ABSTRACT
The space measured by inulin distribution, the ‘inulin volume’, has been determined, and represents approximately 20% of the body weight in crabs ranging in size from 20·0 to 57·2 g.
After the injection of labelled inulin into crabs, the increase in activity of the medium is equal to the fall in blood inulin in all dilutions of sea water. Clearance of inulin from the blood is due only to urine production, and therefore the molecule can be used for quantitative investigations of antennal gland function.
Urine production in various concentrations of sea water has been determined by measuring the clearance of inulin from the blood and the rates at which the tracer appeared in the external media. By these methods the mean rate of urine production in 100% sea water was estimated to be 4·4% body weight per day. In dilute sea water the rate of urine production increases ; for example, in 50 % sea water the urine flow is four times greater than in normal sea water.
INTRODUCTION
Estimates of urine flow rates are necessary in order to assess the role of the antennal gland in regulating the composition of the internal medium of the animal. Urine production rates in Crustacea have been estimated by measuring (i) increases in weight of animals with antennal gland openings blocked, (ii) rates of elimination of injected dyes. Details of flow rates measured by these methods are given in a review by Parry (1960). More recently, urine flow has been determined by calculating the rate of production necessary to maintain the blood sulphate ion concentration lower than that of normal sea water (Shaw, 1961).
Labelled inulin is detectable in the surrounding medium a short time after being injected into crabs. If inulin is inert when injected into Carcinus, measurement of the rate of loss of the molecule from an animal affords a means of estimating urine production. In this paper the suitability of the inulin molecule for quantitative investigations of antennal gland function in Carcinus is examined and urine production is estimated in various concentrations of sea water. The work also gives some information about the blood volume of Carcinus.
METHODS AND MATERIALS
Details of general procedure, methods of sampling and preparation of body-fluid samples for counting have been given previously (Binns, 1969).
Within 2 hr. after the injection of labelled inulin into Carcinus it can be detected in the external medium. The loss of the tracer from crabs in various concentrations of sea water has been investigated.
Crabs were injected with a known amount of inulin and an initial blood sample was taken after hr. The animals were then placed individually into 250 ml. of the selected medium, samples of which were taken over a period of about 40 hr. At the end of this time a second blood sample was taken and counted. Ideally, samples of media would be de-salted before counting, since crystals remaining on planchettes after drying even dilute sea water will reduce the activity recorded. De-salting was considered impracticable, but it was found that the relationship between activity of a standard sample and apparent activity of that sample when treated with 1 ml. of 25 % sea water was fairly constant. This being the case, 1 ml. samples of all external media were diluted to 25 % sea water and 1 ml. samples of this diluted sea water were dried and counted. Making corrections for the effect of salt on planchettes and the dilution of the original samples, the rates of loss of inulin from Carcinus in different dilutions of sea water could be compared.
For comparison with increases in medium activity, changes in total blood activity over the same period were calculated knowing the initial and final blood counting rates and the inulin volume (see below).
RESULTS
1. Blood volume in Carcinus
Taking the blood volume of Carcinus to be 36% of the body weight (Webb, 1940), preliminary comparisons indicated that the amount of inulin lost from the blood of a crab exceeded the increase in activity of the surrounding medium. This meant that either inulin was being metabolized by the animal or that the space occupied by the molecule was less than had been assumed.
From the dilution of injected tracer, the distribution space of inulin was determined in twenty-eight crabs ranging in size from 20·0 to 57·2 g. This space will be referred to as the inulin volume (Fig. 1, Table 1).
The mean inulin volume of approximately 20 % body weight was considerably less than Webb’s estimate of blood volume in Carcinus. Inulin volumes for individual crabs were used in all determinations of urine production involving the clearance of inulin from the animal.
2. Recovery of injected inulin
The correspondence between activity changes in blood (inulin volume) and medium was good, the loss of activity from the inulin volume being balanced by the increase in activity of the medium. In most cases the discrepancy between the two figures was small (Table 2).
Carcinus is probably impermeable to inulin and therefore loss via the urine must account for the clearance of the molecule from the blood. Over relatively short periods inulin is apparently inert when injected into Carcinus. With this proviso, it is a suitable molecule for use in the study of antennal gland function.
3. Urine production rates
(a) Clearance of blood inulin
Since it has been shown that inulin is lost due to urine production alone, clearance of the tracer from the blood can be equated with urine production. When inulin was injected into crabs in different media, blood activity in each decreased exponentially, according to the equation y = yoe-l/T; where y is the recorded activity at the time t, ya is the initial activity and T is the time constant. Plotting log. blood activity against time, the time constant T was found for each animal. This gives the time that would be necessary for the blood to be cleared of inulin at the rate of clearance at y0. Rates of urine production were taken to be equal to this clearance rate. Knowing individual inulin volumes, urine flow rates were calculated as a percentage of body weight per day (Table 3).
(b) Increase in activity in the medium
The measurement of the appearance of isotope to determine urine production rates would be most satisfactory if the concentrations of inulin in blood and urine could be conveniently maintained constant. However, continuous infusion of the tracer and the taking of regular samples to check blood activity may themselves affect urine flow. In the present determinations only two samples of blood were taken, one at the beginning and one at the end of an experiment.
It has been shown previously that urine is produced by filtration (Binns, 1969) and in the present paper that urine production accounts for any increase in the activity of the medium following the injection of tracer into Carcinus. This being so, it is possible to estimate rates of urine flow by measuring changes in the activity of the medium. Because urine is produced by filtration, the urine in the antennal gland will have approximately the same concentration of inulin as the blood at any moment. Taking this into account, the average activity of the urine for an experiment was taken as equal to the final blood count plus half the difference between the initial and final blood counts. The change in activity of the medium (Table 2) divided by this average activity of the urine gave a volume of urine produced per day, from which the rate of urine production was calculated. Though this may be a somewhat arbitrary calculation, for a particular animal the urine production rate given by this method was generally close to the rate determined from the clearance of blood inulin.
DISCUSSION
Measurements of blood volumes in two crayfish, Procambarus clarkii and Orconectes virilis, show that an increase in body weight is correlated with a decrease in relative blood volume measured by the dilution of injection inulin. It is considered that this may be due to allometric growth of the animals and it is suggested that a similar phenomenon might be found in other Crustacea (Riegel & Parker, 1960). Figure 1 shows that in Carcinus there is no such relationship between inulin volume and body weight.
Webb (1940) calculated the blood volume of Carcinus by determining total body chloride, which was assumed to be confined to the blood. Analyses of muscle fibres of Carcinus (Shaw, 1958) and Nephrops (Robertson, 1961) show that this is not the case in Crustacea. Therefore the space estimated by Webb in Carcinus, which is almost double the inulin volume, cannot be the blood volume.
Comparison of the dilution of large and small molecules injected into three species of crab has shown that the larger inulin molecule occupies only about two-thirds of the distribution space of sodium thiocyanate. This indicates that in these crabs there may be a closed circulation of inulin within a larger extracellular compartment (Flemister, 1958). Whether the inulin volume in Carcinus is the effective blood space is not known, though it is possible that the factors which limit the distribution of inulin will also affect the distribution of large protein molecules in the blood.
In all dilutions of sea water any reduction in blood inulin in Carcinus ‘was balanced by an increase in the activity of the medium surrounding the crab. It was necessary to establish that inulin is not metabolized by the animal before using it to measure urine production rates.
The mean rate of urine flow in 100% sea water was 4·4% body weight per day. This is close to Webb’s estimate of 4·7%, but is slightly higher than the 3·6% body weight per day estimated by Shaw (1961). Urine production rates can be expected to be variable and therefore it is difficult to state an exact figure for the rate of urine production in a particular medium. From estimates made using three different methods (weighting after blocking the excretory opening; estimation of the rate of excretion of sulphate ions; elimination of injected inulin), it is quite clear that in 100% sea water urine is produced at roughly 5 % body weight per day.
Carcinus is able to maintain its blood hypertonic to dilute media, and in response to the osmotic influx of water into the animal under these conditions the rate of urine production is increased (Nagel, 1934; Shaw, 1961). Measurements of urine flow using labelled inulin generally agree with rates estimated by previous workers and show a gradual increase in urine production as the medium becomes more dilute (Fig. 2). Urine flow rates calculated from the clearance of blood inulin and from changes in activity of the medium correspond in all media except 40% sea water. The available data indicate that urine production rates in this concentration of sea water are particularly variable. Inulin measurements give a range from 14·6 to 30·9% body weight per day and Shaw (1961) found rates from 20·5 to 35·2% body weight per day. If these flow rates reflect marked differences in blood concentrations, perhaps this indicates that, for prolonged exposures after being placed suddenly into this dilute medium, Carcinus is reaching the lower limit of its salinity tolerance in 40 % sea water.
ACKNOWLEDGMENTS
This work is part of a Ph.D. thesis submitted to the University of Newcastle upon Tyne. I wish to thank Professor J. Shaw for his interest, advice and supervision. I am grateful to S.R.C. for providing a postgraduate studentship.