SWITCH FROM METABOLIC TO VENTILATORY COMPENSATION OF EXTRACELLULAR ph IN CRAYFISH

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1 J. exp. Biol. 137, (1988) 411 Printed in Great Britain The Company of Biologists Limited 1988 SWITCH FROM METABOLIC TO VENTILATORY COMPENSATION OF EXTRACELLULAR ph IN CRAYFISH BY B. BURTIN AND J.-C. MASSABUAU Laboratoire d'etude des Regulations Physiologiques (associe a Vuniversite Louis Pasteur), Centre National de la Recherche Scientifique, 23 rue Becquerel, Strasbourg, France Accepted 23 March 1988 Summary The mechanisms of extracellular ph regulation were studied in crayfish Astacus leptodactylus under conditions that were either favourable or unfavourable for ionoregulation. Animals in intermoult or premoult stages were kept in normoxic artificial waters at 13 C. In intermoult, acid-base balance (ABB) and ionoregulatory disturbances were induced by increasing the ambient partial pressure of CO 2 (Pw CO2 ), by decreasing the concentration of NaCl in the water ([NaCl] w ) or by associating both changes. In premoult we took advantage of the spontaneously occurring endogenous problems of ionoregulation which are linked to shell shedding. In intermoult, an increase of Pw CO2 alone induced a hypercapnic acidosis compensated by metabolic means, whereas in association with a decrease of [NaCl] w (which induced a decrease of [NaCl] in the haemolymph) it led to a ventilatory compensation. In intermoult a decrease of [NaCl] w alone induced a metabolic acidosis that was compensated by metabolic means, whereas in premoult it was compensated by ventilatory adjustments. It is concluded that when water breathers are facing experimentally induced or spontaneous ionoregulatory problems, compensation for superimposed ABB disturbances can be made by ventilatory adjustments instead of by metabolic means. Introduction In water breathers, regulation of extracellular ph has been thought to be performed only by metabolic means, i.e. mainly through ionic exchanges between extracellular medium and water (see Reeves & Rahn, 1979; Heisler, 1984; Truchot, 1987). However, this has been reconsidered in the freshwater crayfish Astacus leptodactylus. In this animal a ventilatory CO 2 drive has been described (Massabuau et al. 1984) and the presence of peripheral CO 2 chemoreceptors located in the branchial cavities has been reported (Massabuau & Burtin, 1985). j[b analyse the role of this chemosensitivity one must first differentiate between Key words: acid-base balance, crayfish, ventilation, respiration, ionoregulation, acidosis.

2 412 B. BURTIN AND J.-C. MASSABUAU reports in the literature showing the CO 2 partial pressure (PcoO m tne blood as a dependent variable and those showing it as an independent variable by which acid-base regulation can be achieved. Indeed, it has been shown that at constant temperature and metabolic rate the blood P CO2 can vary passively - as a dependent variable - following changes of water P CO2 (Dejours & Armand, 1980), water titration alkalinity (Dejours & Armand, 1980; Truchot, 1984; Thomas & Poupin, 1985) and any ventilatory modifications such as those due to changes of inspired O 2 partial pressure (Dejours, 1973) or stressful conditions (see Heisler, 1984). But the capacity of ventilation for active control of P CO2 in the blood - as an independent variable - has only been observed in three situations in A. leptodactylus. First, during the circadian rhythm there is a continuous regulation of ABB achieved through an adjustment of blood P co, (Sakakibara et al. 1987). Second, during a temperature decrease from 13 to 6 C the ABB changes can be explained only in terms of ventilatory compensation (Gaillard & Malan, 1985). Third, during a decrease of water titration alkalinity (which induces a hypercapnic acidosis, Dejours & Armand, 1980) associated with a decrease of water NaCl concentration ([NaCl] w ) the regulation of ph is performed by a ventilatory adjustment (Burtin et al. 1986). In this last study, we proposed that the decrease of [NaCl] w could disturb ionoregulation so that the mechanisms for metabolic acid-base compensation would be impaired. The aim of the present work was to gain insight into the preceding hypothesis. We present two situations, both associated with an increased load on the ionoregulatory mechanisms, in which A. leptodactylus switches from metabolic to ventilatory compensation of extracellular ph. These situations were: a hypercapnic acidosis associated with a decrease of [NaCl] w in intermoult crayfish and a metabolic acidosis in premoult animals. In the latter, crayfish spontaneously face endogenous problems of ionoregulation which are linked with the future shedding of the calcified exoskeleton (see reviews by Mantel & Farmer, 1983; Truchot, 1987). Materials and methods Animals and ambient conditions Male crayfish Astacus leptodactylus, weighing g, were acclimated in the laboratory for at least 1 month before experiments. They were fed weekly with carrots and fish and maintained under natural light conditions. Crayfish were kept in tanks filled with Strasbourg tap water, TWr, at 13 C [see Table 1; other ions (mmoir 1 + ): NH 4 <0-001, NO 2 ~ = 0-002, NO 3 ~ = 0-058]. Experiments were performed either on intermoult or on premoult crayfish (stage C 4 or D 2 and D 3 from Drach, 1939). Prior to experiments, animals were transferred from TWr into 8- to 15-1 tanks filled with a reference or test artificial water, AWr or AWt (throughout the text AWr and AWt are followed by two numbers, the first of which stands for the titration alkalinity, TAw in mequivt 1, and the second for [NaCl] in mmoll" 1 : see Table 1). They were acclimated for at least 14 days in this w and then exposed to a change in water composition. The water renewal rate,

3 Regulation of ph in crayfish 413 Table 1. Ionic concentrations (in mmolt 1 ) TA Na + Ca 2+ TWr AWr4/0-5 AWt 4/0-15 AWt2/5 AWt 2/ cr K of the different waters used Mg SO 4 2 " [I TA, titration alkalinity, mequivl *; fx, ionic strength, mmoll 1 ; O, osmolarity, mosmoll '. TWr, reference tap water; AWt, artificial test water; the two numbers following the abbreviations are TAw and [NaCl] w, respectively (example: AWt2/5 denotes a test artificial water with a TA value of 2mequivl~ 1 and a [NaCl] w value of 5mmoll" 1 ) lh l, was adjusted to keep the actual values of ion concentrations close to the nominal values (±4% range). Ionic strength was kept constant by replacing NaCl with MgSO 4 since the activity of each ion, as well as pk values, are dependent on the ionic strength (Stumm & Morgan, 1981). The ABB in the water was adjusted by a ph-co 2 stat (Dejours & Armand, 1980). The value of CO 2 partial pressure in the water (Pw co,) was maintained at 0-1 ± 0-01 kpa for normocapnic conditions (for TAw = 4mequivl" 1, ph = 8-32 and for TAw = 2mequivl~ 1, ph = 8-03) and at 0-2kPa for hypercapnic conditions (TAw = 4mequivl~ l and ph = 8-03). Experimental temperature was 13 C and Pw o, was 19-5 ± 0-6 kpa. Animals were fasted for at least 1 week before experiments. Haemolymph was sampled only once between 09.00h and 16.00h. Determination of haemolymph acid-base balance Mixed venous haemolymph samples were collected by puncturing the infrabranchial sinus. Venous haemolymph ph (phv) was immediately measured with a Radiometer 6299A capillary electrode and total CO 2 concentration with a modified Cameron chamber (Cameron, 1971). Bicarbonate plus carbonate concentrations and Pv C o 2 were calculated using a CO 2 solubility of 0-427mmoll" 1 kpa" 1, pk{ = 6-12 and pk 2 ' = (Gaillard & Malan, 1985; throughout the text [HCO 3 ~]v corresponds to [HCO 3 "]- V + 2[CO 2 3 "];). Values of pk' were not corrected for changes of haemolymph ionic strength. Na + concentration in the venous haemolymph ([Na + ];) was determined by atomic absorption spectrophotometry at 589 nm and Cl~ concentration ([Cl~]v) by coulometry (Cotlove's method). The water content of the haemolymph and whole animal were determined by measuring differences between wet and dry mass after dehydration at 70 C for 24 and 72 h, respectively. Determination of oxygen consumption and ventilatory flow rate Oxygen consumption (M O2 ) was determined as described by Massabuau et al. J1984) and ventilatory flow rate (Vw) was calculated from M o, and the simultaneous measurement of the value of O 2 partial pressure in the expired water

4 414 B. BURTIN AND J.-C. MASSABUAU (Saunders, 1962). Crayfish were placed in a respirometer at about 13.00h and measurements were performed the following day between 10.00h and 13.00h. Since for technical reasons only one animal could be examined each day, the measurements ran over 10 days. No significant difference between the beginning and the end of this period was noticeable. Data are reported as mean values ±1 standard error, S.E. Differences were evaluated using a two-tailed Student's r-test. P<0-05 was taken as the fiducial limit of significance. Acid-base balance states were considered to be distinct when two parameters out of three (ph, [HCO 3 ~]v and Pv CO2 ) were different. Results The time course of the ionoregulatory disturbance that occurred when [NaCl] w was decreased from 0-5 to O-lSmmolT 1 is presented in Fig. 1. In the first 24h, both [Na + ]- and [Cl~]v decreased. After 7 days, [Na + ]- v recovered to its reference value. A transient metabolic acidosis was evident after 2h but it was corrected by metabolic means after 24 h. Fig. 2 shows that when a hypercapnic acidosis induced by an increase of Pw CO2 was superimposed on this ionoregulatory disturbance, there was no change in ABB after 24 h. This means that the compensation is achieved by a ventilatory adjustment of blood Pco 2 - A control experiment, h B Pv CO2 (kpa), T L 9 XAWr 4/0-5 A AWt 4/0-15 /. J 24 h Reference? f? OU p 150 Z X Time (days) phv 7-9 Fig. 1. (A) Time course of changes in sodium and chloride concentration in the venous haemolymph, [Na + ]v and [Cl~]v, of intermoult crayfish following transfer from artificial reference water, AWr 4/0-5 (TAw = 4 mequiv 1~' and [NaCl] w = 0-5mmoir 1 ) into artificial test water AWt 4/0-15 (TAw = 4 mequiv I" 1 and [NaCl] w = 0-15mmoir l ). Both [Na + ]; and [C\~]- v decrease during the first 24h and only [Na + ]v recovers after 7 days. (B) Time course of changes in acid-base balance in the venous haemolymph of the same animals. There is a slight metabolic acidosis. During the metabolic compensation, points at 2 and 24h are significantly different. March 1985; means ± 1 S.E.; * values are significantly different from reference values; N= 14 crayfish per point.

5 Regulation of ph in crayfish Pw C o 2 (kpa) X AWr 4/ AWr 4/ OAWt4/ h Pv CO2 (kpa) o u X phv Fig. 2. Acid-base balance in the venous haemolymph of intermoult crayfish exposed to a 24-h hypercapnia (Pw cc, 2 = 0-2 kpa). Animals were either kept in artificial water, AWr 4/0-5 (TAw = 4 mequiv 1~ l and [NaCl] w = 0-5mmoir 1 ) or exposed to a decrease of [NaCl] w in passing from AWr4/0-5 to AWt 4/0-15 (TAw = 4 mequiv r 1 and [NaCl] w = 0-15mmoll~ l ). When the hypercapnic acidosis was associated with a decrease of [NaCl] w, ph was compensated by ventilation instead of metabolic means. February 1987; means ± 1 S.E.; * value is statistically different from the reference value although ph is not different in the three situations; reference, N= 14 crayfish; other points, N = 20. Dashed line, buffer line for Astacus leptodactylus (Dejours & Beekenkamp, 1978). without simultaneous decrease of [NaCl] w, shows the metabolic pathway of compensation as both Pv CO2 and [HCO 3 ~]v increased (Fig. 2). Fig. 3 (control experiment in intermoult) and Fig. 4 (during premoult stage) show how a metabolic acidosis induced by decreasing [NaCl] w from 5 to O-lSmmoll" 1 was compensated when ionic exchanges between extracellular medium and water were modified to prepare for moulting. In intermoult, a highly significant metabolic acidosis developed as both [Na + ]v and [Cl~]v decreased, and there was a partial metabolic compensation between 7 and 14 days. Water content of the haemolymph (95-1 ±0-9%) and whole body (78-1 ±1-1%) remained constant throughout the experiment (N = 5). In premoult stages the mechanism of compensation was different; 14 days after the decrease of [NaCl] w both Pv CO2 and [HCO 3 ~]v were decreased and phv did not differ from the reference value. Table 2 confirms that in this situation the ventilatory requirement significantly increased. When crayfish were transferred back into the water in which [NaCl] w was 5 mmol 1~ ] Pv C o an 2 d [HCC>3~]v tended to recover to reference values (Fig. 4). She metabolic alkalosis observed at that time corresponds to what was reported in le same species during the late premoult stage by Dejours & Beekenkamp (1978).

6 416 B. BURTIN AND J.-C. MASSABUAU Pv CO2 (kpa) "T7 O u X Time (days) phv 7-9 Fig. 3. (A) Time course of changes in sodium and chloride concentrations in the venous haemolymph, [Na + ]; and [CP]v, of intermoult crayfish following the transfer from artificial test water, AWt2/5 (TAw = 2 mequiv 1~' and [NaCl] w = SmmolP 1 ) into AWt2/0-15 (TAw = 2 mequiv P 1 and [NaCl] w = O-lSmmolP 1 ). Both [Na + ]; and [Cl~]v decreased in the first 7 days. Between 7 and 14 days [CP]v did not change significantly and [Na + ]v increased. (B) Time course of changes in acid-base balance in the venous haemolymph of the same animals. There is a large metabolic acidosis which developed in 7 days. It is partially compensated by metabolic means after 14 days. October 1984; means ± 1 S.E.; * values significantly different from reference; iy=14 crayfish per point. AWt 2/5 O AWt 2/0-15 Pv CO2 (kpa) 0-5 s Reference #-i 24 h*^^" T 7 days phv 7-9 Fig. 4. Acid-base balance in the venous haemolymph of premoult crayfish following transfer from artificial test water, AWt 2/5 (TAw = 2 mequiv P 1 and [NaCl] w = 5mmolP') into AWt2/0-15 (TAw = 2mequivP 1 and [NaCl] w = 0-15mmolP') and recovery. The metabolic acidosis is compensated by ventilatory adjustment. Following recovery in AWt 2/5, there is a metabolic alkalosis partially compensated by a decrease of ventilation as Pv CO2 returned to its reference value. September 1985; means ± 1 S.E. ; * value significantly different from reference; N= 10 crayfish per point.

7 Regulation of ph in crayfish All Table 2. Ventilatory changes in crayfish transferred from AWt2/5 into AWt2/0-15 during the premoult stage Pio 2 PE O2 E MO.B- 1 VwB-' VwM o ;' Water (kpa) (kpa) (%) (^molmin" 1 kg" 1 ) (mlmin" J kg" 1 ) (mljumoi~ J ) AWt2/5 AWt 2/ From left to right: oxygen partial pressure in the inspired and expired water, Pio 2, and PEQ 2 ; oxygen extraction coefficient, E; oxygen consumption per unit of body mass, M O,B~'; ventilatory flow rate per unit of body mass, VwB" 1 and ventilatory requirement, VwM O2 ~' in two groups of 10 crayfish acclimated for at least 14 days in both waters (see Table 1). In AWt 2/0-15, VwMo 2 ~ l was double the value in AWt2/5 (P<0-05). The values for animals in AWt 2/5 are from Burtin et al. (1986); mean ± 1 S.E. Discussion Present experiments show that when Astacus leptodactylus faces increased ionoregulatory demand, compensation for superimposed ABB disturbances can be made by ventilatory adjustments. This was demonstrated in two types of experiments: during decreases of [Na + ]; and [Cl~]v brought on by decreasing [NaCl] w, and during the premoult period where iono- and osmoregulatory problems were spontaneously present. When no such ionoregulatory problems existed, the compensation was always achieved by metabolic means (Figs 1-3). It seems that these problems governed the functioning of the ionic exchange system and precluded its role in metabolic compensation of extracellular ph. A consequence of this proposal is that the ionic exchange system should be able to perform metabolic compensation once these ionoregulatory problems are solved. Burtin et al. (1987) recently demonstrated this in A. leptodactylus kept in similar experimental conditions. Animals were exposed to a 24-h hypercapnic period 4 weeks after transfer from Twr to AWt 2/0-05 (in which TA = 2mequivl~ 1 and [NaCl] w = 0-05mmoll~ 1 ) and indeed compensation was metabolic. Moreover, evidence for a key role of [NaCl] w was reinforced as it was also shown that the efficiency of the compensation increased at higher [NaCl] w values. When [NaCl] w was decreased from 5 to O-lSmmoll" 1, [Na + ]- v and [Cl~]v decreased and there was a large metabolic acidosis. The acidosis persisted for 7 days (Fig. 3). Surprisingly, there was no ventilatory compensation, although this is quite feasible in view of the way that compensation was made for the same disturbance in premoult (Fig. 4). It is possible that this lack of compensation, instead of being only passive, could be causally related to a change required to optimize the ionoregulatory mechanisms. For example, one can hypothesize that ihe acidosis could stimulate either directly or indirectly (through a neurohormonal ecretion, see Mantel, 1985) the activity or synthesis of Na + /K + -ATPase. In this view, the 1-week delay required for ph and [Na + ]; to start to recover (Fig. 3)

8 418 B. BURTIN AND J.-C. MASSABUAU would be consistent with an enzymatic adaptation, although chronology of the latter remains controversial in the literature about crustaceans (Pequeux & Gilles, 1988). The idea of an enzymatic adaptation would be also consistent with our observation that the delay of recovery was shortened when [NaCl] w was decreased from 0-5 instead of Smmoll" 1. Indeed, this could be due to an already present higher level of Na + /K + -ATPase activity at [NaCl] w = 0-5 mmol T 1. A relationship between steady-state values of Na + /K + -ATPase activity and acclimation level of [NaCl] w has been reported in Eriocheir sinensis and Carcinus maenas (Pequeux & Chapelle, 1982) and Uca pugnax (Holliday, 1985). The present results further confirm the importance of water ionic composition, especially in the freshwater range, as stressed by Dejours et al. (1982) in determining extracellular ABB. But, in addition, the past history of the water breather with respect to environmental ionic composition can also play a major role. Fig. 5 is a composite figure drawn from fig. 2 in Burtin et al. (1986) and Fig. 3. It illustrates that crayfish in AWt2/0-15 can exhibit different ventilatory flow rates and ABB states depending on the composition of the reference water in which they had previously been acclimated (either AWr4/0-5 or AWt2/5). Indeed, in the first situation, animals must face a decrease of [NaCl] w accompanied by a hypercapnic acidosis (due to a decrease of TAw), whereas in the second situation they have only to deal with a decrease of [NaCl] w. In conclusion, Krogh (1938) proposed that in freshwater animals, Na + and Cl~ co 2 X AWr 4/0-5 AWt2/5 O AWt 2/0-15 PH Fig. 5. Composite figure drawn from fig. 2 in Burtin et al. (1986) and Fig. 3 in the present paper. It illustrates that in a given water composition (AWt 2/0-15) crayfish can exhibit two acid-base states and ventilatory flow rates depending on the composition of their previous environment (either AWr4/0-5 or AWt2/5). In one situation, animals were transferred directly from AWr 4/0-5 into AWt2/0-15 but in the other an exposure to AWt 2/5 was interposed. When animals were transferred from AWr 4/0-5 into AWt 2/5, the hypercapnic acidosis due to the decrease of TAw is compensated by metabolic means whereas it is compensated by a ventilatory adjustment in passing from AWr4/0-5 to AWt2/0-15. Transferring crayfish from AWt2/5 into AWt2/0-15 corresponds to a decrease of [NaCl] w alone; the metabolic acidosis is partially compensated by metabolic means.

9 Regulation of ph in crayfish 419 are taken from the ambient medium and exchanged against the end-products of metabolism, NH 4 + and HCO 3 ~. This was the basic idea of the coupling between respiration, i.e. acid-base regulation, and ionoregulation in water breathers (Maetz, 1971). Our results demonstrate the existence of another relationship between these two functions: the CO 2 exchange system can participate in ph regulation when the mechanisms of ionic exchanges involved in the metabolic compensation of extracellular ABB are already engaged in separate ionoregulatory problems. The authors thank Dr D. C. Jackson for help in preparing the English manuscript. BB was supported by the Ministere de la Recherche et de la Technologic References BURTIN, B., MASSABUAU, J. C. & DEJOURS, P. (1986). Ventilatory regulation of extracellular ph in crayfish exposed to changes in water titration alkalinity and NaCl concentration. Respir. Physiol. 65, BURTIN, B., MASSABUAU, J. C. & DEJOURS, P. (1987). Influence de la concentration de NaCl dans l'eau sur l'efficacite de la compensation metabolique d'une acidose hypercapnique chez l'ecrevisse. C.r. hebd. Seanc. Acad. ScL, Paris 305, CAMERON, J. N. (1971). Rapid method for determination of total carbon dioxide in small blood samples. /. appl. Physiol. 31, DEJOURS, P. (1973). Problems of control of breathing in fishes. In Comparative Physiology (ed. L. Bolis, K. Schmidt-Nielsen & S. H. P. Maddrell), pp Amsterdam: North- Holland. DEJQURS, P. & ARMAND, J. (1980). Hemolymph acid-base balance of the crayfish Astacus leptodactylus as a function of the oxygenation and the acid-base balance of the ambient water. Respir. Physiol. 41, DEJOURS, P., ARMAND, J. & BEEKENKAMP, H. (1982). The effect of ambient chloride concentration changes on branchial chloride-bicarbonate exchanges and hemolymph acid-base balance of crayfish. Respir. Physiol. 48, DEJOURS, P. & BEEKENKAMP, H. (1978). L'equilibre acide-base de Phemolymphe au cours de la mue chez l'ecrevisse. C.r. hebd. Seanc. Acad. Sci., Paris 286, DRACH, P. (1939). Mue et cycle d'intermue chez les crustaces decapodes. Annls Inst. Oceanogr. 19, GAILLARD, S. & MALAN, A. (1985). Intracellular ph-temperature relationships in a water breather, the crayfish. Molec. Physiol. 7, HEISLER, N. (1984). Acid-base regulation infishes.in Fish Physiology, vol. XA (ed. W. S. Hoar & D. J. Randall), pp London: Academic Press. HOLLIDAY, C. W. (1985). Salinity-induced changes in gill Na,K-ATPase activity in the mud fiddler crab, Uca pugnax. J. exp. Zool. 233, KROGH, A. (1938). The active absorption of ions in some freshwater animals. Z. vergl. Physiol. 25, MAETZ, J. (1971). Fish gills: mechanisms of salt transfer in fresh water and sea water. Phil. Trans. R. Soc. Ser. B 262, MANTEL, L. H. (1985). Neurohormonal integration of osmotic and ionic regulation. Am. Zool. 25, MANTEL, L. H. & FARMER, L. L. (1983). Osmotic and ionic regulation. In The Biology of Crustacea, vol. 5 (ed. L. H. Mantel), pp New York: Academic Press. MASSABUAU, J. C. & BURTIN, B. (1985). Ventilatory CO 2 reflex response in hypoxic crayfish k Astacus leptodactylus acclimated to 20 C. /. comp. Physiol. 156B, MASSABUAU, J. C, DEJOURS, P. & SAKAKIBARA, Y. (1984). Ventilatory CO 2 drive in the crayfish: influence of oxygen consumption level and water oxygenation. /. comp. Physiol. 154B,

10 420 B. BURTIN AND J.-C. MASSABUAU PEQUEUX, A. & CHAPELLE, S. (1982). (Na + + K + )-ATPase activity and phospholipids in two euryhaline crabs as related to changes in the environmental salinity. Mar. Biol. Letts 3, P QUEUX, A. & GILLES, R. (1988). NaCl transport in gills and related structures. Part I: Invertebrates. In Advances in Environmental and Comparative Physiology "NaCl Transport in Epithelia" (ed. R. Greger). Berlin, Heidelberg: Springer-Verlag (in press). REEVES, R. B. & RAHN, H. (1979). Patterns in vertebrate acid-base regulation. In Evolution of Respiratory Processes. A Comparative Approach (ed. S. C. Wood & C. Lenfant), pp New York: M. Dekker. SAKAKIBARA, Y., BURTIN, B. & MASSABUAU, J. C. (1987). Orcadian rhythm of extracellular ph in crayfish at different levels of oxygenation. Respir. Physiol. 69, SAUNDERS, R. L. (1962). The irrigation of the gill in fishes. II. Efficiency of oxygen uptake in relation to respiratory flow activity and concentrations of oxygen and carbon dioxide. Can. J. Zool. 40, STUMM, W. & MORGAN, J. J. (1981). Aquatic Chemistry. 780pp. New York: Wiley-Interscience. THOMAS, S. & POUPIN, J. (1985). A study of the effects of water carbonate alkalinity on some parameters of blood acid-base status in rainbow trout (Salmo gairdneri R.). /. comp. Physiol. 156, TRUCHOT, J. P. (1984). Water carbonate alkalinity as a determinant of hemolymph acid-base balance in the shore crab Carcinus maenas: a study at two different ambient Pco 2 and PQ 2 levels. J. comp. Physiol. 154B, TRUCHOT, J. P. (1987). Comparative Aspects of Extracellular Acid-Base Balance. Berlin, Heidelberg: Springer-Verlag. 248pp.