The effects of ambient pH on postmoult net fluxes of Ca, acidic/basic equivalents (H+, NH4+/OH, HCO3), Na and Cl, total body Ca, haemolymph pH and electrolyte status were assessed in the freshwater crayfish Procambarus clarkii (Girard). Variables were monitored for 5 days postmoult in acidic (pH5.2; H2SO4) or alkaline (pH9.2; KOH) artificial tap water (ATW) and compared with those in control (pH7.4) tap water. In control ATW there was an initial net influx of Ca (+2700 μmolkg−1 h−1) and titratable basic equivalents (+5000 μmolkg−1 h−1) that declined with time. Calcium uptake accounted for 40% of total body Ca (0.49mmol g−1); haemolymph Ca concentration remained constant. Haemolymph pH was initially relatively alkalotic (7.7) but recovered within 24h. A 20% haemolymph dilution by water uptake at ecdysis necessitated uptake of Cl and Na for the first 2–3 days postmoult (+1000 μmolkg−1 h). In acidic ATW, Ca and basic equivalent uptake were both 60% reduced during the first 3–4 days and total body Ca was reduced by 37%. Chloride and Na uptake and haemolymph [Cl] were decreased. In alkaline ATW, Ca and basic equivalent uptake were elevated by 30% for the first 2 days and haemolymph alkalosis was maintained. Sodium and Clbalance were unaffected. Thus, ambient pH affects Ca and basic equivalent fluxes associated with postmoult calcification. Regulation of Na and Cllevels is also impaired in acidic ATW.

Poorly buffered fresh waters are susceptible to pH alteration occurring either naturally or as a result of man’s activities. Atmospheric acids are formed when industrial gaseous emissions are precipitated as sulphuric, nitric and hydrochloric acids (Graedel and Crutzen, 1989). Acid rain has become a major environmental problem, especially below pH5.0 (Sprules, 1975). Environmental alkalization occurs when alkaline coal ash wastes rich in calcium oxide are discharged along river banks (Shaw, 1981; Peters et al. 1985). In certain athalassohaline lakes (major ionic constituents are MgSO4, Na2SO4 and NaHCO3/Na2CO3) the pH is naturally around 9.5 (Cooper et al. 1987).

The moulting cycle of crustaceans is critical for their survival and growth. Postmoult crustaceans are particularly susceptible to environmental water chemistry because they depend heavily on external electrolytes to calcify the new cuticle with calcium carbonate and to restore extracellular electrolyte and acid–base imbalances (Wheatly, 1993). According to Cameron (1985), calcification of the postmoult cuticle occurs via the overall reaction:
formula
Although modest amounts of Ca are stored between moults in freshwater crustaceans, the majority of the required Ca originates from the external water (Greenaway, 1985; Wheatly, 1990). Since HCO3 is required for calcification and protons are produced, the process is directly affected by ambient pH. Postmoult crayfish must also take up Na and Cto correct a haemolymph dilution caused by uptake of fresh water at ecdysis (Wheatly and Ignaszewski, 1990). For detailed reviews of electrolyte regulation in postmoult crustaceans, see Cameron and Wood (1985), Cameron (1985, 1989, 1990), Wheatly and Ignaszewski (1990) and Wheatly (1993).

Freshwater crustacean populations appear to be very sensitive to acidification (Leivestad et al. 1976; Havas et al. 1984) and alkalization (Shaw, 1981). Crayfish populations have been observed in naturally acid (pH5.8; Huner and Barr, 1991) and alkaline waters (pH10). While certain ecological aspects of pH sensitivity have been addressed in crayfish (France, 1981, 1987a,b; Berrill et al. 1985; Davies, 1989), the underlying physiological responses to extreme pH have only been examined during intermoult (Morgan and McMahon, 1982; Järvenpää et al. 1983; McMahon and Stuart, 1985, 1989; Hollett et al. 1986; Wood and Rogano, 1986; Mauro and Moore, 1987; Patterson and deFur, 1988). Postmoult crayfish are less resistant to low pH (LC50 3.5; Malley, 1980) than are intermoult crayfish (LC50 2.5; Morgan and McMahon, 1982). When maintained at pH5.0 (Malley, 1980), crayfish typically have poorly calcified exoskeletons as a result of reduced postmoult Ca uptake. There are no existing studies on the physiology of intermoult or postmoult crayfish exposed to basic water. In fish, mortality due to exposure to low and high pH is associated with failure of electrolyte and acid–base regulation (McDonald and Wood, 1981; McDonald, 1983; Wright and Wood, 1985; Wilkie and Wood, 1991).

The goal of the present study was to assess the effect of sublethal acidic (pH5.2) and alkaline (pH9.2) pH exposure (compared with control, pH7.4) on postmoult fluxes of Ca and associated ions, total body Ca and haemolymph pH and electrolyte status in the crayfish Procambarus clarkii.

Maintenance of crayfish

Crayfish, Procambarus clarkii (Girard), of both sexes were obtained from Louisiana State University Agricultural Centre. They were kept in groups of 4–8 in 30l aquaria under natural photoperiod at 21°C and fed three times a week with chopped liver or shrimp pellets (Hartz Mountain Corporation). The water was recycled and aerated through a bottom filter and replaced once a week with dechlorinated thermo-equilibrated tap water. Gainesville tap water has the following electrolyte composition (in mmol l−1):

Na, 0.55; K, 0.04; Ca, 0.58; Mg, 0.43; Cl, 0.73; titratable alkalinity, 1.8 and pH7.8. The crayfish were allowed to acclimate for at least 2 weeks before experiments started (Morgan and McMahon, 1982).

Experimental protocol

The experiments were carried out in artificial tap water (ATW) adapted from Greenaway (1970, 1974a,b) to resemble the electrolyte concentrations in local tap water. It contained (in mmol l−1): NaHCO3, 1.18; KHCO3, 0.03; CaSO4.2H2O, 0.57; MgCl2.6H2O, 0.52; and control (neutral) ATW had a pH of 7.4–7.6 and a titratable alkalinity of 1.1. For experimental solutions, the pH of ATW was modified by gradual titration with either 1mol l−1 H2SO4 to reach pH5.2 (acidic ATW) or 1mol l−1 KOH to reach pH9.2 (alkaline ATW). Water pH and total carbon dioxide content were measured.

Net electrolyte fluxes with experimental water

Experiments were performed on 30 juvenile postmoult crayfish (1.32±0.10g). Immediately after ecdysis had occurred naturally, animals were placed in up to 500ml (this was adapted according to size) of control, acidic or alkaline ATW (using 10 animals for each treatment). The ATW was continuously aerated and renewed every 24h. Whole-animal net fluxes of Ca, Na, Cl, titratable alkalinity and ammonia (NH3+NH4+) with the experimental water were monitored over 24h flux periods for a total of 5 days following ecdysis. A water sample was removed at the start (initial, i) and end (final, f) of each 24 h flux period and kept at 4°C until analyzed. Since no buffer was added to the ATW, pH did vary during the course of the flux period, but only by 0.3–0.5 units.

Total body calcium

In a second experimental series, 28 immediately postmoult crayfish (1.25±0.08g) were exposed to control, acidic or alkaline ATW following the procedure outlined above for a period of 5 days. They were then killed and frozen for subsequent determination of total body calcium.

Haemolymph pH and electrolyte levels

In a third experimental series, postmoult crayfish were sampled for haemolymph pH and electrolytes. Different series of crayfish were used for flux studies and haemolymph sampling because of the known disturbing effects of handling on whole-animal electrolyte fluxes. For this experiment, 24 crayfish of mean mass 12.4±0.5g were used, to avoid haemolymph depletion by repetitive sampling (total haemolymph volume around 0.28ml g−1 bodymass; Wood and Rogano, 1986). The volume of ATW was correspondingly increased to around 1.0–1.8l (depending upon individual animal size) so that the ion uptake mechanisms remained above their saturation concentrations as previously determined (see Wheatly and Ignaszewski, 1990).

On the day of ecdysis (day 0) a prebranchial haemolymph sample was rapidly collected with a 500 μl gas-tight Hamilton syringe from the base of a walking leg. Haemolymph pH was measured immediately after collection (200 μl) and the remaining sample (100 μl) was frozen for subsequent electrolyte analysis. Crayfish were then exposed to control or experimental pH for 5 days (N=8 in each treatment) and sampled daily.

Analytical procedures and calculations

Water pH was measured using a combination pH electrode and pH meter (Radiometer PHM 84) calibrated with Fisher buffers (pH7.0, 4.0 and 10.0). The water was measured using a Capni-Con analyzer (Cameron Instruments Co.) adapted for use with water samples as outlined by Cameron and Wood (1985). Water concentrations of Na and Ca were determined after appropriate dilution (1:40 in 0.1% CsCl2 for Na and 1:20 in 0.1% LaCl3 for Ca) using an atomic absorption spectrophotometer (Perkin Elmer 5000).

Chloride concentration was measured by coulometric titration (Radiometer CMT 10) using standards of 1 and 2mmol l−1. Titratable alkalinity was determined by titrating an air-equilibrated 5ml sample with 0.02mol l−1 HCl either to pH4.0 (ATW and alkaline ATW) or to pH3.5 (for acidic ATW; McDonald and Wood, 1981). Ammonia was measured using the phenolhypochlorite method of Solorzáno (1969). Net flux of each electrolyte X was calculated in μmolkg−1 h−1 as:
formula
where i and f refer to initial and final water concentration (mmol l−1), V is flux volume (l), t is elapsed time (h) and W is mass (kg). By convention, a negative value indicates net electrolyte loss and a positive value net uptake by the animal. The apparent net basic equivalent flux was calculated as the sum of the titratable alkalinity and ammonia fluxes, taking consideration of signs (discussed by McDonald and Wood, 1981). This variable does not distinguish between uptake of basic equivalents and excretion of acidic equivalents or vice versa. It is an operational term that includes flux of the following acidic or basic equivalents: H+/NH4+or OH/HCO (Wood and Rogano, 1986).

For total body calcium measurements, crayfish were thawed and weighed. Samples were then ashed for 12h at 450°C in a muffle furnace (Lindberg model 51894). The ashes were then digested in concentrated HCl, diluted so that Ca concentration could be read on the atomic absorption spectrophotometer, and back-calculated to total body Ca content (mmol g−1 wet mass).

Haemolymph pH was measured using an IL pH capillary electrode (20982) attached to an IL 213 blood gas analyzer, calibrated with precision buffers of pH6.840 and 7.384 and thermostatted to 22°C. Haemolymph electrolytes were measured using modified analytical procedures outlined above for water. Dilutions for haemolymph were 1:8000 for Na samples and 1:400 for Ca samples. Chloride concentration was measured on a 5 μl subsample against a standard of 150mmol l−1.

Statistical analyses

Data are expressed as means ± S.E.M. with number of observations in parentheses ( N). Effects within (days of exposure) and between (pH) treatments were compared using repeated-measures analysis of variance (ANOVA) (Ott, 1988). Whenever there was a significant interaction between days and pH exposure, a Student–Newman–Keuls’ test (Sokal and Rohlf, 1969) was used for multiple comparisons. Flux measurements were also compared with zero by means of a modified t-test enabling a data set to be compared with a single point (Bailey, 1981). For total body calcium content, means were compared between pH treatments using a one-way ANOVA and a multiple comparisons test was performed using a Student-Newman-Keuls’ test. Haemolymph electrolytes and pH were analyzed using a repeated-measures ANCOVA (Abacus Concepts, Super Anova). Again, if there was a significant interaction between days and pH exposure, a multiple comparisons test was performed using a Student–Newman–Keuls’ test. Statistical significance was accepted at P<0.05 throughout.

Control ATW (pH=7.4–7.6) had a of 1.13±0.01mmol l−1 (N=3). In acidic ATW (pH=5.2), decreased to 0.83±0.13mmol l−1 (N=3), whereas in alkaline ATW (pH=9.2) increased to 1.36±0.08mmol l−1 (N=3).

Net electrolyte fluxes with experimental water

Postmoult crayfish in control ATW showed an initial net uptake of Ca (around +2700 μmolkg−1 h−1; Fig. 1A), which decreased steadily with time to around +500 μmolkg−1 h−1 by day 5. This was accompanied by a net uptake (+5000 μmolkg−1h−1) of basic equivalents (Fig. 1B) that was 80% attributable to net titratable base uptake (Fig. 1C). The remaining 20% originated from net ammonia excretion (Fig. 1D). All three variables (basic equivalents, titratable base and ammonia) decreased linearly over the 5 days postmoult but remained significantly above zero.

Fig. 1.

Whole-animal net flux of (A) calcium, (B) basic equivalents, (C) titratable base and (D) ammonia for the first 5 days postmoult in Procambarus clarkii (mean mass 1.32±0.10g) at 21°C. Bars represent mean values±S.E.M. for three different pH treatments. Control ATW had a pH of 7.2–7.6 (N=10; mean mass 1.46±0.25g); experimental treatments were either pH5.0–5.2 (acidic ATW; N=10; mean mass 1.36±0.08g) or pH9.0–9.2 (alkaline ATW; N=10; mean mass 1.10±0.12g). Asterisks denote differences from the control value on the appropriate day (repeated-measures ANOVA, P<0.05). Dots represent values that are significantly different from zero. By convention, positive values indicate net uptake and negative values net output by the animal.

Fig. 1.

Whole-animal net flux of (A) calcium, (B) basic equivalents, (C) titratable base and (D) ammonia for the first 5 days postmoult in Procambarus clarkii (mean mass 1.32±0.10g) at 21°C. Bars represent mean values±S.E.M. for three different pH treatments. Control ATW had a pH of 7.2–7.6 (N=10; mean mass 1.46±0.25g); experimental treatments were either pH5.0–5.2 (acidic ATW; N=10; mean mass 1.36±0.08g) or pH9.0–9.2 (alkaline ATW; N=10; mean mass 1.10±0.12g). Asterisks denote differences from the control value on the appropriate day (repeated-measures ANOVA, P<0.05). Dots represent values that are significantly different from zero. By convention, positive values indicate net uptake and negative values net output by the animal.

In acidic ATW there was a 55–65% reduction in Ca net uptake (averaging+800 μmolkg−1h−1; Fig. 1A), mainly on the first 3 days postmoult (see statistical summary in Table 1). Net basic equivalent influx was similarly 70% inhibited on the first 4 days postmoult (Fig. 1B). The titratable base uptake was 75% lower for the first 3 days postmoult, with mean values around +1000 μmolkg−1 h−1 (Fig. 1C). Meanwhile, net ammonia efflux was reduced (−550 μmolkg−1h−1) on the first day (Fig. 1D). Under acid conditions, ammonia and titratable base flux contributed 33% and 67%, respectively, to the total basic equivalent flux.

Table 1.

Summary of statistical analysis (repeated-measures ANOVA) for electrolyte net fluxes with external water under three different pH treatments

Summary of statistical analysis (repeated-measures ANOVA) for electrolyte net fluxes with external water under three different pH treatments
Summary of statistical analysis (repeated-measures ANOVA) for electrolyte net fluxes with external water under three different pH treatments

In alkaline ATW, Ca net influx exhibited a 30% increase on the first 2 days postmoult (averaging +3550 μmolkg−1 h−1; Fig. 1A). Similarly, influxes of net basic equivalents and net titratable base were higher on the first 2 days postmoult (Fig. 1B,C, values averaging +6500 μmolkg−1 hv1) but net basic equivalent uptake was significantly lower than control on day 4. Ammonia net efflux was reduced on day 1 postmoult (Fig. 1D), and the flux measured was not significantly different from zero. In alkaline ATW, ammonia net efflux only contributed 5–10% to the net basic equivalent influx.

In control ATW, postmoult crayfish exhibited net uptake of Cl and Na (Fig. 2A,B), initially at rates of +1350 and +900 μmolkg−1 h−1 respectively. Within 3 days for Cl and 2 days for Na, influxes had dropped to rates that were not significantly different from zero, suggesting that ion balance had been re-established. In acidic ATW, the pattern of net Cl uptake over 5 days was not significantly different from the control pattern (Fig. 2A), and no significant differences were found between days. Net uptake of Na, however, was not significantly different from zero on any of the 5 days postmoult (Fig. 2B), unlike controls, which showed significant net uptake for the first 2 days postmoult. Net uptake of Cl was significantly higher than the control on day 1 in alkaline ATW (Fig. 2A). Alkaline pH had a significant effect on Na net uptake, which was reduced compared to the control (Fig. 2B). Starting on the second day, the net Na efflux was not significantly different from zero, whereas controls showed significant net uptake for 2 days.

Fig. 2.

Whole-animal net flux of (A) chloride and (B) sodium for the first 5 days postmoult in Procambarus clarkii. For additional details, consult legend to Fig. 1.

Fig. 2.

Whole-animal net flux of (A) chloride and (B) sodium for the first 5 days postmoult in Procambarus clarkii. For additional details, consult legend to Fig. 1.

The total flux of each electrolyte with the experimental water was summed over the entire 5 days of measurement (Table 2).

Table 2.

Total electrolyte flux (mmolkg 1) with the experimental water over 5 days in postmoult crayfish under three different pH treatments

Total electrolyte flux (mmolkg 1) with the experimental water over 5 days in postmoult crayfish under three different pH treatments
Total electrolyte flux (mmolkg 1) with the experimental water over 5 days in postmoult crayfish under three different pH treatments

Total body calcium

After 5 days in control ATW, total body Ca was 0.49mmol g−1 wetmass (Table 3). In acidic ATW, total body Ca was significantly lower than control values (0.31mmol g−1 wetmass), whereas it was unchanged in alkaline ATW.

Table 3.

Total body calcium content (mmol g 1) and total whole-animal Ca influx (mmolg 1 from Table 2) after 5 days postmoult at three different pH treatments

Total body calcium content (mmol g 1) and total whole-animal Ca influx (mmolg 1 from Table 2) after 5 days postmoult at three different pH treatments
Total body calcium content (mmol g 1) and total whole-animal Ca influx (mmolg 1 from Table 2) after 5 days postmoult at three different pH treatments

Haemolymph pH and electrolyte levels

Crayfish in control ATW had a haemolymph pH of 7.7 at ecdysis (Fig. 3A) that declined to 7.5 after 24h and remained at that level. An identical trend was observed upon transfer to acidic ATW (Fig. 3A; statistical summary in Table 4). On day 1, crayfish exposed to alkaline ATW (Fig. 3A) had a haemolymph pH of 7.75, which was significantly higher than control values. Haemolymph pH remained at that level for the next 2 days. On day 4, pH declined to 7.6, which was still significantly elevated compared with control values. By day 5, haemolymph pH was similar to control values.

Table 4.

Summary of statistical analysis (repeated-measures ANCOVA) for haemolymph electrolytes under three different pH treatments

Summary of statistical analysis (repeated-measures ANCOVA) for haemolymph electrolytes under three different pH treatments
Summary of statistical analysis (repeated-measures ANCOVA) for haemolymph electrolytes under three different pH treatments
Fig. 3.

Changes in haemolymph pH (A), calcium (B), chloride (C) and sodium (D) as a function of days postmoult under three different pH treatments. Control treatment represents pH7.4 exposure and experimental treatments were pH5.2 or pH9.2. Day 0 refers to ecdysis, when haemolymph was sampled in control pH prior to transfer of the crayfish to experimental pH. Bars represent mean values ± S.E.M., N=8. Asterisks indicate significant differences from control for corresponding days.

Fig. 3.

Changes in haemolymph pH (A), calcium (B), chloride (C) and sodium (D) as a function of days postmoult under three different pH treatments. Control treatment represents pH7.4 exposure and experimental treatments were pH5.2 or pH9.2. Day 0 refers to ecdysis, when haemolymph was sampled in control pH prior to transfer of the crayfish to experimental pH. Bars represent mean values ± S.E.M., N=8. Asterisks indicate significant differences from control for corresponding days.

Following ecdysis, crayfish in control ATW had a haemolymph Ca level of 9.6mmol l−1 Haemolymph Ca concentration did not change significantly over 5 days postmoult in crayfish in control, acidic or alkaline ATW (Fig. 3B). Following ecdysis, haemolymph Cl concentration was initially reduced to 128mmol l−1 (Fig. 3C) but subsequently recovered to 157mmol l−1 on day 1, reaching values around 168mmol l−1 after 5 days. Crayfish exposed to acidic ATW (Fig. 3C) had Cl levels of 116mmol l−1 at ecdysis. After 1 day, Cl levels had increased to 151mmol l−1, exhibiting the same trend as in control animals. On days 4 and 5, Cl levels exhibited a secondary drop compared with the control. Exposure to alkaline ATW (Fig. 3C) had no effect on postmoult Cl concentration. Immediately following ecdysis, Na concentration was 122mmol l−1 (Fig. 3D); this increased to 137mmol l−1 on day 1, with values levelling off around 146mmol l−1 on days 2–5. Exposure to acidic and alkaline ATW (Fig. 3D) had no effect on this trend.

Control (neutral) ATW

Postmoult mineralization in Procambarus clarkii in neutral ATW was characterized by a net uptake of Ca and basic equivalents (Fig. 1A,B) agreeing with previous studies (Wheatly and Ignaszewski, 1990). Comparable Ca net influxes have been reported for the crayfish Austropotamobius pallipes (Greenaway, 1974b) and Orconectes virilis (Malley, 1980). In sea water, blue crabs with net Ca and basic equivalent influxes as high as +6000 μmolkg−1 h−1and +12500 μmolkg−1 h−1,respectively, have been reported (Cameron, 1985; Cameron and Wood, 1985), possibly relating to increased availability of Ca and HCO3 in sea water as opposed to fresh water. In both cases, the stoichiometry between Ca and basic equivalent uptake rates is 1:2, as one might predict from the calcification equation. Greenaway (1974b) similarly reported that Ca influx rate dropped over time, although it remained significant for several weeks in 10g crayfish. A decrease in ammonia excretion with time (Fig. 1D) has been observed in other studies (Mangum et al. 1985a; Wheatly and Ignaszewski, 1990). Muscle atrophy during late premoult is believed to facilitate exuviation from constricting regions of the old exoskeleton (Mangum et al. 1985a) and may account for elevated ammonia excretion in the early postmoult period. Alternatively, it may be indirectly related to the exchange of counterions such as sodium that are taken up in the postmoult period (see below).

Postmoult crayfish also exhibited significant net uptake of Cl and Na (Fig. 2A,B) for 2–3 days to correct the haemolymph dilution created by uptake of fresh water at ecdysis. Net Cl influx was greater in magnitude, which agrees with another study (Wheatly and Ignaszewski, 1990). Unidirectional flux analysis using radiotracers (Wood and Rogano, 1986; Wheatly, 1989) has demonstrated that the crayfish integument has a greater permeability to Cl than to Na.

Allometric relationships derived for total body Ca of intermoult Austropotamobius (Greenaway, 1985) and Procambarus clarkii (Wheatly, 1990) predict values of 0.452 and 0.491mmol g−1, respectively, for a 1.25g crayfish. Perfect correspondence between these and the values measured in day 5 postmoult crayfish (Table 3) suggests that calcification was virtually completed. The net Ca influx integrated over 5 days postmoult in a different group of similarly sized crayfish (Table 3, values taken from Table 2) suggests that about 40% of total body Ca originated from the external medium. The remaining 60% was presumably stored in various body tissues (Greenaway, 1985).

Intermoult extracellular pH of Procambarus at 22°C is typically 7.5 (Wheatly and Henry, 1992). The alkalosis observed at ecdysis (Fig. 3A) is probably associated with elevated haemolymph [HCO3] resulting from premoult exoskeletal CaCO3 reabsorption as well as HCO3 influx from the medium. However, since haemolymph [HCO3] was not measured in the present study, one can only speculate that the alkalosis was of metabolic not respiratory origin. The alkalosis was compensated within 24h, agreeing with previous studies on crayfish (Dejours and Beekenkamp, 1978) and blue crabs (Mangum et al. 1985a,b; deFur et al. 1988).

In this study, the haemolymph data are not strictly comparable with the flux data since they were obtained from a separate series of crayfish that were an order of magnitude greater in mass. Separate studies in our laboratory on the allometry of postmoult calcification in neutral ATW (Wheatly et al. 1991) concluded that diffusional and active flux rates are both greater in smaller crayfish, commensurate with an increased surface area to volume ratio. Haemolymph data for crayfish ranging in mass from 3 to 30 g (Wheatly, 1993; M. G. Wheatly, unpublished observations; and the present study), however, showed no differences between circulating electrolyte levels in either intermoult crayfish or immediately after ecdysis, with the exception of Cl. Postmoult Cl values were initially lower in larger crayfish, suggesting that the values presently reported for 12g crayfish may have overestimated the changes expected in the group used for flux experiments.

The present study agrees with Wheatly (1993) that haemolymph [Ca] remains constant during the postmoult period (Fig. 3B) and at essentially intermoult levels (around 10mmol l−1, Morgan and McMahon, 1982), suggesting a delicate balance between Ca uptake and deposition into the new exoskeleton. The fact that extracellular Ca concentration remained constant in the face of dilution of other major circulating electrolytes suggests that total circulating Ca actually increased around ecdysis. Greenaway (1974a) confirmed that total extracellular Ca was elevated in postmoult Austropotamobius, primarily in the bound moiety; ionized calcium remained constant. Intermoult Cl and Na levels in Procambarus are typically around 190mmol l−1 (Morgan and McMahon, 1982; Wheatly, 1993). A 30% drop in both upon ecdysis (Fig. 3C,D) indicates a substantial haemodilution. Restoration of ion balance coincided with significant influx from the external medium (Fig. 2).

Acidic ATW

The fact that Ca and basic equivalent net uptake were both similarly reduced in acidic ATW (Fig. 1A,B) indicates that their uptake mechanisms are linked, as previously suggested (Cameron, 1985; Cameron and Wood, 1985; Wheatly and Ignaszewski, 1990). Malley (1980) similarly showed in postmoult Orconectes virilis that Ca influx was impaired in acid water and completely inhibited below pH4.0. Low pH combined with high aluminium concentration reduced Ca uptake further (Malley and Chang, 1985). Cameron (1985) demonstrated that Ca and basic equivalent uptake in postmoult blue crabs were both virtually eliminated in acid sea water. This effect could not be reproduced in high-CO2 sea water (which lowered pH by a similar amount), leading him to the conclusion that the response in acidified sea water was due to the lowering of ambient rather than an effect of pH per se. In the present study, ambient was approximately halved by acid titration, as was basic equivalent influx rate. In a related study (Zanotto and Wheatly, 1990) we repeated these measurements in decarbonated water and found that Ca and basic equivalent uptake were reduced in neutral decarbonated ATW compared with uptake in neutral non-decarbonated ATW. Flux rates were the same in neutral and acidic decarbonated ATW, confirming Cameron’s (1985) conclusions. Other studies have demonstrated that removing external HCO3 lowers Ca uptake in postmoult crayfish by 60% (Greenaway, 1974b) and virtually eliminates the basic equivalent uptake (M. G. Wheatly and A. T. Gannon, unpublished observations). The fact that Ca uptake decreases in all these studies suggests that the Ca uptake mechanism is HCO3-dependent.

Total body Ca was one-third reduced after 5 days in acidic ATW compared with control values (Table 3). Crayfish naturally exposed to acidified lakes have a decreased exoskeletal calcium content (Malley, 1980; Appelberg, 1985; France, 1987a). In either case this could result from reduced postmoult Ca uptake (as demonstrated above) and/or erosion of existing exoskeletal CaCO3 under acid conditions. Extracellular acidosis has been shown to induce exoskeletal dissolution in intermoult crustaceans (see review by Wheatly and Henry, 1992). In a separate experiment we observed a sizeable Ca efflux from isolated shed exuviae into acidic ATW (F. P. Zanotto and M. G. Wheatly, unpublished observations). Direct erosion of CaCO3 at the carapace/water interface could oppose branchial uptake, contributing to reduced net influx under acid conditions.

In acidic ATW, postmoult crayfish exhibited an identical haemolymph pH and Ca profile to those in neutral ATW (Fig. 3A,B). Two studies on acid exposure (H2SO4; pH4.0) of intermoult crayfish (Procambarus clarkii, Morgan and McMahon, 1982; Orconectes propinquus, Wood and Rogano, 1986) report acid–base and ion responses that are exactly counter to those observed in postmoult. A progressive metabolic acidosis, originating from H+ entry at the gills, was accompanied by an elevation in circulating [Ca] and net Ca efflux (Wood and Rogano, 1986), both originating from exoskeletal erosion. McMahon and Stuart (1989) reported that the carapace Ca content decreased significantly after 21 days of acid exposure.

The pronounced reduction in net basic equivalent uptake in acidic ATW (Fig. 1B) primarily reflected changes in net titratable base uptake (Fig. 1C). In addition, on the first day postmoult there was a significant reduction in ammonia excretion (Fig. 1D). Recent reviews indicate that ammonia excretion in aquatic animals can occur by ionic or non-ionic diffusion as well as by Na+/NH4+ exchange and NH4Cl+NaCl cotransport (Kormanik and Cameron, 1981; Evans and Cameron, 1986). Based on the analysis of Wright and Wood (1985), acid exposure should raise the diffusional gradients for both NH3 and NH4+, thereby increasing ammonia excretion. Since a reduction in Na influx accompanied the decrease in ammonia excretion (Fig. 2B), Na+/NH4+ exchange may have been inhibited. In acid-exposed intermoult crayfish, ammonia excretion is either unaffected (Wood and Rogano, 1986) or increases (Mauro and Moore, 1987).

Haemolymph Cl levels were significantly reduced on days 4 and 5 (Fig. 3C), when whole-animal uptake had essentially ceased. This would suggest that extracellular Cl was being sequestered in a body fluid compartment other than the haemolymph. Haemolymph pH increased on the same days that [Cl] dropped, implicating the operation of a Cl/HCO3 exchanger. Acid-exposed intermoult crayfish similarly showed a reduction in circulating [Cl] (Appelberg, 1985; Wood and Rogano, 1986), although in this case it was attributed to net efflux due to reduced unidirectional influx. Other studies (Morgan and McMahon, 1982; Hollett et al. 1986) report no effect of acid exposure on haemolymph [Cl] in intermoult crayfish.

Net sodium uptake was reduced in acid ATW (Fig. 2B and Table 2). Shaw (1960) reported that low external pH reduced unidirectional Na influx in intermoult crayfish while passive efflux remained unchanged. A net efflux of Na was observed in acid-exposed intermoult crayfish (Wood and Rogano, 1986) caused initially by a reduction in the unidirectional influx (competition between H+ and Na for a common carrier), followed by an increase in diffusive efflux. Recent experiments (M. G. Wheatly and A. T. Gannon, unpublished observations) indicate that postmoult Na uptake may be linked to Ca uptake, since removal of external Na reduces Ca uptake by 50%.

In the present study, Na fluxes were not significantly different from zero during acid exposure, explaining why haemolymph Na levels remained constant (Fig. 3D) and agreeing with studies on intermoult Procambarus (Morgan and McMahon, 1982) and Cambarus robustus (Hollett et al. 1986). In acid-exposed Orconectes (Wood and Rogano, 1986), however, a net Na efflux was correlated with reduced circulating levels.

The differences between physiological responses to acute acid in postmoult and intermoult crayfish may be attributable to the degree of acidification (generally lower in intermoult studies, pH4 versus pH5.2) as well as to differences in water hardness and the species under study. In fish, ambient Ca concentration affects acid toxicity (McDonald, 1983). In hard water, the major physiological effect is a metabolic acidosis that originates from Na loss in excess of Cl; in soft water, H+influx does not occur but equimolar losses of Na and Cl result in rapid mortality due essentially to ion failure. Havas et al. (1984) similarly showed that low pH had more debilitating effects on Na regulation in Daphnia in soft water than in hard water. The present study was conducted in water of intermediate hardness (Ca=0.5mmol l−1), as was the study by Morgan and McMahon (1982; 1.1mmol l−1), which may explain why the electrolyte disturbances were less pronounced. Studies conducted by Malley (1980) and Wood and Rogano (1986), however, were both in soft water (0.7 and 0.1mmol l−1respectively).

Laboratory and field studies indicate that certain crayfish genera, such as Procambarus and Cambarus, are far more resistant to low pH than other genera, such as Orconectes (Berrill et al. 1985; Hollett et al. 1986; Davies, 1989; McMahon and Stuart, 1989) because of their evolutionary history. The genus Procambarus is commonly found in swampy areas and therefore may have acquired resistance to acid conditions. The evolution of orconectoid and cambaroid lines from a Procambarus-like ancestor has been under different conditions of water chemistry. Cambarus originated in mountain streams under soft water conditions and so may have become preadapted to withstanding low pH stress. Differing acid sensitivity may also be related to the seasonal timing of reproductive and moulting events.

Recent research (McMahon and Stuart, 1989) has shown that the associated anion is also an important factor in the physiological responses to acid exposure. Both acute and chronic (7–60 days) exposure to nitric acid in Procambarus produce less pronounced disturbances in ion/acid–base status than does H2SO4. The initial haemolymph acidosis is less severe and more rapidly compensated, and exoskeletal CaCO3 mobilization does not occur.

Alkaline ATW

The increased basic equivalent uptake observed in alkaline ATW (Fig. 1B) may be attributed to higher ambient . Cameron (1985) showed that increased ambient HCO3 in sea water caused an increase in HCO3- influx into postmoult blue crabs, although a corresponding change in Ca influx was not observed. In the present study, net Ca uptake increased initially in proportion to the titratable base uptake (Table 2 and Fig. 1A), suggesting again that the uptake mechanisms are linked and possibly that the increase in Ca uptake is secondary to the HCO3 uptake. Over the entire 5-day period, total Ca uptake was only 30% increased over control levels (Table 2), which was insufficient to be reflected in the total body Ca content (Table 3) or haemolymph [Ca] (Fig. 3B).

There was a significant decrease in net ammonia efflux on day 1 postmoult (Fig. 1D). A corresponding reduction in net Na influx was not observed in this case (Fig. 2B). Since high external pH would increase the external and reduce external [NH4+] (Wright and Wood, 1985), the observed reduction in ammonia excretion could be interpreted as evidence that there is an NH3 diffusional component to the net ammonia excretion, at least during the initial postmoult period when the cuticle is soft. Subsequently (day 2–5), ammonia excretion recovered to control levels (Fig. 1D). Without resolving the net Na flux (Fig. 2B) into its unidirectional components, it is difficult to say whether the ammonia excretion occurring at this time was via Na+/NH4+ exchange or NH4+ diffusion. However, recent experiments (M. G. Wheatly and A. T. Gannon, unpublished observations) indicate that postmoult ammonia excretion persists in Na+-free medium, suggesting the predominant role of diffusional routes in ammonia excretion. Collectively, the acid and alkaline experiments suggest that ammonia excretion in the postmoult crayfish occurs by a combination of Na+/NH4+ exchange and diffusion of NH3 or NH4+.

Sodium and Cl regulation were generally unaffected by exposure to alkaline water. Net Cl uptake was initially elevated (Fig. 2A) and both Cl and Na balances were re-established a day earlier than in control conditions (Fig. 2A,B). The latter effect may be related to the increased rate of calcification (Fig. 1). However, when summed over the 5-day postmoult period (Table 2), net fluxes of Na and Cl-were similar to controls. Likewise, circulating [Cl] and [Na] showed similar postmoult trends to those control crayfish (Fig. 3C,D). Even in fish, plasma ion disturbances are less pronounced in alkaline water than in acid water.

The haemolymph alkalosis following ecdysis persisted for several days postmoult in alkaline ATW, whereas it had recovered within 24h in control and acid-exposed crayfish (Fig. 3A). Without measuring additional indices of acid–base status, one can only speculate on the origin of this. The maintained alkalosis may be attributed to increased basic equivalent uptake from water (Fig. 1C). Cameron (1985) reported that haemolymph pH increased in postmoult blue crabs experiencing increased basic equivalent uptake in high-HCO3 water.

To conclude, this study has outlined the effect of acidic or alkaline exposure on electrolyte regulation during the postmoult period in freshwater crayfish. By affecting the ability to calcify, environmental pH can potentially affect crustacean population dynamics in the wild. To elucidate how electrolyte-transporting mechanisms are affected by ambient pH, it will be necessary to measure unidirectional ion fluxes, which is the focus of our continued studies. The uptake mechanisms for Na and Cl in freshwater crustaceans have been reviewed by Ehrenfeld (1974) and Mantel and Farmer (1983). The uptake mechanism for Ca has been modelled for freshwater teleost fish (Fenwick, 1978; Flik et al. 1983), although there have been some preliminary studies using crustacean models (Roer, 1980).

This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brazil) and Sigma-Xi Grant 97921 to F.P.Z. and by National Science Foundation grants DCB 8415373 and 8916412 to M.G.W. We are indebted to Drs D. H. Evans and L. R. McEdward for helpful discussions during the early stages of this work, to Drs B. R. McMahon and C. M. Wood for valuable comments on the manuscript, to Evan Chipouras and Dr C. Martínez del Rio for statistical help and to Grace Kiltie for preparing the manuscript.

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