Holthuisana transversa (von Martens), a freshwater/land crab from arid areas of Australia, is an efficient bimodal breather. In water, resting (1·65μmolg-− h−1 ) and (15·6mlg−1 h−1) at 25°C were lower than in other aquatic crabs whilst percentage extraction of oxygen was quite high (47 %). was not regulated at low ambient PO2 but could be increased at least three times during exercise. Normoxic was low (18 Torr) compared with other water-breathing crabs. The haemolymph contained haemocyanin which had a moderate affinity for oxygen (P50 = 80 Torr) and carried over 90% of the oxygen transported. Oxygen content of post-branchial haemolymph was low (346μmol l−1). There was a small positive Bohr shift (log P50/pH-0·33). The strategy of gas exchange in water is discussed and compared with that of aquatic crabs.
Recent studies have revealed the basic patterns involved in gas exchange in aquatic crabs although the details vary considerably between species due to morphological differences between taxa and adaptation to habitat. Circulation of blood and water through the gills is counter-current in Carcinus (Hughes, Knights & Scammell, 1969) and, given the common functional design of crab gills, this is possibly true of most aquatic crabs. Oxygen extraction in resting crabs is quite high e.g. Callinectes and Ubinia (approx. 50%) (Batterton & Cameron, 1978; Burnett, 1979), Cancer (>60%) (McMahon & Wilkens, 1977), although values are much lower during hyperventilation. Resting oxygen consumption is regulated over a wide range of external oxygen tension (Arudpragasam & Naylor, 1964; Johansen, Lenfant & Mecklenburg, 1970; Taylor, 1976; Burnett, 1979). In response to hypoxia, ventilation of the gills increased and, in Libinia, there is increased cardiac output. However, Callinectes an oxygen conformer at rest, although ventilation and oxygen consumption are both elevated during exercise (Batterton & Cameron, 1978). Haemocyanin is normally saturated with oxygen in postbranchial haemolymph (Mangum, 1980), but the total oxygen capacity is variable because of interspecific variation in the quantity of pigment and the PO2 levels of haemolymph. The oxygen affinity of the pigment is relatively high (P50 at normoxic pH = 10–16Torr) (Mangum, 1980), and much oxygen is transported in simple solution by crabs in aerated water (Burnett, 1979; McMahon, McDonald & Wood, 1979). The oxygen gradient across the gill is high in the species examined, typically 60–100 Torr (Johansen et al. 1970; Taylor, 1976; McMahon et al. 1979). The PCO2 levels of postbranchial haemolymph are considered to be uniformly low (< 5 Torr) in aquatic crabs (Rahn, 1966; Cameron, 1979).
The physiological and morphological requirements for gas exchange in water and air are very different (Rahn, 1966; Rahn & Howell, 1976) and adaptations which facilitate gas exchange in air may reduce efficiency in water. Thus it is of great interest to examine gas exchange in a species, such as Holthuisana transversa, which breathes effectively in both media. Holthuisana is a freshwater/land crab from the arid-zone of Australia. For long periods it breathes air under severe terrestrial conditions; to this end it has developed tidally ventilated lungs (Greenaway & MacMillen, 1978 ; Greenaway & Taylor, 1976; Taylor & Greenaway, 1979). In the brief wet periods, the crab forages actively in water (Greenaway, 1981). In this study the features of gas exchange in water by Holthuisana are examined and compared with the pattern established for aquatic crabs, whilst the accompanying paper is concerned with gas exchange in air.
MATERIALS AND METHODS
Holthuisana (Austrothelphusa) transversa (von Martens) were collected from Bourke, Gulargambone and Lightning Ridge in western N.S.W. and maintained in the laboratory as described previously (Greenaway & MacMillen, 1978). Only large crabs (20–30 g) were used in experiments, which were carried out at 25 °C.
The rate of oxygen consumption , rate of ventilation and extraction of oxygen (% Ext) were measured in an overflow apparatus similar to those described by earlier workers (e.g. Larimer, 1961). This was a Perspex box of about l·41capacity (14 × 10 × 10cm). Masks were made from balloons attached to the carapace with Eastman 910 contact cement. The overflow method may underestimate slightly due to increased resistance to water flow (Johansen et al. 1970). It also suffers from the disadvantage that the crabs are tethered and cannot move freely about the chamber. The use of electromagnetic flow probes, which reduces these problems, was investigated but proved to be unsuitable due to the small size of Holthuisana and the low conductivity of fresh water.
The overflow technique will also underestimate if reversals of the direction of ventilatory flow occur during the measurement period. In several preliminary experiments the occurrence of reversals of scaphognathite beat were tested for with a Statham P23 AA pressure transducer connected to the mask by plastic cannula tubing. Scaphognathite activity was recorded on a Beckman Dynograph for crabs exposed be range of PO2, over a period of several hours. No pressure changes due to reversals were observed and it was concluded that reversals were infrequent in Holthuisana and unlikely to cause significant error in measurement of
Preliminary experiments showed that, and % Ext settled to stable, reproducible levels within a few hours of placing crabs in the overflow apparatus. An example is shown in Fig. 2. Subsequent measurements were made on crabs which had been in the apparatus for 24 h.
Samples of postbranchial haemolymph were taken anaerobically from the pericardial cavity in 1 ml plastic syringes in which the dead-space was filled with mineral oil. To avoid delay and disturbance at the time of sampling, holes were drilled in the carapace over the pericardium about 5 h before haemolymph was taken. Care was taken to avoid puncturing the hypodermis during drilling. Samples of prebranchial haemolymph were taken from the ventral thoracic sinus at the base of the chelae or pereiopods. All samples were taken whilst the crabs were submerged.
The PO2 and PCO2 of the haemolymph were measured separately on 0·2 ml samples using a Radiometer blood-gas analyser (pH27 plus PHM927B) equipped with separate water-jacketed electrodes maintained at 25 °C. For determinations of PCO2, the electrode was calibrated with 2·9 % and 0·44 % CO2, and output was measured on a mV recorder. Oxygen content was measured with a Lex O2 Con TL oxygen analyser (Lexington Instrument Corporation). Both pH and [HCO3] of haemolymph were measured on the same 0·3 ml sample. Immediately after sampling, the needle of the syringe was detached and haemolymph from the centre of the syringe was aspirated into a Radiometer G297/G2 capillary electrode and its pH determined. The remainder of each sample was used for the determination of the bicarbonate + carbonate concentration with a Radiometer autotitration system (PHM64, TTT80, TTA80, ABU 80). A measured volume (0·2 ml) of haemolymph was added to a titration vessel containing 0·1 ml of 0·1 mmol 1−1 HC1 and 1 ml of saline. The samples were evacuated in a vacuum desiccator to remove the liberated CO2 from solution and then titrated with 0·01 mmol 1−1 NaOH until the original pH of the haemolymph was reached. Values obtained were corrected with blank samples containing only HC1 and saline.
Oxygen-binding curves were determined at several different pH values at 25 °C. Haemolymph samples (1–2 ml) were taken from the ventral sinuses of five crabs and allowed to clot. The clots were removed by centrifugation (35000 r.p.m.) and the supernatant containing the haemocyanin was retained. A few μl of each sample were used for electrophoresis (acrylamide gel) and, as no evidence of polymorphism between subunits of haemocyanin was apparent, we pooled the samples for the oxygen binding experiments, keeping the pooled haemolymph of male and female crabs separate. The pooled samples were diluted with saline containing the same major ions as the haemolymph (270mmol 1−1 NaCl, 15 mmol 1−1 CaCl2, 5 mmol l−1 MgCl2) (Greenaway & MacMillen, 1978) and were buffered with 50 mmol 1−1 Tris (pH 7·68 and above) or 50 mmol 1−1 Bis-Tris buffer. A vacuum-spectrophotometric method, similar to that of Riggs & Wolbach (1956) was used to determine the binding curves.
Values of the percentage saturation of the haemocyanin with oxygen were calculated from absorption spectra collected over the wavelength range 400–300 nm using a scanning spectrophotometer (Cary 14).
Further samples of haemolymph from each crab were diluted with saline as described above and examined with a Beckman model E analytical ultracentrifuge at 60 000r.p.m. to provide information on the molecular species of haemocyanin present.
The symbols used in the text follow Dejours (1981). Values are given as means ±1 × S.E.M..
The in air-saturated water was 1·65 μmolg−1 h−1. In response to declining oxygen tension in the water, fell in a more or less linear fashion for each crab tested (Fig. 1). Regression analysis was performed on the pooled data for MO2 of all crabs and a significant linear relationship was found between and PO2 of the water (, μmolg−1h−1, P< 0·001). Holthuisana is an oxygen conformer, at least whilst at rest.
Rate of ventilation
The mean value of for resting crabs in air-saturated water was 15·6 ± 2·0mlg−1 h−1. Individual crabs showed a small increase in when the PO2 of the water fell below 100 Torr (Fig. 1) but no general response was apparent when the data were pooled for linear regression. The mean value for at the lowest PO2 tested (22·2 ml g−1 h−1 at 26 Torr) was, however, significantly higher than that obtained in air — saturated water (0·02>P>0·05 using a paired ‘t’ test), an increase of 42%.
Extraction of oxygen from the respired water varied considerably between individuals (21·9–73·4%) but in all cases was relatively high with a mean in air saturated water of 46·4 % ± 5·1 S.E.M.. The individual response to reduced PO2 was rather variable but the % Ext was generally maintained until low PO2 was reached. The level of individual variability prevented any statistical demonstration of responses to low PO2, and, indeed some crabs showed elevation of % Ext whilst others showed decreased % Ext at the lowest PO2 levels tested (Fig. 1).
The PaO2 values for crabs maintained in a resting state in aerated water (155 Torr) were very low with a mean of only 17·8Torr (Table 1). Crabs which retained air bubbles in their branchial chambers had much higher values of PaO2, similar in fact to the values found for crabs breathing air (Greenaway, Taylor & Bonaventura, 1983). Care was taken to ensure that all air was expelled from the gill chambers well before samples of haemolymph were taken.
The pH of the haemolymph of Holthuisana (Table 1) was low compared with the of other crabs (Mangum & Schick, 1972; McMahon et al. 1978; Aldridge & Cameron, 1979; Taylor & Wheatly, 1979). At these pH levels the CO32− concentration was negligible (Truchot, 1976) and CO2 in the haemolymph was present as HCO3− and dissolved CO2. Dissolved CO2 in the haemolymph was calculated from PCO2, using a value of 0 ·041 mmol 1−1 Torr−1 for the solubility coefficient of CO2 taken from the data for Carcinus at 12%o and 25 °C (Truchot, 1976). At this salinity the osmotic concentration of the haemolymphs of Carcinus and Holthuisana was similar and their respective solubility coefficients for CO2 would have been very close. Both and PvCO2 of haemolymph samples were higher than recorded in other aquatic crabs (McMahon et al. 1978; Taylor & Wheatly, 1979). However, PvCO2 values were almost certainly too high. Several minutes necessarily elapsed between removing a sample of haemolymph and obtaining its PvCO2. In this time, even in the absence of carbonic anhydrase, a new equilibrium would have been established between CO2 and HC03− in the sample with a resultant increase in PCO2 from the expected low level of post-branchial haemolymph and a slight decrease in the concentration of HCO3−. This is discussed in more detail below. Values for PvCO2are likely to be more accurate, erring on the low side if at all, as equilibrium between CO2 and HCO3− would have been more complete. These considerations have no effect on the values for CCO2 and it was apparent that the total loss of CO2 across the gills was about 4 % of that carried in prebranchial haemolymph.
The effect of disturbance
Immediately after crabs had been placed in the overflow apparatus and were very high (Fig. 2). These parameters declined steadily from their initial values and became stable well within a 24 h period. The initial for crab 748 (Fig. 2) was 40·6 ml g−1 h−1, five times the level seen in resting metabolism (24 h). Clearly the capacity existed to increase ventilation substantially, although this ability was largely unused by resting crabs during hypoxia. Initial for crab 748 (3·08μmolg−1 h−1), was about 3·5 times the resting level.
In a separate experiment, crabs were kept moving for a 5 min period and their PaO2 was measured immediately afterwards. The mean value obtained (22·2 ± 2·6 Torr) was not significantly different from that found in resting crabs (0·4>P>0·2).
The haemocyanin had a moderate affinity for oxygen at 25 ±C with a P50 of 8·0 Torr at normoxic pH, in the absence of CO2.A small positive Bohr effect was apparent with a log P50/ pH value of −0·33 (Fig. 3).
The data for oxygen binding (Fig. 3) revealed that the pigment was nearly saturated with oxygen at approximately 20TorrPO2, at normoxic pH and in the absence of CO2. The exact PaCO2was not known but was likely to have been about 2·5 Torr and thus had little effect on oxygen binding. Thus, haemocyanin was about 90% saturated with oxygen at (17·8 Torr) and about 80% saturated at (13·0Torr), which indicated a large venous oxygen reserve.
Cooperativity of the haemocyanin was calculated from Hill plots and lay between 2 and 3 over the range of pH studied (Fig. 3). These values are characteristic of decapod crustaceans (Mangum, 1980). Ultracentrifugation of the haemolymph gave Schlieren peaks at 23–24s and 15·5–16·5 s which corresponded to dodecameric and hexameric aggregation states of haemocyanin molecules respectively. This was again characteristic of decapod haemocyanins and similar to values for other freshwater crabs (BonaventuraetaZ. 1979). In female crabs, a third peak was evident at 10–11s but did not represent haemocyanin.
CaO2 was measured in five crabs kept in aerated water and gave a mean value of 46± 3·6μmolo2l−1 (0·774 vol %). Using the mean values for PaCO2and CaO2 and treating the haemolymph as half strength sea water, the oxygen carried in simple solution was calculated to be 26μmol1−1, 7·5% of CaO2. The haemocyanin was approximately 90% saturated at and carried the remaining 92·5 % (320μmol 1−1) of the measured CaO2. Thus saturated haemocyanin had a capacity of 355 μmol 1−1.
Weight-specific log decreases linearly with increasing log body mass within crab species (Taylor & Wheatly, 1979; MacMillen & Greenaway, 1978; Kotaiah & Rajabai, 1975) and this might be expected to hold interspecifically for crabs generally. Thus a small species should have a relatively high but, in practice, Holthuisana has a lower than any of the larger species studied (Table 2). Indeed, was three times lower than that found in Carcinus of similar body weight (25 g) at the same temperature (25 °C) (Taylor & Wheatly, 1979). The of Holthuisana was also the lowest of the species studied (Table 2), even allowing for an expected decrease in with increasing body size. Comparison with slightly larger Carcinus at 18 °C yielded a value 2·2 times lower in Holthuisana (Table 2). The % Ext of oxygen by Holthuisana was quite high, similar to that of the aquatic crabs Callinectes, Cancer and Libinia and higher than in Carcinus (Taylor, Butler & Al-Wassia, 1977). The emergent pattern for Holthuisana was of an animal with a low and a very low but with efficient extraction of oxygen from the water. Like Callinectes, Holthuisana was an oxygen conformer at rest but was capable of increasing (5 times) and (at least 3 times) during exercise.
In the aquatic crabs studied, haemocyanin generally carried most of the oxygen transported by the haemolymph (Mangum, 1980). Although the PaO2 maintained in aerated water differed widely between species of crabs, the haepiocyanin was nearly saturated with oxygen in each case and PaCO2largely reflected the P95 of the pigment (Mangum, 1980). In Holthuisana, (17 · 8 Torr) was lower than that found in any other aquatic crab and lower than that found in freshwater crayfishes e.g. Astacus leptodactylus (28Torr) and Austropotamobius pallipes (33 TOIT) (Angersbach Decker, 1978; Wheatly & Taylor, 1981). However, given the affinity of the haemocyanin at normoxic pH, near-saturation was achieved at and the pigment was responsible for more than 90% of oxygen transport. Removal of oxygen by the tissues was quite small, and a relatively large venous reserve was present in the resting crab.
The very low PaCO2 requires some further comment because a substantial gradient of PO2 existed between post-branchial haemolymph (17 · 8 Torr) and water leaving the gills (71 · 5 Torr). The simplest explanation was that % Ext from the water actually passing over the gill lamellae was very high (and the gradient of PO2 much lower) but much of the water bypassed the gills so that the measured extraction was much lower. The low may be seen as minimizing energy expenditure on ventilation as it was adequate to permit a high level of saturation of the haemocyanin, which carried most of the oxygen used, and higher energy expenditure on would not have correspondingly increased oxygen content. Additionally, the affinity of the haemocyanin would enable saturation of the respiratory pigment at the low ambient PO2 which the crabs may frequently encounter in their water-filled burrows and in the warm, shallow temporary pools which they inhabit. In shallow water, crabs were frequently observed to augment water breathing by taking air into the branchial chambers and this probably represented a normal respiratory pattern in shallow water. This behaviour elevated PaCO2 and would act to increase delivery of oxygen to the tissues. The PCO2 of the haemolymph of aquatic decapods is believed to be controlled largely by physical factors. Thus the high solubility of CO2 and the low oxygen content of water would ensure that the PCO2 of exhaled water would not exceed about 5 Torr at 25 °C and 100% extraction of oxygen (Rahn, 1966). In the absence of a significant barrier to diffusion of CO2 across the gill, PaCO2 should be close to that of exhaled water and indeed this has been demonstrated in certain fish, cephalopods and crabs (Rahn, 1966). In Holthuisana the maximum PaCO2 should have been about 2 · 5 Torr, given the mean % Ext measured, and the measured value was clearly erroneous as discussed above. Using an estimate of 2 ·5 Torr for PaCO2, about 40 % of total CO2 loss at the gills originated in the dissolved CO2 pool and 60% from the bicarbonate pool.
Dissolved CO2 is lost passively across the gill epithelium, but the bicarbonate must be exchanged for a counterion or be converted to CO2 (Cameron, 1979). The uncatalysed conversion of HCO3− to CO2 is too slow to contribute significantly to loss of CO2 during the residence time of haemolymph in the gills (Aldridge & Cameron, 1979) and, given the low fluxes of ions in Holthuisana, adequate excretion of HCO3− by ion exchange alone seems unlikely. It follows that most of the HCO3− lost must firstly be dehydrated by carbonic anhydrase, which is reported to be present in the gill epithelium of crabs (Burnett, Woodson, Rietow & Vilich, 1981; Maren, 1967; Aldridge, 1977). The pH of the haemolymph of Holthuisana was lower than values recorded for marine crabs at similar temperatures (Mangum & Schick, 1972; Aldridge & Cameron, 1979; Taylor & Wheatly, 1979) and lower than those found in freshwater crayfish which are mofe similar in haemolymph chemistry (Angersbach & Decker, 1978; Wheatly & Taylor, 1981).
Gas exchange by Holthuisana in water was characterized by very low and P 2O 2 values and low . Resting , although lower than in other aquatic crabs, we higher than in Holthuisana breathing air. However, behaviour patterns differ in air and water; Holthuisana is normally inactive and very low in air in order to conserve food reserves (MacMillen & Greenaway, 1978) whilst in water the crab forages actively. Maximum values were similar in both media. Holthuisana was evidently successful at gas exchange in both media although in absolute terms was low in both (Greenaway, et al. 1983).
We are grateful to G. Ferruzzi, S. Tesh, T. Brouwer, G. Godette and D. Hair for technical assistance. K. Schmidt-Nielsen and Barbara Grubb made the measurements of oxygen content possible. The work was supported by the Australian Research Grants Committee through grant no. D17615225R to P. Greenaway and by grants from the U.S. National Institute of Health (HL15460) and N.S.F. (PCM79-06462).