O2 uptake was determined for periods of 23–46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10°C. These media were replaced by artificial lakewater media (ALW) containing 0·20–6 mM Na and O2 uptake was again determined for periods of 24–66 h.
During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0·1–15·5% (mean 6·4 (15)14·4% s.D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22·0–66·7%, but some of this increase may have been due to locomotor activity.
The calculated values for thermodynamic work involved in ion transport were 0·056–0·268 J/10 g.h at 10°C, or 1·5–7·2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. ± 7 %). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.
Active transport of ions against an electrochemical potential gradient is dependent on additional energy derived from anaerobic glycolysis and aerobic respiration (see papers in Keynes, 1971; Stobbart, 1974), and it is therefore generally presumed that osmoregulatory processes make some demand on the metabolic activity of an animal. Numerous measurements of respiration in relation to the salinity of the external medium, using both whole animals and tissue preparations, indicate that although many marine and brackish-water animals respond to a change in salinity by altering the rate of respiration, some do not, and with a few possible exceptions, e.g. Rao (1968), it is debatable how much of the observed changes in respiration rate are directly attributable to osmoregulation (Kinne, 1964; Potts & Parry, 1964). In the case of freshwater animals, theoretical considerations suggest that a series of adaptive changes in body permeability, urine concentration, and affinity for ions in transporting systems, have markedly reduced the energetic cost of maintaining salt and water balance so that less than 10% and possibly only 1 % of the total metabolism may be sufficient for osmoregulatory purposes (Potts, 1954; Shaw, 1959 a; Potts & Parry, 1964). Supporting evidence for these theoretical predictions was recently obtained by Bielawski (1971). Using isolated gill preparations from the crayfish Astacus leptodactylus Esch, Bielawski found no difference in the respiration rate under circumstances where the rate of active ion uptake by the gills was varied. However, the minimum amount of energy used for ion transport was reckoned to be about 13·5 % of the total energy obtained from respiration.
This paper is a further attempt to relate crayfish respiration rates to active ion uptake from dilute media, using whole animals. In order to maximize both the rate of ion uptake and possible differences in respiration rate, emphasis was placed on comparing O2 uptake in salt-depleted animals before and after placement in artificial lakewater media (ALW). Following salt-depletion in deionized water (DW) to remove some 5–10% of the internal sodium, the rate of sodium uptake increases three to fivefold when crayfish are subsequently placed in dilute media containing sodium ions (Shaw, 1959b; Bryan, 1960a, b, c). The maximum rate of sodium uptake is dependent on external sodium concentrations below ca. 1 mM, so that the ion transporting system sited in the gills is half-saturated at concentrations of about 0·25 mM Na. In our experiments we have used ALW containing 0·20–0 ·60 mM Na. At these concentrations most if not all of the net uptake of sodium ions can be regarded as active uptake in salt-depleted crayfish. The observed net sodium uptake rates in our salt-depleted crayfish at 10°C were 2–3 μmol/h and 3–5 μmol/h respectively in ALW containing 0·25 and 0·6o mM Na. At the same time there may have been active uptake of other ions present in the external medium, e.g. chloride and potassium (Shaw, 1960a, b, c, 1964; Bielawski, 1964; Ehrenfeld, 1974). For comparison we have also measured O2 uptake in steady-state animals acclimated to dilute ALW, and then altered the sodium chloride concentration of the medium to see if O2 uptake changed during the process of reaching a new steady-state with respect to sodium.
Specimens of Austropotamobius pallipes Lereboullet were collected from a local stream at High Borrans. They were kept individually in small tanks through which a slow flow of Windermere lake water was maintained. The temperature of the lake water was fairly constant from day to day but it fluctuated during the course of a year, reaching a minimum of ca. 5 °C in February and rising to a maximum of ca. 15 °C in August. The crayfish were fed weekly on live earthworms and Gammarus pulex.
Some individuals were used for experiments over a period of 2 years. The crayfish moulted in mid-summer, when an increase in wet weight was recorded. However, postmoult calcification of the exoskeleton was very slow in Windermere water, which is a relatively soft water compared with High Borrans stream water (Table 1), and some specimens died after moulting. The calcium concentration of Windermere water is probably close to the minimum tolerated by A. pallipes (see Greenaway, 1972, 1974). Specimens that moulted in lake water were allowed to recover for a period of at least 8 weeks before they were used again in experiments.
Nine crayfish were used, with body wet weights ranging from 5·6 to 13·3 g.
(1) Experimental procedures affecting sodium balance in crayfish
Early experiments were carried out on crayfish salt-depleted in DW (pH ca. 5·7) and then exposed to ALW I with a low alkalinity and pH = 6·6 (Table 2). In later experiments salt-depleted crayfish were exposed to ALW II with an increased alkalinity and pH = 7·2 (Table 2). Low pH values may affect sodium influx (Shaw, 1960c). In addition it was thought possible that when a crayfish is exposed to an abrupt change from DW with pH < 6·0 to ALW II with pH = 7·2, the higher pH might lead to increased locomotor activity, e.g. increased movement of sensory appendages. If sustained, such activity might lead to a slight increase in metabolic demand. To avoid this possibility (or at least to minimize it) salt-depletion in later experiments was carried out in a Na-free medium with pH = 7·0 (Table 2). The relatively high potassium concentration in this Na-free medium also had the added advantage that potassium ions were continually available to the crayfish, minimizing changes in the internal (cellular) potassium level during the salt-depletion process. Details of the media employed in each experiment are given with the experimental results in Tables 3 and 4.
Salt-depletion was effected by keeping each crayfish without food in 3 1 of DW or Na-free medium at 9·0 ± 1·0°C for 72 h. During this time some 36–75 μmol Na was lost into the external medium. Crayfish were then placed in the respirometer and were exposed to further salt loss in DW or Na-free medium for periods of 20–48 h (Expts 1–13, 23–27). The loss rate was approximately 2μmol Na/h. Using data in Shaw (19596) the total amount of sodium lost during salt-depletion was estimated to represent 10–20% of the internal sodium content. The removal of a relatively large proportion of the total body sodium was done to maximize the subsequent net sodium uptake from ALW, and to ensure that the maximum rate would be maintained for a period of at least 10 h (Bryan, 1960b), so that the animal’s respiration rate could be measured over an adequate period of maximum net ion uptake. In practice, O2 uptake in ALW was measured for periods ranging from 20–66 h, to see if there was any alteration in the O2 uptake rate which might be related to a gradual return towards the normal internal steady-state as the net uptake of sodium and other ions proceeds. In one instance, after 24 h in DW and 41 h in ALW containing 0·25 mM sodium (i.e. 0·25 ALW), crayfish A1 was then exposed to a second period of salt depletion in DW, whilst measuring the respiration rate (Expts 1 and 22). On another occasion with the same crayfish (A2), O2 uptake was determined in DW for 24 h, followed by 0·25 ALW for 45 h, and 0·5 ALW for 22 h (Expts 2 and 14).
Eight experiments were carried out on four crayfish in which O2 uptake was first measured in steady-state animals, and then the external sodium concentration was altered. In five cases (Expts 15–18 and 28) crayfish were initially held at the steadystate without food in 0·2 ALW at 9°C for 72 h. This sodium concentration is the same as in Windermere water (Table 1). Crayfish were then placed in the respirometer (at 10°C), and were exposed to 0·2 ALW for periods ranging from 20–44 During this time, if the internal steady state with respect to sodium and other ions had remained unaltered, there would have been no net uptake of ions. But some active uptake of ions would normally occur in order to balance the continuous loss of ions that occurs via permeable areas of the body surface and in the urine (Shaw, 1959b ; Bryan, 1960a, b, c). In the present case, at 10°C the active uptake rate required to balance sodium losses was reckoned to be approximately 1 μmol Na/h. After O2 uptake had been determined at the external steady-state concentration for sodium, the latter was raised to 0·6 mM Na. At this new level the active uptake of sodium is increased (the rate would probably be almost doubled), resulting in an active net uptake of sodium which progressively declines until a new internal steady-state is reached. At the same time respiration rates were measured for periods of 20–30 h.
In three further experiments (Expts 19–21) crayfish were first brought to a steadystate in 0·6 ALW and O2 uptake in this medium was determined for periods of 20–28 h. The external sodium concentration was then lowered to 0·2 mM and O2 uptake was again measured for another 24–44 h. In these experiments the initial net loss of sodium from the crayfish, induced by lowering the external sodium concentration, again stimulated the sodium transporting system to operate at a faster rate, so that the rate of active ion uptake was increased.
(2) Measurement of O2 uptake
A detailed account of methods and experimental errors is given by Sutcliffe, Carrick & Moore (1975). Briefly, O2 uptake was measured on individual crayfish by continuously monitoring the O2 tension in the medium in a closed respirometer. The latter was immersed in a constant-temperature water bath at 10 ·0±0 ·02 °C. During each closed period, lasting ca. 2–4 h, the O2 tension in the experimental medium fell from ca. 135 Torr to ca. 105 Torr (i.e. from 90–70% of the air saturation level). Fresh medium was then automatically flushed through the respirometer until the O2 tension was raised back to 135 Torr. At these levels, O2 uptake by the crayfish is independent of the external O2 tension.
In the majority of experiments, crayfish settled down in the respirometer within 1–2 h and then remained relatively quiescent. O2 uptake by these animals represent standard metabolism. But some individuals became restless after changing the experimental medium, e.g. when ALW replaced DW, and other individuals were almost continually restless when in the respirometer. O2 uptake in these cases represents active metabolism. Some examples of both standard and active metabolism are described by Sutcliffe et al. (1975). That paper makes a distinction between assessments of the mean metabolic rate that are based on successive hourly rates of O2 uptake, and assessments that are based on averaged rates of O2 uptake (μg/ animal. h) for each closed period of 2–4 h. Averaged rates are employed in the present study because they covered some 90 % of the total O2 uptake by the animal during the course of an experiment (ca. 10 % was not monitored during the flushing periods). On the other hand, hourly rates accounted for only 75–85% of the total, as the duration of closed periods did not always coincide with convenient multiples of 1 h.
Table 3 presents the results from 22 experiments on crayfish where the O2 uptake rates represent standard metabolism measured over periods ranging from 65 h (crayfish D2, Expt 8) to 98 h (crayfish A1, Expts 1 and 22). During this time the sodium concentration in the medium was normally altered once, but it was altered twice in experiments on crayfish A1, A2, A8 and D4.
In Expts 1–13, active ion uptake presumably occurred when salt-depleted crayfish were exposed to ALW containing sodium, and if active ion uptake requires additional energy then some increase in the rate of O2 uptake might be expected to occur during the 24–66 h periods of exposure to ALW. In fact, in eight experiments there was an increase in the mean rate of O2 uptake in ALW, and in three of these the increased rate was significantly different (P<0 ·05) from the mean rate previously measured in DW or Na-free media. However, in two cases (Expts 12 and 13, Table 3) the variances of the means were also significantly different (P<0 ·05); hence the significance of the differences between the means is questionable. Moreover, in Expt 13 the mean O2 uptake rate by crayfish G1 in 0 ·6 ALW had a relatively large standard deviation (± 89 ·47 μg O2/h) which may have been associated with locomotor activity by the animal. The substantial increase of 67 ·7% in the metabolic rate of crayfish G1 following salt-depletion in a Na-free medium (Table 3) therefore may not be linked with ion transport alone. This leaves seven increases in standard metabolic rate, ranging from 0 ·1–22 ·0%, that may be directly linked with ion transport.
On the other hand, in five experiments there was a decrease in the mean O2 uptake rate in ALW, ranging from 1 ·1–15 ·5% the mean rate in DW or Na-free media (Table 3). In one instance (Expt 4) there was a significant difference (P<0 ·05) between the mean O2 uptake rate measured first in DW and then in 0 ·5 ALW.
Thus the standard metabolic rate was both elevated and depressed to a similar extent, and changes in both directions were observed on different occasions in the same crayfish, i.e. crayfish A, B, D and G (Table 3). For example in crayfish A, on three occasions there was an increased rate of O2 uptake and on two occasions there was a decreased rate of O2 uptake in ALW, following salt-depletion in DW or Na-free media.
In Expts 14–18 some active ion uptake by crayfish would normally occur when the sodium concentration of ALW was raised from 0 ·20 or 0 ·25 mM to 0 ·5 or 0 ·6 mM (Table 3), although the rates of net ion uptake by crayfish in these experiments were probably smaller than the rates of net ion uptake by the salt-depleted crayfish in Expts 1–13. Again the O2 uptake rate was both elevated and depressed at the higher sodium concentrations, although the differences between the means were not significant (P>0 ·05).
In Expts 19–21, steady-state animals in 0 ·6 ALW were exposed to a reduced external sodium concentration in 0 ·2 ALW. Crayfish A, D and G all showed a small decrease in the standard metabolic rate, ranging from 8 ·2–12 ·9 %. The decrease of 12 ·9% in Expt 21 was highly significant (P<o ·001).
Excluding Expts 12, 13 and 18 where there were exceptionally large increases ranging from 22 ·0–67 ·7%, in the remaining 19 experiments shown in Table 3 the standard metabolic rate altered by 0 ·1–15 ·5 %, and the mean percentage change was 7 ·2±4 ·4% S.D. (N=19). This value is close to the combined estimates for experimental errors (< 7 %) and calibration errors (< 2 %) in the method for measuring O2 uptake (Sutcliffe et al. 1975).
Table 4 shows the results from five experiments on crayfish where the O2 uptake rates represent active metabolism, and one experiment (Expt 33) where crayfish D3 moulted after approximately 80 h in the respirometer. In another eight experiments, prolonged periods of active metabolism occurred after the sodium concentration in the medium was altered; in six instances this occurred after replacing DW or Na-free media with ALW. The results from these eight experiments are not presented in this paper as they cannot be related directly to the energetic cost of ion uptake.
Returning to Table 4, in four experiments the metabolic rate increased when crayfish B3, B4, C2 and H1 were exposed to 0 ·6 ALW after salt-depletion in DW or Na-free media. The increases ranged from 4 ·8–42 ·6% of the mean O2 uptake rates in the salt-depleted animals. But only one of the mean differences was significant (P<0 ·o5), in crayfish B4 (Expt 25). In crayfish C6 the mean O2 uptake rate in 0 ·6 ALW decreased by 12 ·9% compared with the mean O2 uptake rate at the steadystate in 0 ·2 ALW, but the difference was not significant (P > 0 ·05).
Following Potts (1954), Shaw (1959a) and Bielawski (1971) the minimum thermodynamic work required to transport sodium ions was calculated from the relation W= URT In Na1/Na0, where Na1 and Nar respectively are the internal and external sodium concentrations (moles), U is the uptake rate of sodium (mol/g. h) and W is the work done (J/g-h). R is the universal gas constant, T is the absolute temperature, and 4 ·148 Joules = 1 ·0 thermochemical calorie. Estimated minimum values for W at various combinations of Na1 and Na0 are given in Table 5, where it is assumed that the simultaneous uptake of sodium and chloride ions needs double the amount of work required to move sodium ions alone. For transporting NaCl the minimum values for W range from 0 ·028–0 ·134 J/10 g.h. However the ion transporting system(s) is unlikely to be 100% efficient, and some additional thermodynamic work may also be required to transport other ions apart from NaCl. A rough approximation to the work performed when moving several ions simultaneously in a 50 % efficient transporting system was therefore obtained by doubling the estimated values of W for NaCl (Table 5). The amount of real work utilized during ion transport in a steadystate crayfish at Nao=0 ·6mM is therefore estimated to be approximately 0 ·056 J/10 g.h at 10 °C. This increases to approximately 0 ·268 J/10 g.h in a salt-depleted crayfish (with a lowered Na,) exposed to Nao=0 ·6mM, at least when the rate of NaCl transport is about 5 μmol/10 g.h.
The total amount of energy utilized by a crayfish may be estimated from the mean standard metabolic rate of 273 μg O2/10 g.h at 10 °C (Sutcliffe et al. 1975). Various estimates are given in the literature for the energy equivalents of carbohydrates, fats and proteins; we chose a value of 13 ·56 J /mg O2 suitable for carnivores that utilize ammonia as the chief excretory product (see review by Elliott & Davison, 1975). The total amount of energy available to the crayfish is then 3 ·70 J/h, and the calculated energy requirement for ion transport in the present study represents some 1 ·5–7 ·2% of the standard metabolic rate.
The calculated energy requirement for ion uptake, equivalent to 1 ·5–7 ·2% of the mean standard metabolic rate in a 10 g crayfish, can now be considered in relation to the experimental errors for the measurements of O2 uptake. Calibration errors were less than 2% and the background component due to microbes was reckoned to produce an error of less than 7 % in most of the experiments. Nevertheless it is clear that practically all of the calculated energy requirement for ion uptake might we indistinguishable from the experimental errors. Therefore the experimental results must be considered with two points in mind; (a) does the metabolic rate appear to increase in a consistent manner when there is an increased net uptake of ions, and (b) does the metabolic rate increase substantially when compared with the small increase expected from the above calculations ?
Both points are summarized in Table 6. During net ion uptake the standard metabolic rate increased by 0 ·1–11 ·0% in eight experiments, but it decreased by 1 ·1–15 ·5% in seven experiments. In three other experiments which are not included in Table 6, the metabolic rate showed a relatively large increase ranging from 22 ·0 – 67 ·7 % of the standard rate. This increase might be directly associated with net ion uptake, but it might also be due to more locomotor activity during the second part of each experiment. Certainly there was a larger variability (standard deviation) in the averaged rates of O2 uptake when the animals were exposed to 0 ·6 ALW (Table 3, Expts 12, 13, 18). With these three exceptions, there was no consistent increase in the standard metabolic rate during net ion uptake, and the relative change was small, equivalent to a mean percentage change of 6 ·4±4 ·4% (s.D.) (N=15). In active animals there were two relatively large increases in the metabolic rate during net ion uptake, but in three experiments the increases only ranged from 4 ·8–14 ·9%. None of the increases was significant at the 5 % level (Table 4).
In four experiments where crayfish incurred a net loss of ions the standard metabolic rate consistently decreased by 7 ·5–12 ·9% (Table 6). The decrease was significant in only one instance (Expt 21, Table 3), and this was also the only highly significant (P<0 ·001) change in metabolic rate that was measured during 36 experiments.
The results summarized above and in Table 6 strongly suggest that the energetic cost of ion uptake in A. pallipes is so small that it is practically impossible to measure any direct change in the metabolic rate of intact crayfish. Hence the calculated value of 1 ·5–7 ·2% of the standard metabolic rate in a 10 g crayfish appears to be substantially correct for the amount of thermodynamic work involved in active ion uptake.