Electrolyte and Gas Exchange During the Moulting Cycle of a Freshwater Crayfish

While Ca²⁺, HCO₃⁻ and H⁺ fluxes are important in mineral reabsorption/ deposition, moulting will also indirectly affect the regulation of other ions, more so in freshwater than in marine species. The increase in size during ecdysis is accomplished by uptake of external water. For freshwater species such as the crayfish, which normally hyperregulate extracellular osmolality and ion concentrations, this will cause haemolymph dilution. Thus during postmoult, in addition to accumulating CaCO₃, circulating levels of major electrolytes such as Na⁺ and CP must be restored. In fact, there is preliminary evidence of coupled Na⁺/Ca²⁺ exchange across mammalian basolateral membranes (Taylor and Windhager, 1979) and in an isolated cuticular crab hypodermis (Roer, 1980); however, it has not yet been demonstrated in branchial Ca²⁺ transport in fish (Flik et al. 1985). Another feature of moulting that could potentially affect ion balance is the increase in cuticular permeability around the time of ecdysis, which has been demonstrated for respiratory gases (Mangum et al. 1985) and would be expected to increase diffusional ion efflux.

Calcium balance at various stages in the moulting cycle has been documented in the freshwater crayfish by McWhinnie (1962) and Greenaway (1974a,b,c). Greenaway (1974c) alluded to the necessity for Na⁺ and Cl⁻ uptake during postmoult. He also hypothesized that the HCO₃⁻ for calcification originated both externally and from metabolic CO₂, since removal of external HCO₃⁻ reduced but did not eliminate Ca²⁺ uptake. This was confirmed by Cameron and Wood (1985), who demonstrated apparent H⁺ excretion in postmoult blue crabs which matched a net CO₂ deficit (negative CO₂ excretion). CO₂ excretion has not been measured during the moulting cycle in the freshwater crayfish, although Scudamore (1947) measured variations in the rate of O₂ uptake.

The purpose of the present study was to correlate net Ca²⁺ fluxes throughout the moulting cycle of the freshwater crayfish with fluxes of acidic/basic equivalents, major haemolymph electrolytes and respiratory gases.

Measurements were made on 32 juvenile crayfish (mean mass±s.E.M., 1·58±0·18g) Procambarus clarkii, which were obtained from Louisiana State University Agricultural Centre, Baton Rouge. In Gainesville they were kept 10 to a 30–1 aquarium under a 12 h light: 12 h dark cycle at 21 °C. The water was recycled through a bottom filter and was replaced every 3 days with dechlorinated thermoequilibrated tapwater. Local tapwater had the following ionic composition (in mmolI⁻¹): Na⁺, 0·55; K⁺, 0·04; Ca²⁺, 0·58; Mg²⁴,0·43; Cl⁻, 0·73; phosphate, 0·003; titration alkalinity 1·80; pH7·8.

Under these laboratory conditions, this size class moulted naturally approximately every 21 days. Whole-body net electrolyte fluxes and gas exchange rates were measured as a function of time before (t= —7, –3 or —1 days; premoult) or after (r=l–6 days; postmoult) ecdysis (t=0 days) and compared with intermoult values (more than 10 days prior to ecdysis). Premoult was determined by viewing the degree of development of new setae on the uropods and telson (Stevenson, 1985). [For comparison with Drach’s (1939) moulting stages, t= –3 and —1 days in this study correspond to stage DI-3, 1= + l day to stage A, t=+2 to +4 days to stage B and t=+6 days to stage C; however, these stages tend to be somewhat arbitrary.]

To determine net whole-body electrolyte fluxes, appropriately staged crayfish were placed individually in experimental chambers containing a fixed volume of tap water. Chambers were individually aerated and visually shielded. Crayfish remained in the flux water for a total of 24 h and electrolyte concentrations were determined in water samples removed at the start and end of this period. Fluxes were initially determined on 12 crayfish whose mean mass was 1·66±0·18g. (Mass increased by 7·2±2·1 % after ecdysis in this size class.)

Intermoult flux rates of all ions dictated a flux volume of 50 ml. However, it became apparent after these initial experiments that postmoult Ca²⁺ uptake was potentially limited in such a small volume, since ambient concentration fell below the saturation level for the uptake mechanism (Greenaway, 1974c). Rather than reduce the length of the flux period, thereby increasing handling stress, we increased flux volume to 500 ml in a second group of postmoult crayfish (mean mass 1·74±0·20g, N=8) and remeasured Ca²⁺ and acidic equivalent fluxes. In a third series we remeasured postmoult fluxes of other ions (Na⁺, Cl⁻, etc.) in a volume of 150 ml to determine whether these were affected by external Ca²⁺ or flux volume. Since no effect could be demonstrated we have reported the original postmoult data for these ions.

Whole-animal rates of O₂ uptake and CO₂ excretion were determined by continuous-flow respirometry as outlined in Wheatly (1989a) on 12 crayfish of mean mass 1·71±0·14g. (In this group mass increased by 9·5±1·4% after ecdysis.) Respirometers (volume 10ml) were manufactured from 20ml disposable syringes. These were fed with air-equilibrated water from a Gilson minipulse 2 pump at a constant flow rate of 1ml min⁻¹. Incurrent (inc) and excurrent (exc) flows were sampled for O₂ tension and total CO₂ content (⁠⁠). Crayfish were placed in the experimental apparatus at least 6h before sampling to allow the animal to settle and the respirometer to equilibrate. Five successive pairs of incurrent and excurrent samples were then removed over a period of 3 h to compute a mean value. All respirometers were tested for diffusive gas entry and adequate mixing.

Water total ion concentrations of Na⁺, K⁺, Ca²⁺ and Mg²⁺ were measured using an atomic absorption spectrophotometer (Perkin Elmer model 5000) and chloride ion concentration by coulometric titration (Radiometer CMT10). Sulphate concentration was determined by the turbidometric method of Jackson and McCandless (1978) and phosphate concentration by the phosphomolybdate method of Atkinson et al. (1973). Titration alkalinity was determined by titrating an air-equilibrated 10 ml sample to pH 4 with 0·02 mol l⁻¹ HC1 (McDonald and Wood, 1981) and ammonia (NH₃+NH₄⁺) using the phenolhypochlorite method (Solorzáno, 1969).

Water was measured using a thermoequilibrated O2 electrode (IL 20984) connected to an IL213 blood gas analyser. Flow rate through the respirometer was adjusted so that the drop in never exceeded 4kPa. was measured using the Capni-Con (Cameron Instruments Co.) adapted for use with water samples, as outlined by the manufacturer and Cameron and Wood (1985).

Data are expressed throughout as mean±s.E.M. (number of observations). Flux measurements were compared with zero by means of a modified t-test enabling a data set to be compared with a single point (Bailey, 1981). Statistical differences among treatment means were determined by one-way analysis of variance (dependent variablextime). When a significant F ratio was obtained, multiple comparisons were performed using Fisher’s least significant difference (Ott, 1988). Statistical significance was accepted at P < 0·05.

Electrolyte fluxes and gas exchange rates are both typically expressed per unit wet mass. Comparing values during the moulting cycle presents a unique problem since mass does not remain constant in individuals. Water loading at ecdysis results in increased mass which does not necessarily reflect an increase in metabolizing tissue. To give some idea of the potential magnitude of this problem, female blue crabs (Callinectes sapidus) undergo a 47% mass increase as they enter their terminal moult (Lewis and Haefner, 1976). Solutions commonly employed to circumvent this problem are to report individual whole-animal rates (Scudamore, 1947) or to correct for dry mass (Lewis and Haefner, 1976; Mangum et al. 1985). Whole-animal dry mass is not a good index to use since premoults will have a greater amount of inert material, some of which is shed at the moult. For the size of crayfish used in the present study, the mean increase in mass after ecdysis was only 8·4%, which is within the S.E.M. for the range of mass for which data are reported. Therefore, we feel justified in expressing mass-specific data using the mass at the time measurements were obtained.

The small net efflux of Ca²⁺ in intermoult crayfish (Fig. 1) was insignificantly different from zero (P>0·05), suggesting that they were in Ca²⁺ balance. This continued up until around 4 days premoult when a significant (P=0.003) net Ca²⁺ efflux of around —800μmolkg⁻¹ h⁻¹ was measured. Following ecdysis, this changed vector to a substantial (P < 0·001) net influx initially approaching +2500μmol kg⁻¹ h⁻¹. In most crayfish, uptake commenced after ecdysis; however, in some it was measured prior to shedding, explaining the large error bar on day t= — 1. As postmoult progressed, the influx rate decreased. From day 3 onwards, influx rates had dropped significantly (P < 0·00l) below day 1 postmoult rates; however, they remained significantly (P < 0·001) above intermoult rates for a further 2–3 days.

Intermoult crayfish exhibited a significant (Pμ0·00l) net apparent H⁺ output (or base uptake) of around —300μmolkg⁻¹ h⁻¹ (Fig. 1), consisting primarily of titratable acidic equivalents with only 3 % attributable to ammonia efflux. Around 3–4 days before ecdysis there was a significant (P < l) net H⁺ influx (or base efflux) approaching +2000 μmol kg⁻¹ h⁻¹, which persisted in all crayfish until shedding occurred. The flux then promptly changed vector to a significant (P < 0·00l) H⁺ efflux at initial rates exceeding —4000μmolkg⁻¹ h⁻¹. These rates were significantly reduced by day 3 (P < 0·003) although they continued to remain above intermoult levels for a further 2 days (P < 0·044). By the end of day 6, intermoult fluxes had been re-established. Identical trends were observed for titratable acidity fluxes. Ammonia excretion was significant (P < 0.05) in intermoult crayfish (Fig. 1) and did not change significantly during the premoult stages (P>0.05). However, during immediate postmoult there was a significant increase from around –120 to -500μmol kg⁻¹ h⁻¹. After 2 days postmoult, the rates had fallen significantly (P < 0·02) but remained above intermoult values for an additional 2 days (P < 0·00l). Six days after the moult, control (intermoult) levels of ammonia excretion had been restored (P=0·189).

The small net influxes of Na⁺ and Cl⁻ in intermoult crayfish (Fig. 2) were both insignificantly different from zero (P < 0·05), again suggesting ion balance. This persisted throughout the premoult phase, although there was less uniformity in the flux rates, especially in the day immediately preceding ecdysis. Individuals that had begun Ca²⁺ uptake prior to ecdysis also displayed net influx of both Na⁺ and Cl⁻ (around +500μmolkg⁻¹ h⁻¹), whereas those crayfish that continued to excrete Ca²⁺ immediately prior to shedding also exhibited net efflux of Na⁺ and Cl⁻. Immediately following ecdysis there was a significant net influx of both ions (P < 0·00l), and the influx remained elevated above control for an additional 2 days. While Na⁺ influx remained at uniformly high levels (P>0·21), Cl⁻ influx decreased significantly on the second day (P>0·0l), remaining at that level for the third day (P=0·46). In absolute terms, postmoult Cl⁻ influx rates were virtually double those of Na⁺ (+833 and +434μmol kg⁻¹ h⁻¹, respectively). After 3 days postmoult, Na⁺ and Cl⁻ balance was re-established.

Intermoult crayfish were similarly in K⁺ balance (Fig. 2) until 3–4 days preceding ecdysis, when a significant (P < 0·00l) efflux at rates of around —70μmol kg⁻¹ h⁻¹ was measured until ecdysis. Immediately after ecdysis, there was a significant net uptake (+14μmolkg⁻¹ h⁻¹) for 1 day (P < 0·04). Thereafter, K⁺ balance was restored.

Intermoult crayfish excreted significant (P < 0·0l) amounts of Mg²⁺ (—21·4±5·9/zmolkg⁻¹ h⁻¹) and sulphate (—75·4±15·6μmolkg⁻¹ h⁻¹). Multiple comparisons failed to reveal any significant changes in either throughout the moulting cycle. Phosphate excretion (Fig. 3) was negligible (P < 0·10) in inter- and premoult crayfish. During postmoult, there was a significant (P < 0·005) phosphate excretion for the first two days.

Intermoult crayfish had a mean of 46 μmol kg⁻¹ min⁻¹ and a corresponding Of 57μmolkg⁻¹ min⁻¹, producing an R of 1·27 (Fig. 4). Analysis of variance revealed significant changes in all three during the moulting cycle. was unchanged until 2 days prior to ecdysis, when it increased to around 80μmol kg⁻¹ min⁻¹, remaining elevated (P < 0·008) throughout the shedding process and into the first day postmoult, when it returned to intermoult levels. After 8 days postmoult, the values were significantly (P < 0·02) below those of the previous intermoult.

similarly exhibited a doubling (P < 0·001) of intermoult values in the immediate premoult period. During the entire postmoult period, was significantly (P < 0·034) depressed below intermoult values. The combined effect of these changes was that R was significantly (P < 0·011) lowered during the first 4 days of postmoult, although it had recovered by 8 days.

This study has shown that the net fluxes of several major body electrolytes vary as a function of moulting stage in the freshwater crayfish. As crayfish approached premoult they switched from Ca²⁺ balance to net efflux (Fig. 1) at identical rates to those reported by Greenaway (1974b) in the crayfish Austropotamobius pallipes. In that study Greenaway calculated that 83 % of total body Ca²⁺ was lost at ecdysis; of this 56% was in the exuviae and 27% in soluble form. Thus Ca²⁺ storage in haemolymph and gastroliths (Travis, 1960, 1963) would appear to be relatively unimportant (17 % of total Ca²⁺) compared with postmoult uptake from external sources.

In previous whole-animal studies (crayfish, Greenaway, 1974c; blue crab, Cameron, 1985,1989) Ca²⁺ influx prior to shedding was never reported. However, Henry and Kormanik (1985) did report premoult Ca²⁺ uptake in isolated crab cuticle and Porcella et al, (1969) have reported that this occurred in Daphnia. Postmoult Ca²⁺ uptake rates were of the same order of magnitude as reported for crayfish in Greenaway (1975c). However, calcification was accomplished more rapidly in the present study (5 as opposed to 10 days) since the crayfish were smaller (2 as opposed to 10 g) and were maintained at a higher ambient temperature (21 compared to 10°C). Based on the existing chemical gradient, Ca²⁺ influx into the crayfish is necessarily by active uptake (Greenaway, 1985) and is elicited literally within minutes of shedding (Greenaway, 1974c). The uptake mechanism has not been identified but probably involves a Ca²⁺-ATPase, if it is analogous with Ca²⁺ uptake across freshwater fish gills (Fenwick, 1978, modelled by Flik et al. 1985). By comparison, postmoult influx into the marine crab Callinectes (≃200 g wet mass) was of the order of +8000 μmol kg⁻¹ h⁻¹ (Cameron, 1985) and did not commence until 12–24 h after ecdysis. Cameron (1989) has recently shown that this occurs by passive diffusion not active uptake; so the two processes do not appear to be analogous.

The apparent H⁺ fluxes (Fig. 1) mirrored the net Ca²⁺ fluxes, suggesting that the transport mechanisms are linked. A small intermoult efflux of H⁺ (alternatively HCO₃⁻ uptake) became a sizeable H⁺ uptake (or HCO₃⁻ output) immediately prior to ecdysis. Resorption of cuticular CaCO₃ from the old skeleton prior to ecdysis should produce Ca²⁺ and basic equivalents in the ratio of 1:2 (Cameron, 1985), which is the observed ratio. While it is tempting to conclude that the acid-base flux is a base efflux (probably HCO₃⁻), the measurement technique does not differentiate between acid influx and base efflux or identify the ion. In the day immediately preceding ecdysis, the Ca²⁺ and acidic equivalent fluxes were not strongly correlated. All crayfish exhibited an influx of acidic equivalents even though Ca²⁺ uptake had commenced in certain individuals. During postmoult the observed fluxes were in the expected direction (i.e. Ca²⁺ uptake; H⁺ output/HCO₃⁻ uptake) and similarly obeyed the stoichiometry of the calcification equation given in the Introduction. The fluxes also followed a similar time course, accomplishing calcification in approximately 5–6 days. The acid-base flux was attributable mainly to the titratable component; ammonia contributed little to the net flux. The postmoult increase in ammonia efflux may have been due to an increase in cuticular permeability or increased protein metabolism.

Intermoult crayfish exhibited a small net uptake of Na⁺ and Cl⁻ (Fig. 2), as demonstrated in previous studies (Shaw, 1964; Ehrenfeld, 1974; Wheatly, 1989b); during most of premoult they remained in ion balance with respect to both ions. Net fluxes of Na⁺ and Cl⁻ were variable on the day preceding ecdysis, yielding zero mean values. Uptake of Na⁺ and Cl⁻ occurred in those individuals in which Ca²⁺ uptake had commenced (see above). Wherever Ca²⁺ efflux persisted, Na⁺ and Cl⁻ net effluxes were also measured. This is strong evidence that part of whole-animal Ca²⁺ uptake is linked to uptake of Na⁺ and Cl⁻. This is in accordance with the view that Na⁺ and Ca²⁺ fluxes across epithelial cells are related by a Na⁺/Ca²⁺ exchanger (see review by Taylor and Windhager, 1979). Along these lines, Roer (1980) demonstrated that Ca²⁺ transport across an isolated crab cuticular hypodermis was Na⁺-dependent.

Following ecdysis, there were large net influxes of both Na⁺ and Cl⁻, presumably to correct the haemodilution resulting from water uptake. Ion balance was restored within 3 days while calcification was still in progress. Although these fluxes were numerically only half the net Ca²⁺ influx rate, they are far larger than any recorded in response to acid-base disequilibria in the literature (Wood and Rogano, 1986; Wheatly, 1989b). Furthermore, assuming that unidirectional Na⁺ efflux remained at intermoult values (—300 μmol kg⁻¹ h⁻¹ in the crayfish Pacifastacus; Wheatly, 1989b), unidirectional influx can be estimated as + 1000 molkg⁻¹ h⁻¹, which is a substantial rate. Postmoult net Cl⁻ fluxes were higher than Na⁺ fluxes, possibly reflecting greater diffusional permeability. The evidence from unidirectional flux studies on other crayfish species (Orconectes, Wood and Rogano, 1986; Pacifastacus, Wheatly, 1989b), indicates that the integument is more permeable to Cl⁻ than to Na⁺. Again, assuming a control unidirectional Cl⁻ efflux of — 500μmolkg⁻¹ h⁻¹, postmoult Cl⁻ influx rate can be estimated as +1300 μmol kg⁻¹ h⁻¹. In the likely event that diffusional efflux increases at ecdysis, unidirectional influxes of both ions would be even greater than predicted.

The switch from K⁺ balance to a sizeable net efflux (Fig. 2) in the 3 days prior to ecdysis deserves explanation. The extracellular fluid compartment in the carapace selectively concentrates electrolytes, including K⁺ (M. G. Wheatly, unpublished observations). Little is known of the carapace fluid dynamics during the moult but it is likely that fluid is reabsorbed as the old skeleton is degraded. In addition, atrophy of various somatic muscle cells (Skinner, 1985) could contribute to high circulating K⁺ levels which would result in increased diffusional loss. The initial postmoult uptake is insufficient to make up for the premoult loss, from which one must conclude that K⁺ is obtained over the long term in the diet.

The significant postmoult phosphate excretion (Fig. 3) may have been attributable to enhanced diffusional permeability before the new exoskeleton had hardened. Alternatively it may have resulted from increased urinary flow. While this has never been measured in freshly moulted decapods, the increase in hydrostatic pressure at ecdysis should increase filtration pressure (Mykles, 1980; deFur et al. 1985) and thereby urine flow. Because of large variability between animals, similar trends could not be demonstrated for Mg²⁴⁻ and sulphate, which are also prominent components of both haemolymph and urine (Wheatly and Toop, 1989).

Intermoult and were not significantly different (Fig. 4). The fact that R exceeded unity (1.27) may have arisen from aeration of the flux water which would have reduced ambient ⁠, thus facilitating CO₂ excretion. The only R value in the literature for which had been measured and not estimated was 0·6, which was calculated for the land crab Cardisoma by Wood and Randall (1981). They attributed the low value to retention of respiratory CO₂ for carapace formation. However, during exercise R did rise above 1 in Cardisoma, confirming that this can occur in decapods. Owing to the small size range of the crayfish used in this study, absolute values for and cannot be compared with the majority of existing data. The values were of the same order of magnitude as values measured in similarly sized Pacifastacus leniusculus (Wheatly, 1989a).

The rate of O₂ consumption has been measured during the moulting cycle in the crayfish (Scudamore, 1947) as well as certain marine decapods (Lewis and Haefner, 1976; Penkoff and Thurberg, 1982; Mangum et al. 1985). Common to all the studies was a doubling of around the time of ecdysis, although the precise timing of the peak rate varied. The present finding that is highest immediately premoult (Fig. 4) agrees with certain studies (Scudamore, 1947; Penkoff and Thurberg, 1982). Some (Scudamore, 1947; Lewis and Haefner, 1976) have suggested that the rate drops during the actual shedding process, based on measurements made using closed respirometers. Some authors have reported elevated postmoult rates for up to 2 days (Scudamore, 1947; Penkoff and Thurberg, 1982; Mangum et al. 1985). In the present study the mass-specific returned to control levels within a day. By 8 days postmoult, had dropped significantly below the previous intermoult value as reported by Penkoff and Thurberg (1982); when converted to whole-animal rates there was no difference in successive intermoult rates, suggesting that this was an artefact of the increase in mass due to water loading.

dropped significantly below for the first 4 days postmoult, possibly representing the net result of HCO₃⁻ uptake countering CO₂ elimination. Assuming that R remained at 1 for cellular respiration, then an deficit of 1178μmolkg⁻¹ h⁻¹ (2356μrequivkg⁻¹ h⁻¹) can be calculated. This is of the same order of magnitude as the uptake of basic equivalents illustrated in Fig. 1, given that measurements were on separate experimental series. The ‘apparent’ respiratory exchange ratio meanwhile dropped (Fig. 4) while calcification was underway. In the 24 h preceding ecdysis, the excess was 1800 μmol kg⁻¹ h⁻¹, which is similar to the measured output of basic equivalents (Fig. 1). Cameron and Wood (1985) measured a net CO₂ uptake from sea water in postmoult blue crabs, concluding that the CO₂ source for calcification was at least partly external. Subsequent measurements of failed to reveal that the carapace was a sink for metabolic CO₂ (Cameron, 1985). This suggests that the external water provided the majority of the CO₂ in the form of HCO₃⁻. Uptake of HCO₃⁻, like Ca²⁺ uptake, proceeds at a relatively reduced rate in crayfish compared with the blue crab; this may reflect the lowered external HCO₃⁻ concentration or some direct or indirect limitation to the uptake mechanism. Postmoult ion uptake mechanisms are examined in greater detail in a separate paper (M. G. Wheatly and A. T. Gannon, in preparation).

This study was supported by NSF grant no. DCB-8415373 to MGW. We thank Dr Robert Romaire from Louisiana State University Agricultural Centre for supplying crayfish, Leanne Yow for technical assistance, Grace Kiltie for preparing the manuscript and Daryl Harrison for drawing the figures.