The Role Of CaCO₃ Dissolution as A Source of HCO₃⁻ for the Buffering of Hypercapnic Acidosis in Aquatic and Terrestrial Decapod Crustaceans

Both aquatic and terrestrial animals respond to a respiratory acidosis by increasing the levels of blood HCO₃⁻ to bring blood pH back toward the normal resting value (Cameron & Randall, 1972; Reeves, 1977). In water-breathing animals there is evidence that the mechanisms responsible for controlling blood HCO₃⁻ are branchial ion exchanges (Cl⁻ for HCO₃⁻ and/or Na⁺ for H⁺). This work has been done on ‘teleost fish (Maetz & Garcia-Romeu, 1964; Randall & Cameron, 1973; Cameron, 1976) and decapod crustaceans (Krogh, 1938; Truchot, 1975, 1979; Cameron, 1978) as well as other invertebrates. Air-breathing vertebrates are known to employ ventilatory control of ⁠, HCO₃⁻ reabsorption via the kidney and mobilization of CaCO₃ in bone (Davenport, 1974), for pH compensation.

The terrestrial decapod crustaceans are interesting because although they show a ventilatory response to increased CO₂ (Cameron & Mecklenburg, 1973; Cameron, 1975) their mechanism for long-term acid-base regulation is unknown. They lack the opportunity for branchial ion exchange available to aquatic crabs, and in aquatic crabs the antennal gland apparently does not play a role in acid-base balance (Cameron & Batterton, 1978; Truchot, 1975, 1979). Fully terrestrial crabs do attempt to compensate a hypercapnic respiratory acidosis by raising blood HCO₃⁻ (Cameron, 1981) but where the HCO₃⁻ originates is not known.

Since it has been shown that some other organisms, bivalve molluscs in particular, utilize CaCO₃ as a source of HCO₃⁻ to buffer pH disturbances in the blood (Dugal, 1939; Mangum, Henry & Simpson, 1979) it is reasonable to suggest that terrestrial crabs may do the same. We have investigated that possibility by following the concentrations of blood Ca in a terrestrial and an aquatic crab during hypercapnic acidosis.

Adult intermoult individuals of C. sapidus and G. lateralis were collected respectively by trawl from Lydia Ann Channel, Port Aransas, Texas, and by hand from the back dunes of Boca Chica, Texas. C. sapidus were held in 500 1 aerated, running seawater tanks at 900 mOsm/kg and 26 °C. G. lateralis were maintained in sand-filled tubs at 25 °C provided with dechlorinated tap water and fed on dry dog food, lettuce, and melon. Animals were kept for from 2 to 30 days before use.

Blue crabs weighing between 200 and 300 g were put in a flow-through chamber, the water either being normocapnic or being made hypercapnic (⁠ torr) by equilibration in a stripping column with a mixture of CO₂ in air provided by a Wösthoff gas-mixing pump. G. lateralis (80–100 g) were placed in individual plexiglas chambers into which either humidified air or 3 % CO₂ in humidified air was pumped. About 5 ml of 80 % seawater were placed in each chamber to prevent desiccation.

The remaining blood was allowed to clot in the syringe ; the clot was then disrupted and the blood centrifuged. Calcium concentrations were determined on an atomic absorption spectrophotometer (Perkin Elmer 303). The blood osmolality was measured using a vapour-pressure osmometer (Wescor Model 5130B).

When exposed to hypercapnia both species exhibited a typical respiratory acidosis and compensation. In Callinectes the blood pH dropped 0-3 unit over the first how and then rose approximately 0·15 unit to a partially compensated value of 7·61 by 8 h (Fig. 1). No further compensation occurred beyond 8 h. The partial restoration of normal (resting) blood pH was accomplished by a significant (P < 0·05, F test) elevation of blood HCO₃⁻ between 1 and 8 h of hypercapnia (Fig. 1). During both the uncompensated and the compensated phases of the acidosis, the blood Ca concentration did not change significantly from the resting value (Fig. 1 ; P > 0·05, F test).

G. lateralis underwent a less severe acidosis, with the pH dropping about 0·18 unit during the initial 4 h of hypercapnia (Fig. 1). However, compensation was slower and less complete than in C. sapidus. By 24 h the pH had risen only about 0·07 unit to a value of 7.30, and there was no further increase in HCO₃⁻ or pH between 24 and 48 h. The major point of interest is that the mean blood Ca concentration increased significantly during the period of compensation, and the Ca concentration increased for every individual crab (Fig. 1; P < 0·01, paired t test). From a resting value of 0·45 mm, the mean Ca concentration increased by about 3 mm at 24 h, and by almost 6 mm at 48 h. The relationship between base excess (BE) and the increase in Ca for individual crabs was, unfortunately, variable, so no good estimate of the stoichiometry could be made. Both BE and Ca increased in every individual, however, and the ratio of means was roughly 2:1 (BE to ΔCa; Table 1).

The blood osmolality did not vary more than 2 % from the resting mean of 976 ± 11 mOsm/kg in G. lateralis during the experiments, and the mean values at 24 h and 48 h were not significantly different from those at rest.

The responses of the blood acid-base parameters to hypercapnic acidosis in both C. sapidus and G. Lateralis were typical of crabs, and of water-breathers in general (see review by Reeves, 1977). The rapid and relatively large compensation seen in C. sapidus agrees well with published data on freshwater-adapted individuals of this species (Cameron, 1978); and the relatively slow and small compensation observed in G. lateralis agrees fairly well with data from Carcinus maenas in air (Truchot, 1975) and the terrestrial coconut crab Birgus latro (Cameron, 1981). The two species’ responses were quite different in one significant aspect, however ; there was no change at all in the Ca concentration in C. sapidus, and a significant rise in every individual of G. lateralis.

The data for the aquatic C. sapidus are consistent with the current theory, which holds that ionic exchanges between blood and water, usually via the gills, are responsible for the maintenance of pH in aquatic animals. For example, Cameron (1976, 1978) found changes in the relative rates of Na and Cl fluxes in response to hypercapnia that were consistent with (hypothesized) changes in Na⁺/H⁺ and Cl⁻/ HCO−3 exchanges that would regulate pH in response to hypercapnic acidosis. De Renzis & Maetz (1973) and others have reported a link between ionic exchange and blood pH in fish. Truchot (1975, 1979) has reported large branchial acid/base fluxes in response to various disturbances.

Ionic exchange in the gills is clearly not an option for G. lateralis, nor for any fully terrestrial crab. Neither is the antennal gland likely to provide the needed route for acid excretion, since studies done on antennal glands have shown either no significant acidification capability (Cameron & Batteron, 1978) or very low flow (Harris, 1977), or both. Since compensation does occur, as shown in Fig. 1 and Table 1, The most likely source appears to be the large CaCO₃ reservoir of the shell, which will yield Ca²⁺ and HCO₃⁻ upon acidification. The observed increases in Ca and HCO₃⁻ concentrations were independent of any change in blood osmolality, so the results were not a spurious consequence of dehydration.

The shell has been implicated in the buffering of pH disturbances in other organisms: Dugal (1939) reported a visible erosion of the shell, along with elevated mantle fluid Ca²⁺ and HCO₃⁻ concentrations during several days of anaerobiosis in the bivalve Venus mercenaria; and a similar rise in Ca²⁺ and HCO₃⁻ was seen during valve closure in the bivalve Rangia cuneata (Magnum et al. 1979). These water-breathing molluscs appear to utilize their shells only during periods of valve closure, when they are cut off from contact with the large ion pool in the ambient sea water.

Not surprisingly, terrestrial gastropod molluscs exhibit the same pattern of elevation of blood and HCO₃⁻ during hypercapnia. Burton (1976), in discussing the appearance of Ca²⁺ and HCO₃⁻ in Helix pomatia during hypercapnia, proposed that the ions could originate either from acidification of CaCO₃ in the shell, or from bound intracellular Ca²⁺. If the origin of the Ca²⁺ is the shell, then a 1:1 correspondence between BE and ACa²⁺ would be expected. If the mechanism is replacement of bound intracellular Ca²⁺ by H⁺ ions, the same relationship would be expected, so the present study does not allow a choice. Intracellular Ca²⁺ in crustaceans is low, however, and it would have to be virtually exhausted to account for the 3–6 mm rise seen in the blood of G. lateralis.

Regardless of the variability of the data for G. lateralis (Table 1), which is distressing, the argument for CaCO₃ dissolution for buffering purposes remains strong. The increase in blood Ca occurs only in the terrestrial species in which acid-base regulation via branchial ion exchange is not an option. The increase begins during the period of compensatory pH adjustment, 8–24 h (Fig. 1), and continues through 48 h, during which time blood pH remains stable despite an increase in the ⁠. The relative times of compensation in G. lateralis and C. sapidus indicate that a much slower process is occurring in the former : this is compatible to the idea of the slow dissolution of CaCO₃ by a weak acid (H₂CO₃) as compared to the more rapid ion exchange mechanisms of C. sapidus. We believe the most obvious source of CaCo₃ is the shell, although other sources such as CaCO₃ granules in digestive gland (Becker et al. 1974) cannot be ruled out.

How important the source of CaCO₃ is in nature has yet to be determined. In most cases an increase in ventilation is probably adequate to relieve internal acidosis brought about by exercise, for example (Smatresk et al. 1979). Only when the ventilatory control mechanism is insufficient to restore pH, such as in prolonged hypercapnia or exhaustion, or during temperature variation, might CaCO₃ become important in acid-base regulation.

Supported by NSE Grant No. PCM 77-24358 to James N. Cameron.