars’ in body fluids estimated by such methods as the reduction of ferricyanide should therefore be regarded with caution.

The estimations of T.R.S. are useful because they indicate that the urine of Carcinus contains large amounts of reducing substances, even though these are at present unidentified. Ramsay (1961) has pointed out that although the antennal glands of Crustacea may not be important in terms of total nitrogen excretion, until some of the unknown compounds in invertebrate urine are identified the role of the ‘émonctoire’ in eliminating, say, complex excretory products will remain in doubt. In Maia, 51% of the urine nitrogen is unidentified (Delaunay, 1931) and it is possible that some of the unknown reducing substances detected in the urine of Carcinus may be nitrogenous.

Aeration of experimental media appears to be necessary if animals are to be considered in good condition, particularly in dilute sea water. Animals which died in non-aerated 50% sea water showed a considerable increase in blood glucose concentration, and it seems likely that this was a stress reaction caused by the unfavourable conditions of the experiment. Increases in blood sugar concentration caused by keeping crustaceans in sea water with low oxygen content can be reversed and the blood sugar brought back to normal when an adequate supply of air is made available (Stott, 1932). Hyperglycaemia occurs in Libinia emarginata, a marine spider crab, during asphyxia caused by exposure to air for long periods. The reaction is dependent on the eyestalks being intact and probably mediated by the sinus gland (Kleinholz & Little, 1949). It is possible that a similar hormonal response in Car anus caused increases in blood glucose in those animals which died in 50% sea water.

The observed blood glucose threshold of approximately 150 mg. % for Car anus in normal sea water is close to those found in Homarus (Burger, 1957) and in freshwater crayfish (Riegel & Kirschner, 1960). It is apparent that Forster & Zia-Walroth (1941) were unable to demonstrate reabsorption in the lobster because in their experiments glucose concentrations well above the threshold value were used.

The concept of the threshold value as a fixed parameter of glucose reabsorption, below which there is complete conservation of the molecule and above which glycosuria occurs, is an over-simplification. For instance, glucose is often present in the urine of freshly captured crabs with low blood glucose concentrations. Glucose could have passed into the bladder at a time when the blood concentration was above the threshold level, as might be the case soon after a meal, though at present there is no information to show that this happens. Alternatively, glucose reabsorption may not always be complete, even at low blood glucose concentrations. In support of this, glucose was detected in the urine in almost 75% of the freshly captured crabs tested. However, if animals were starved for several days and then sampled, glucose was never present in the urine, even though blood glucose fell only slightly below the level found in animals direct from the field. This suggests that if adequate sources of carbohydrate are available, glucose passing through the antennal gland is not completely reabsorbed. Should carbohydrates become limited or unavailable, there is then complete reabsorption. Although some glucose is always reabsorbed, the degree of reabsorption is probably related to the demand for, and the availability of, the metabolite at a particular time.

Further adaptability of the process of glucose reabsorption is shown by the demonstration that the threshold level in Carcinus is reduced when the rate of urine flow increases. Because of this lower threshold, in dilute media there is a greater tendency for glucose to be lost in the urine. In this respect consideration of the threshold value alone gives a misleading impression of the reabsorptive capacity of the antennal gland under these conditions. Considering previously starved animals, just below the threshold level the glucose load presented to the antennal gland (blood glucose concentration × rate of urine flow) is completely reabsorbed. Knowing the rates of urine production, glucose reabsorption rates at the respective threshold concentrations for animals in 100 and 50% sea water can be estimated roughly (Table 2).

In 50% sea water the rate of glucose reabsorption at the threshold is almost doubled, even though in this medium the glucose threshold is lower and the rate of urine flow is four times that in 100% sea water. Therefore, the normal threshold may define roughly the upper limit for complete reabsorption in 100% sea water, but it does not indicate the maximum rate of glucose reabsorption. In view of this, it seems unlikely that the uptake mechanism will be saturated at the increased rate of reabsorption shown in 50% sea water. An estimate of the maximum rate of glucose reabsorption by Carcinus was not made nor are figures available for other Crustacea. A maximum reabsorptive rate of 3−4·2 mg. glucose/hr./100 g. animal has been recorded for Achatina fulica (Martin, Stewart & Harrison, 1965). This is six to eight times the rate found for Carcinus in 50% sea water at a blood concentration of 75 mg. glucose %. In the field the antennal gland may be only seldom subjected to the heavy metabolite loads used experimentally, and its reabsorptive capacity is probably sufficient to ensure that glucose is adequately conserved.

Inhibition of glucose reabsorption after treatment with phloridzin corresponds with the situation found in Homarus (Burger, 1957), freshwater crayfish (Riegel & Kirschner, 1960), Achatina (Martin et al. 1965), Octopus (Harrison & Martin, 1965) and in vertebrates (Smith, 1951). This indicates that, even in such diverse forms, a common metabolic pathway for the active reabsorption of glucose is present. Furthermore, the tendency for the glucose U/B ratio in Carcinus to approach unity after the administration of phloridzin lends further support to the suggestion that the primary urine is produced by a filtration of the blood into the antennal gland (Binns, 1969a).

Amongst the Crustacea glucose reabsorption has now been studied in three physiologically distinct types: a stenohaline lobster (Homarus), a euryhaline crab (Carcinus) and some freshwater crayfish. In all of them there is an apparent glucose threshold level at 150−200 mg. glucose/100 ml. blood and inhibition of reabsorption by phloridzin, indicating that in each animal essentially the same type of mechanism is responsible for glucose reabsorption. To date there is no experimental evidence to indicate in which part of the antennal gland reabsorption occurs. Morphologically, the coelom sac and labyrinth regions are common to the antennal glands of the three animals, the crayfish having an extra segment between the labyrinth and the bladder. The coelom sac is thought to be the region where filtration of the blood takes place to produce the primary urine. If this is its only function, cells in the labyrinth may perhaps be of importance in actively transporting glucose from urine passing through the antennal gland.

This work constitutes part of a Ph.D. thesis submitted to the University of Newcastle upon Tyne. It is a pleasure to thank Professor J. Shaw for his encouragement and guidance during the course of the work. I am grateful to S.R.C. for providing a postgraduate studentship.