Carcinus maenas (L.) were exercised using a novel design of aquatic treadmill respirometer. Tethered exercise was performed in sea water at 5.8mmin−1 for 5 min. The rate of oxygen consumption and the heart and scaphognathite beat rates increased at the onset of exercise, reaching a steady state within 180 s. The estimated haemolymph flow rate rose 2.6-fold during exercise, achieved by a 1.8-fold increase in heart rate and a 1.5-fold increase in the estimated cardiac stroke volume. The haemolymph total oxygen content difference increased significantly during exercise. Haemolymph total carbon dioxide content did not change significantly during exerise, but haemolymph pH deceased as a result of an L-lactate-induced metabolic acidosis. The acidosis may also have led to a reduced Bohr shift. It is concluded that O2 and CO2 exchange were not impaired during exercise and that C. maenas relied primarily upon O2 to fuel underwater running at 5.8 m min−1.

Underwater locomotion, often involving slow-speed walking, is one of the characteristic features of marine Brachyura. Despite this, physiological changes during such activities have been studied in only a few brachyuran species (Houlihan and Innes, 1984), including Carcinus maenas (Houlihan et al. 1984; Houlihan and Mathers, 1985). These studies were primarily concerned with locomotory energetics, so many of the physiological changes occurring during exercise remain unknown. In these studies, a constant walking speed could not be induced as a probe was used to stimulate walking.

The present study was therefore designed to provide new information about changes in the physiology of Carcinus maenas during quantified submaximal exercise. Accurate control of the intensity and duration of exercise was achieved using a novel design of aquatic treadmill respirometer.

In addition, the data in the present study allow a better quantitative comparison to be made with the much larger volume of literature available on land crabs exercised in air. The evolution of crab air-breathing has resulted in a transition from diffusion-limited gills to perfusion-limited respiratory structures. This change, combined with the problems of desiccation and CO2 excretion that land crabs potentially face (Innes and Taylor, 1986c), could conceivably lead to major differences in the response to exercise between aquatic and terrestrial crab species.

### Animals

Carcinus maenas (L.) of both sexes were collected from around the coast of Aberdeen, Scotland. The animals were held in recirculating seawater aquaria at the experimental temperature of 15°C (salinity 31‰) for a maximum of 1 week. A short captivity time was desirable as the exercise performance of C. maenas declines with an increase in captivity time (Houlihan and Mathers, 1985). All animals had a complete complement of legs and chelae and were at the intermoult stage (Drach and Tchernigovtzeff, 1967). The mean dry mass of the animals was 12.01±0.76g (34.38 ±3.09g live mass; N=137).

### The respirometer

Animals were exercised in a closed-system treadmill respirometer filled with sea water (total volume 2.971) (Fig. 1). During the observation periods, water was continuously circulated within the respirometer by the pump and the stirrer. The effect, if any, that turbulence may have had upon the exercise performance of the animal was not assessed. Following observation periods, a rubber bung in the roof of the respirometer could be opened, allowing the apparatus to be flushed with fresh, filtered sea water.

Fig. 1.

Diagram of the treadmill respirometer. The respirometer was made of clear Perspex. The entire unit was submerged in a bath of sea water at 15°C. A, outer respirometer wall; B, removable roof; C, oxygen sensor housing containing oxygen sensor (D) and magnetic stirring bar (not shown). A submerged stirrer rotated the bar underneath the sensor, ensuring good mixing of water. Mixing was enhanced by circulating water from the output (E) to the input (F) using an external pump (not shown). The circulating circuit could be altered to flush fresh sea water through the respirometer as required. The animal was exercised on a ridged rubber belt (G) driven by the hand-turned handle (H) via a system of gears. The animal was tethered to a balance arm (I), which allowed limited vertical and horizontal rotation. The arm position could be altered vertically using the counterbalance (K). The rubber bung (J) was removed when flushing the respirometer with fresh sea water.

Fig. 1.

Diagram of the treadmill respirometer. The respirometer was made of clear Perspex. The entire unit was submerged in a bath of sea water at 15°C. A, outer respirometer wall; B, removable roof; C, oxygen sensor housing containing oxygen sensor (D) and magnetic stirring bar (not shown). A submerged stirrer rotated the bar underneath the sensor, ensuring good mixing of water. Mixing was enhanced by circulating water from the output (E) to the input (F) using an external pump (not shown). The circulating circuit could be altered to flush fresh sea water through the respirometer as required. The animal was exercised on a ridged rubber belt (G) driven by the hand-turned handle (H) via a system of gears. The animal was tethered to a balance arm (I), which allowed limited vertical and horizontal rotation. The arm position could be altered vertically using the counterbalance (K). The rubber bung (J) was removed when flushing the respirometer with fresh sea water.

The animals were tethered to a balance arm and the counter-balance was adjusted so that the animals ran with a gait stereotypical of freely moving C. maenas (Bethe, 1930; Clarac and Coulmance, 1971). Free walking proved repeatedly unsuccessful as the animals allowed themselves to be dragged along by the treadmill belt.

Mixing time was assessed by injecting dye into the respirometer at a position close to where the animal’s exhalant opening would normally be. After injection, 1ml water samples were withdrawn from the sensor housing (Fig. 1) at 15 s intervals. During the experiment, water was continuously circulated within the respirometer by the pump and stirrer. The absorbance of the samples was measured at 580nm (peak absorbance) and plotted against time. These data suggested that complete mixing was achieved within 50 s.

### Oxygen consumption measurement

The output from the oxygen sensor was fed into the analogue-to-digital converter of a BBC microcomputer (Acorn Computers Ltd; see Hamilton, 1987, for computer program). The computer was used to calculate and store the value of for each 1 min period of observation. Sensor drift was checked at the end of the experiment by comparing the computer-calculated value to the reading of the respirometer water measured using an independently calibrated oxygen measurement cell. No problems with oxygen sensor drift were noted. At the end of each experiment, was determined for 15 min without the animal in the respirometer (blank run). The blank value of was then subtracted from the measured of the animal. As filtered, fresh sea water was flushed through the respirometer prior to determination during each experimental period, the value of the blank was found to be negligible and was not considered to be a source of measurement error.

### Experimental regimes

The animals were allowed to rest, tethered, in the respirometer for a minimum of 12 h before determining the of the resting animal over a 60 min period (preexercise value). The animal was then exercised at 5.8 m min−1 for 5 min (sub-burst speed walking for C. maenas;Houlihan et al. 1984). Following exercise, was monitored until the pre-exercise value was reached. Fresh filtered sea water was flushed through the respirometer between observation periods to maintain the respirometer above 80% saturation. Experiments in which the animals were active for more than 2 min in any 5 min time block during the pre-exercise or postexercise periods were aborted. Any animal that hesitated during exercise for more than 60s was discarded.

Each animal was only used once. In some cases , heart rate or scapho-gnathite beat rate alone was monitored. In others, combinations of heart rate measurement, scaphognathite beat rate measurement, ventilatory water flow rate estimation and haemolymph variables were measured. The data from these different combinations have been pooled as no statistically significant differences were found when the same variables were compared between groups (P>0.01 in each case).

### Oxygen consumption and mass relationships

In another series of experiments the relationship between mass and resting or peak of C. maenas was investigated. Prior to determination, the animals were allowed to rest as described above. Resting was then determined with the animals untethered. Peak oxygen consumption rates were considered to represent maximal oxygen consumption rates and were induced by tethering the animals to the balance arm and repeatedly stimulating the animals with a probe. was considered to have been achieved when the three highest readings were observed over three consecutive minutes.

### Ventilation and circulation

#### Heart and ventilation rates

Impedance techniques were used to measure heart (FH) and scaphognathite (FSCAPH) beat rates (Ansell, 1973; Depledge, 1978). Using measurements of and inspired and expired water values, ventilatory water flow rate was estimated using the Fick principle. Expired water was sampled using a respiratory mask made from a commercially available rubber balloon (Butler et al. 1978; Hamilton, 1987).

#### Haemolymph gas analysis

Haemolymph was sampled after 12 h of tethered rest or at the end of the fifth minute of the exercise period. Postbranchial haemolymph samples were taken via a hole previously drilled above the pericardial sinus on the dorsal side of the carapace. Prebranchial samples were taken via the arthrodial membrane at the base of the pereiopods. Each sample (approximately 0.7 ml) was withdrawn into a chilled plastic syringe, sub-sampled and then the various haemolymph gas variables were measured immediately. Sampling time was less than 60s.

Haemolymph O2 tension and pH were measured using a BMS3 blood gas analysis system (Radiometer, Copenhagen). Total O2 content was measured using a Lex-O2-Con TL O2 content analyzer (Lexington Instruments). 40μl haemolymph samples were used to avoid underestimation of O2 content (Wood et al. 1979). Total CO2 content was determined by injecting duplicate haemolymph samples (80μl each) into a reaction chamber containing 0.01 moll−1 HC1 saturated with n-octanol and the increase in CO2 units noted on a PHM 73 meter (Radiometer, Cophenhagen), pre-calibrated with 10 mmol I−1 NaHCO3. was then calculated as described by Cameron (1971) and Houlihan et al. (1982).

was calculated using the equation:
where X is the antilogarithm of (pH–apparent first dissociation constant of carbonic acid) and αCO2 is the solubility coefficient of CO2 (Truchot, 1976b). The values of the constants were for C. maenas at 15 °C at a salinity of 35 ‰.

was estimated using the values of and substituted into the Fick equation. Estimates of obtained this way must be treated with caution because of the absence of a sampling site that yields a truly mixed venous haemolymph sample (Taylor, 1982; Taylor and Greenaway, 1984).

#### Oxygen equilibrium curves

Haemolymph oxygen equilibrium curves were determined using pooled (10 animals), declotted, mixed prebranchial and postbranchial haemolymph. Curves were determined for resting, untethered C. maenas or for tethered C. maenas exercised for 5 min at 5.8m min−1 by mixing various ratios of oxygenated and deoxygenated haemolymph (BMS2 blood tonometry system, Radiometer; Copenhagen and Wöstoff gas-mixing pumps, Wöstoff, Germany). and the pH of each mixed sample were determined as described above. The oxygencarrying capacity of the haemolymph was determined for each pooled sample after equilibration with air at 15 °C. The lactate concentration was also measured (see below). P50 values were derived from Hill plot regression equations.

#### Lactate concentration measurements

Whole-body L-lactate concentration was measured in untethered animals, animals that had been tethered for 20 min and animals that had been exercised at 5.8mmin−1 for 5 min. Haemolymph L-lactate concentrations of resting and exercised animals was also measured in some cases. L-Lactate concentrations were determined using a specific spectrophotometric assay (Sigma diagnostic kit no. 826-UV; Sigma Chemical Co. Ltd), modified as suggested by Full and Herreid (1984).

#### Statistical analysis

Parametric statistical tests were performed according to formulae outlined by Snedecor and Cochran (1972) and Sokal and Rohlf (1969). The 5% level of statistical significance has been used throughout.

### Response to exercise

At the onset of exercise, , fH and fscAPH increased until a steady state was achieved (Table 1, Fig. 2). The half-times for these responses were 91±8s, 73±4s and 72±6 s, respectively. The mean half-time of the response was significantly slower (one-tailed t-test; P<0.05) than the mean half-times of the heart and scaphognathite response for the same animal. This discrepancy was probably due to inherent delay in the measuring system.

Table 1.

Measured variables (±S.E.M.) for Carcinus maenas prior to exercise and during steady-state exercise at 5.8 m min−1

Fig. 2.

Mean (±S.E.M.) $M˙O2$, fH and FSCAPH of Carcinus maenas exercised in the treadmill respirometer at 5.8 m min−1 for 5 min, plotted as a function of time. The preexercise and post-exercise periods were divided into 5-min time blocks. Each block consisted of five 1-min observations made for each animal. The observations in a time block were pooled and the mean was plotted against the third minute in the time block. In the case of the exercise period, the time blocks span 1min $M˙O2$or 30 s (FH and FSCAPH), with each block consisting of one observation per animal.

Fig. 2.

Mean (±S.E.M.) $M˙O2$, fH and FSCAPH of Carcinus maenas exercised in the treadmill respirometer at 5.8 m min−1 for 5 min, plotted as a function of time. The preexercise and post-exercise periods were divided into 5-min time blocks. Each block consisted of five 1-min observations made for each animal. The observations in a time block were pooled and the mean was plotted against the third minute in the time block. In the case of the exercise period, the time blocks span 1min $M˙O2$or 30 s (FH and FSCAPH), with each block consisting of one observation per animal.

At steady state, the had increased 3.4-fold (N=10 animals). was estimated to have increased by 3.5-fold (Table 2,N=4 animals), resulting from the 3.7-fold increase in fSCAPH (N=8 animals). The animals wearing the mask was not significantly different from that of unmasked animals (P>0.01). As the scaphognathite of C. maenas is known to be a variable-volume pump (Wilkens et al. 1984; Mercier and Wilkens, 1984), scaphognathite stroke volume (Vs) changes may also have contributed to the increase.

Table 2.

Calculated variables and indices for Carcinus maenas prior to exercise and during steady-state exercise at 5.8 m min− 1

#### Ventilation

The increase ensured a large gradient between the water and haemolymph. This diffusion gradient into the haemolymph would have been further enhanced by the 0.59 kPa decrease in the prebranchial haemolymph O2 tension (N=7 animals). The convection requirement for water was maintained during exercise (N=4 animals), and no significant change in the percentage of O2 extracted from the ventilatory current (%Extrw) was noted (Table 2). This suggests that any increase in the physiological, anatomical or diffusional dead space resulting from the increase in did not create an O2 transport problem during exercise.

#### Circulation

The observed 1.8-fold increase in fH, coupled with a calculated 2.6-fold increase in , suggests that cardiac stroke volume increased 1.5-fold during steady-state exercise. The ventilation perfusion ratio increased slightly during exercise, indicating that perfusion of the gills was increased to facilitate the increase in available O2 at the gills. The efficiency of O2 uptake by the haemolymph increased and the convection requirement for haemolymph decreased as a result of changes in the amount of O2 transported and released by the haemolymph (Table 2). The increase in coupled with the maintenance of a high , resulted in an increase in the amount of O2 transported by the haemolymph to the tissues during exercise, as indicated by the capacity rate increase .

#### Haemolymph gas

Postbranchial O2 content and tension did not change significantly during exercise (P>0.05 in each case; N=7 and 8 animals, respectively; see Table 1). In contrast, prebranchial O2 content and tension values after exercise were significantly different from the pre-exercise values (P<0.002 in each case; N=6 and 7 animals, respectively). The difference therefore increased during exercise (P<0.002).

Under pre-exercise conditions, the haemocyanin transported 78 % of the O2 to the tissues. During exercise, the haemocyanin transported 84% of the O2 to the tissues; however, this is not a statistically significant difference (P>0.05).

Of the O2 made available to the tissues, 71.8% diffused from the haemolymph to the tissues (%Extrb) during the pre-exercise period, rising to 96.6% during exercise. An increase in %Extrb, despite an increase in as noted in C. maenas, is a typical crustacean response to exercise (McMahon, 1981; McMahon et al. 1979).

#### Haemolymph pH

Both post-and prebranchial pH values (N=4 animals in each case) decreased significantly as a result of exercise (P<0.01 and P<0.05, respectively). CO2 content did not change significantly as a result of exercise (N=4 animals in each case). The estimated pre-exercise values for postbranchial and prebranchial CO2 tension were 0.21 kPa and 0.24 kPa, respectively. There was no significant change in these values following exercise .

#### L-Lactate concentrations

Five minutes of exercise resulted in a significant 1.4-fold increase in whole-body L-lactate concentration (P<0.01; N=15 animals) and a significant 2.5-fold increase in haemolymph L-lactate concentration (P<0.05; N=4 animals; see Table 1). Anaerobiosis accounted for 29% of the total ATP production during exercise, calculated using ATP equivalents (Bennett and Licht, 1972).

#### Oxygen equilibrium curves

Prior to exercise, haemolymph P50 was 1.68kPa (12.6Torr) at a pH of 7.798. After 5 min of exercise, P50 had increased to 2.08 kPa (15.6Torr) at a pH of 7.733, resulting in a Bohr shift of –1.43 (Fig. 3). The estimated values at P50 of the haemolymph were 0.23kPa (1.7Torr) for the pre-exercise samples and 0.24kPa (1.8 Torr) for the samples from exercised animals. Cooperativity decreased from 4.8 to 3.9. No Root effect was exhibited by either of the pooled haemolymph samples.

Fig. 3.

In vitro haemolymph oxygen equilibrium curves for Carcinum maenas expressed as percentage saturation. (†) Pre-exercise conditions; (○) exercised for 5 min at 5.8mmin−1. Two overlapping data points are indicated by an asterisk.

Fig. 3.

In vitro haemolymph oxygen equilibrium curves for Carcinum maenas expressed as percentage saturation. (†) Pre-exercise conditions; (○) exercised for 5 min at 5.8mmin−1. Two overlapping data points are indicated by an asterisk.

#### Post-exercise response

After exercise, , fH and fSCAPH decreased, eventually reaching the original pre-exercise value. The half-times for these responses were 752±67s, 771±63s and 1030±245s, respectively (each significantly longer than the corresponding half-time at the beginning of exercise, P<0.002; paired t-test).

#### Oxygen consumption and live mass

The relationship between the rate of oxygen consumption and live mass has been expressed in units of ml O2 animal−1 h−1 to allow easy comparison with the relationships noted in the established literature. The values of resting and maximally exercised animals were linearly related to live mass (Fig. 4). The slopes of the two lines are not significantly different (P>0.05; F slope=0.033) whereas the elevations are significantly different (P<0.05; F elevation=337.47). The relationship between resting and mass was: log (ml O2 animal−1 h−1)=0.655 × log(live mass in grams) – 0.706; (r=0.85; N=32; P<0.01).

Fig. 4.

$V˙O2$ of resting and maximally respiring Carcinus maenas at 15°C plotted as a function of live mass. Lines were fitted by least-squares regression analysis. (•) Resting animals (N=32); (○) maximally respiring animals (N=12).

Fig. 4.

$V˙O2$ of resting and maximally respiring Carcinus maenas at 15°C plotted as a function of live mass. Lines were fitted by least-squares regression analysis. (•) Resting animals (N=32); (○) maximally respiring animals (N=12).

The relationship between maximal and mass was: log (mlO2animal−1 h−1) = 0.704 × log(live mass in grams) – 0.116; (r=0.88; N=12; P<0.05). The aerobic expansibility (ratio of ) was 4.6 for the mean live mass of animal used in the exercise experiments.

### Response to exercise

#### Oxygen consumption

increased at the onset of exercise and rapidly reached a steady state (Fig. 2). This is characteristic of primarily aerobically dependent decapod crustaceans running in air (Full, 1987; Full and Herreid, 1983; Herreid, 1981; Herreid et al. 1979) and primarily aerobic vertebrates and insects (Brackenbury and Avery, 1980; Cerretelli et al. 1977; Chadwick and Gilmour, 1940; Krough and Weis-Fogh, 1951; Morton, 1985). The latter animals usually have high aerobic expansibility values ranging up to 20 times resting value (Brett, 1964; Pasquis et al. 1970; Priede, 1985; Schmidt-Nielsen, 1984; Thomas, 1975). Like vertebrates, primarily aerobically dependent crustaceans have higher aerobic expansibility values than more anaerobically reliant species (see Hamilton, 1987, for a brief review), although comparison is difficult as truly basal metabolic rates have not been measured in the majority of crustacean studies. Aerially respiring Ocypode spp., renowned as the world’s fastest crustaceans (Hafemann and Hubbard, 1969), have expansibilities between 6.4 to 11.9 times the resting rate (Full, 1987; Full and Herreid, 1983). In contrast, the land crab Sesarma cinereum has an expansibility of just 1.5 times the resting rate, a very limited exercise capability and a heavy reliance upon anaerobiosis during exercise (Full et al. 1985). C. maenas fits somewhere between these two species, with an expansibility of 4.7 for a 50 g live mass animal (Fig. 4).

In C. maenas the expansibility was relatively constant (4.4 – 4.8) over a mass range of 10 g to 100 g live mass. In contrast, aerobic metabolic scope increases progressively with body mass (0.17 mmol O2g−1 live mass h−1 to 0.70 mmol O2 g − 1 live mass h−1 ; 3.9 ml O2g−1 live mass h−1 to 15.5mlO2g−1 live massh−1) over the same mass range (Fig. 4). Consistent with this relationship is the observation that aerobic metabolic enzymes in this species all scale with exponents that are not significantly different from those of the resting and regression lines (N. M. Hamilton and D. F. Houlihan, personal observation). The scaling of aerobic scope has been reported to be mass-dependent in many crabs (Houlihan et al. 1984, 1985; Innes et al. 1986). Such scaling effects must be investigated if meaningful comparisons of crab exercise performance are to be made using as an indication of aerobic capacity.

#### Ventilation

With an increase in there was a concomitant increase in fSCAPH and of C. maenas (Fig. 2). This is a typical response to exercise in Cancer magister (McDonald, 1977; McMahon et al. 1979) and Callinectes sapidus (Booth et al. 1982; McMahon and Wilkens, 1983). In these three species, and in the aerially respiring crabs Cardisoma guanhumi (Herreid et al. 1979) and Cardisoma carnifex (Wood and Randall, 1981a), increases in ventilation volume contribute more to increased O2 provision than do increases in the percentage of O2 extracted from the respiratory medium. In contrast, the %Extra increases much more than the rate of air flow through the gills during exercise in Gecarcinus lateralis (Herreid et al. 1983).

#### Circulation

The cardiac response of submerged C. maenas to exercise, in which FH increases more than Vs (Tables 1 and 2), is the opposite to its cardiac response at rest in progressively increasing external hypoxia (Taylor, 1976; Taylor and Butler, 1978). fH also shows a greater proportional increase than Vs during exercise in Callinectes sapidus (Booth et al. 1982) and in the lobster Homarus vulgaris (Hamilton, 1987). In contrast, the proportional increase in Vs is greater than that of FH during exercise in Carcinus mediterraneus and Pachygrapsus marmoratus (Houlihan and Innes, 1984), Cancer magister (McMahon et al. 1979) and in the land crab Cardisoma carnifex (Wood and Randall, 1981a). A bradycardia in response to exercise in Cardisoma guanhumi (Herreid et al. 1979) is yet another variation that has been reported. The reason for these differences is not clear.

#### Haemolymph gas

No significant decrease in postbranchial O2 tension and content was noted in C. maenas following exercise (Table 1). C. sapidus has a similar response (Booth et al. 1982). Cardisoma carnifex (Wood and Randall, 1981a) and Cancer magister (McMahon et al. 1979; Johansen et al. 1970) have significantly decreased values during exercise; however, shows little change as the haemocyanin remains virtually fully saturated in each case. In Carcinus mediterraneus and Pachygrapsus marmoratus, both haemolymph O2 tension and O2 content decrease significantly as a result of exercise (Houlihan and Innes, 1984).

The change in the of C. maenas during exercise resulted in an increase in the difference (Table 1). Several other authors have noted an increase in the difference during exercise, primarily as a result of a decrease (Johansen et al. 1970; McMahon et al. 1979; Wood and Randall, 1981b). Callinectes sapidus is a notable exception as its shows no significant change after 25 min of swimming (Booth et al. 1982).

The release of O2 to the tissues is enhanced in some species by a normal Bohr shift (see McMahon and Wilkens, 1983). In C. maenas, the Bohr shift probably plays a minimal role in the release of O2. The Bohr shift noted in C. maenas exercised at 5.8m min−1 was – 1.43 (Fig. 3). This is lower than the value of – 1.54 reported by Taylor and Butler (1978). The lower value in the former case may have been caused by the L-lactate accumulation opposing the rightward equilibrium curve shift induced by the haemolymph pH drop. L-Lactate is known to modulate the affinity of C. maenas haemocyanin in vitro (Truchot, 1980). Modulation of O2-binding by other haemolymph cofactors during exercise cannot be ruled out (Lallier et al. 1987; Morris et al. 1985). Cooperativity decreased during exercise. A decrease was also reported in swimming C. sapidus (Booth et al. 1982); however, the relationships between cooperativity, haemolymph pH and lactate ion concentration are not understood.

The post-and prebranchial haemolymph CO2 tensions did not change significantly as a result of exercise, suggesting that gas exchange at the respiratory surface was not seriously impaired. This was possibly facilitated by the reduced Bohr shift, which would assist the maintenance of a high .This, in turn, would aid CO2 unloading as O2 uptake and CO2 excretion are thought to be linked in C. maenas (Truchot, 1976a). As the difference between the prebranchial and postbranchial haemolymph was small (<0.03kPa; 0.2Torr), unloading of CO2 would be aided further by the Haldane effect (Truchot, 1976a). In contrast, Callinectes sapidus and Cancer magister both show increases in haemolymph CO2 tension during exercise (Booth et al. 1984; McDonald et al. 1979).

Assuming the animals were in acid-base equilibrium at the end of the exercise period, the insignificant changes in haemolymph CO2 content and calculated suggest that C. meanas did not experience a respiratory acidosis during exercise. The significant decrease in haemolymph pH would therefore be due largely to a metabolic acidosis induced by the significant increase in L-lactate concentration. The majority of crab species that have been examined show a mixed metabolic/ respiratory acidosis during exercise (McDonald et al. 1979; Smatresk and Cameron, 1981; Smatresk et al. 1979; Waldron et al. 1986; Wheatly et al. 1986; Wood and Randall, 1981a,EXBIO_162_1_37C75b).

The L-lactate concentration increase in C. maenas indicates the anaerobiosis contributed 29% of the total ATP equivalent production, as noted in the Results section. Accumulation of this amount of lactate suggests that the exercise intensity was approaching burst speed levels.

#### Recovery from exercise

The recovery from exercise in crustaceans follows a monoexponential curve (Full and Herreid, 1983). The reason why the half-times of the , fH and fSCAPH responses were each significantly longer at the end of exercise than at the beginning is not clear. The most likely explanation is that the responses during the recovery period were periodically elevated during the observed short bursts of spontaneous activity (duration <1 min). There is no significant difference between these half-times for in Ocypode gaudichaudii (Full and Herreid, 1983).

#### Interspecific differences in response to exercise

The reason why some species of crustaceans show markedly different physiological responses to submaximal exercise remains unclear, largely due to the dearth of information in this field. One contributing factor may be whether the animal relies upon rapid neurophysiological or much slower-acting humoral mechanisms to initiate the ventilatory responses during exercise (Herreid, 1981).

The O2 diffusion rate across the gills will also be a limiting factor during exercise. The amount of O2 that can diffuse into the haemolymph will be a function of the magnitude of the O2 diffusion gradient, the area available for diffusion, the diffusion pathlength and the barrier to diffusion created by the physical structure of the gas exchange apparatus. Callinectes sapidus has a relatively large gill area (Gray, 1957) and short diffusion pathlength (Aldridge and Cameron, 1979) compared to many other species of crab (Gray, 1957), including C. maenas (Butler, 1976; Taylor and Butler, 1978). These factors may explain why C. sapidus has a shorter half-time for , at the beginning of exercise than C. maenas (Booth et al. 1982).

Associated with adaptation to a terrestrial environment is a reduction in gill surface area (Gray, 1957; Cameron, 1981; Hawkins and Jones, 1982) in addition to the evolution of alternative gas exchange structures (Bliss, 1968, 1979; Diaz and Ridriguez, 1977; Innes and Taylor, 1986c, 1987; El Haj et al. 1986; Maitland, 1986). The effect of these morphological adaptations upon exercise performance and the accompanying physiological adjustments are not clear. Ocypode gaudichaudii shows exceptional exercise capabilities in air, has a fast half-time for at the beginning of exercise and reaches a steady state (Full and Herreid, 1983). This may be due to the invaginations of its branchial chamber lining, which act as an accessory respiratory surface (Diaz and Rodriguez, 1977). However, Pseudo-thelphusa garmani has evolved an invaginated ‘lung’, which enables it to achieve the highest values recorded for crustaceans (approximately 18.7 kPa; Innes and Taylor, 1986a, 1987), but this species does not have exceptional locomotory capabilities (Innes and Taylor, 1986b). Tolerance to desiccation may also determine the exercise responses of aerially respiring species (Innes et al. 1986).

The magnitude of phosphate and O2 stores, in addition to the rate of phosphate utilization, is also likely to determine the rate of the response. Unfortunately, accurate information concerning these variables is limited and is of little help in clarifying the reasons for the interspecific differences in response time.

Circulatory response data are also sparse in the literature and there is no clear correlation between physiological responses, habitat and exercise performance. It is worth noting that aquatic crustaceans are diffusion-limited in their O2 transport, whereas aerially respiring crustaceans are perfusion-limited (reviewed by Innes and Taylor, 1986c). Perfusion limitation may explain the extremely limited exercise capabilities of Sesarma cinereum, although the exact nature of the limitation is unknown (Full et al. 1985).

The present study has revealed the major ventilatory and circulatory adjustments that occur in C. maenas in response to controlled submaximal exercise. These results suggest that O2 and CO2 exchange were not impaired during exercise and that C. maenas relied primarily upon O2 to fuel exercise at 5.8mmin−1. C. maenas shows responses that are similar to primarily aerobic land crabs. From the aquatic species that have been studied, it would appear that the aquatic medium does not confer any particular advantage to marine crustaceans in terms of exercise performance when compared to aerially respiring species exercised in air. This implies that the evolution of air-breathing in crabs per se is not necessarily limiting to exercise performance. Owing to a lack of published information on detailed physiological responses under a controlled exercise regime, the reason for interspecific differences in crustacean exercise response remains unknown.

The authors thank Alastair Innes who helped with the blood gas analysis and the oxygen equilibrium curve determinations. Thanks are also given to Robert Hamilton, Roland Smith and Ron Bisset who constructed the treadmill respirometer. This research was funded by an SERC (UK) grant to N.M.H.

Aldridge
,
J. B.
and
Cameron
,
J. N.
(
1979
).
CO2 exchange in the blue crab, Callinectes sapidus (Rathbun)
.
J. exp. Zool
.
207
,
321
328
.
Ansell
,
A. D.
(
1973
).
Changes in oxygen consumption, heart rate and ventilation accompanying starvation in the decapod crustacean
.
Cancer pagurus. Neth. J. Sea Res
.
7
,
455
475
.
Bennett
,
A. F.
and
Licht
,
P.
(
1972
).
Anaerobic metabolism during activity in lizards
.
J. comp. Physiol
.
81
,
277
288
.
Bethe
,
A.
(
1930
).
Studien liber die Plastizitat des Nervensystems. I. Mittelung. Arachnoideen und Crustaceen
.
Pflügers Arch. ges. Physiol
.
224
,
793
820
.
Bliss
,
D. E.
(
1968
).
Transition from water to land in decapod crustaceans
.
Am. Zool
.
8
,
355
392
.
Bliss
,
D. E.
(
1979
).
From sea to tree: saga of a land crab
.
Am. Zool
.
19
,
385
410
.
Booth
,
C. E.
,
Mcmahon
,
B. R.
,
De Fur
,
P. L.
and
Wilkes
,
P. R. H.
(
1984
).
Acid-base regulation during exercise and recovery in the blue crab, Callinectes sapidus
.
Respir. Physiol
.
58
,
359
376
.
Booth
,
C. E.
,
Mcmahon
,
B. R.
and
Pinder
,
A. W.
(
1982
).
Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab, Callinectes sapidus
.
J. comp. Physiol
.
148
,
111
121
.
Brackenbury
,
J. H.
and
Avery
,
P.
(
1980
).
Energy consumption and ventilatory mechanisms in the exercising fowl
.
Comp. Biochem. Physiol
.
66A
,
439
445
.
Brett
,
J. R.
(
1964
).
The respiratory metabolism and swimming performance of young sockeye salmon
.
J. Fish. Res. Bd Can
.
21
,
1183
1226
.
Butler
,
P. J.
(
1976
).
Gas exchange
.
In Environmental Physiology of Animals
(ed.
J.
Bligh
,
J. L.
Cloudsley-Thompson
and
A. G.
Macdonald
).
Oxford
:
Blackwell
.
Butler
,
P. J.
,
Taylor
,
E. W.
and
Mcmahon
,
B. R.
(
1978
).
Respiratory and circulatory changes in the lobster (Homarus vulgaris) during long term exposure to moderate hypoxia
.
J. exp. Biol
.
73
,
131
146
.
Cameron
,
J. N.
(
1971
).
Rapid method for determination of total carbon dioxide in small blood samples
.
J. appl. Physiol
.
31
,
632
634
.
Cameron
,
J. N.
(
1981
).
Acid-base response to changes in CO2 in two Pacific crabs: the coconut crab, Birgus latro, and a mangrove crab, Cardisoma carnifex
.
J. exp. Zool
.
218
,
65
73
.
Cerretelli
,
P.
,
Shlndell
,
D.
,
Pendergast
,
D. P.
,
Diprampero
,
P. E.
and
Rennie
,
D. W.
(
1977
).
Oxygen uptake transients at the onset and offset of arm and leg work
.
Respir. Physiol
.
30
,
81
97
.
,
L. E.
and
Gilmour
,
D.
(
1940
).
Respiration during flight in Drosophila repleta Wollaston: the oxygen consumption considered in relation to the wing rate
.
Physiol. Zool
.
13
,
398
410
.
Clarac
,
F.
and
Coulmance
,
M.
(
1971
).
La marche latérale du crabe (Carcinus). Coordination des mouvements articulaires et régulation proprioceptive
.
Z. vergl. Physiol
.
73
,
408
438
.
Depledge
,
M. H.
(
1978
).
Cardiac activity of the shore crab Carcinus maenas (L
.).
Comp. Biochem. Physiol
.
60A
,
65
67
.
Diaz
,
H.
and
Rodriguez
,
G.
(
1977
).
The branchial chamber in terrestrial crabs: a comparative study
.
Biol. Bull. mar. biol. Lab., Woods Hole
153
,
485
504
.
Drach
,
P.
and
Tchernigovtzeff
,
C.
(
1967
).
Sur la méthode de détermination des stades d’intermue et son application générale aux crustacés
.
Mie et Millieu
18A
,
595
607
.
El Haj
,
A. J.
,
Innes
,
A. J.
and
Taylor
,
E. W.
(
1986
).
Ultrastructure of the pulmonary, cutaneous and branchial gas exchange organs of the Trinidad mountain crab
.
J. Physiol., Lond
.
373
,
84P
.
Full
,
R. J.
(
1987
).
Locomotion and energetics of the ghost crab. I. Metabolic cost and endurance
.
J. exp. Biol
.
130
,
137
153
.
Full
,
R. J.
and
Herreid
,
C. F.
(
1983
).
Aerobic response to exercise of the fastest land crab
.
Am. J. Physiol
.
244
,
R530
R536
.
Full
,
R. J.
and
Herreid
,
C. F.
(
1984
).
Fiddler crab exercise: the energetic cost of running sideways
.
J. exp. Biol
.
109
,
141
161
.
Full
,
R. J.
,
Herreid
,
C. F.
and
,
J. A.
(
1985
).
Energetics of the exercising wharf crab Sesarma cinereum
.
Physiol. Zool
.
58
,
605
615
.
Gray
,
I. E.
(
1957
).
A comparative study of the gill area of crabs
.
Biol. Bull. mar. biol. Lab., Woods Hole
112
,
34
42
.
Hafemann
,
D. R.
and
Hubbard
,
J. I.
(
1969
).
On the rapid running of ghost crabs (Ocypode ceratophthalma)
.
J. exp. Zool
.
170
,
25
32
.
Hamilton
,
N. M.
(
1987
).
Respiratory and circulatory changes accompanying aquatic treadmill exercise of Carcinus maenas (L.) and Homarus vulgaris (M.E
.).
PhD thesis
.
University of Aberdeen, Aberdeen, Scotland
.
Hawkins
,
A. J. S.
and
Jones
,
M. B.
(
1982
).
Gill area and ventilation in the mud crabs, Helice crassa Dana (Grapsidae) and Macrophthalmus hirtipes (Jacquinot) (Ocypodidae), in relation to habitat
.
J. exp. mar. Biol. Ecol
.
60
,
103
118
.
Herreid
,
C. F.
(
1981
).
Energetics of pedestrian arthropods
.
In Locomotion and Energetics in Arthropods
(ed.
C. F.
Herreid
and
C. R.
Fourtner
), pp.
491
526
.
New York, London
:
Plenum Press
.
Herreid
,
C. F.
,
Lee
,
L. W.
and
Shah
,
G. M.
(
1979
).
Respiration and heart rate in exercising land crabs
.
Respir. Physiol
.
36
,
109
120
.
Herreid
,
C. F.
,
O’mahoney
,
P. N.
and
Full
,
R. J.
(
1983
).
Locomotion in land crabs: respiratory and cardiac response of Gecarcinus lateralis
.
Comp. Biochem. Physiol
.
74A
,
117
124
.
Houlihan
,
D. F.
,
Govind
,
C. K.
and
El Haj
,
A. J.
(
1985
).
Energetics of swimming in Callinectes sapidus and walking in Homarus americanas
.
Comp. Biochem. Physiol
.
82A
,
267
279
.
Houlihan
,
D. F.
and
Innes
,
A. J.
(
1984
).
The cost of walking in crabs: aerial and aquatic oxygen consumption during activity of two species of intertidal crab
.
Comp. Biochem. Physiol
.
77A
,
325
334
.
Houlihan
,
D. F.
,
Innes
,
A. J.
,
Wells
,
M. J.
and
Wells
,
J.
(
1982
).
Oxygen consumption and blood gases of Octopus vulgaris in hypoxic conditions
.
J. comp. Physiol
.
148
,
35
40
.
Houlihan
,
D. F.
and
Mathers
,
E.
(
1985
).
Effects of captivity and exercise on the energetics of locomotion and muscle of Carcinus maenas (L
.).
J. exp. mar. Biol. Ecol
.
92
,
125
142
.
Houlihan
,
D. F.
,
Mathers
,
E.
and
El Haj
,
A. J.
(
1984
).
Walking performance and aerobic and anaerobic metabolism of Carcinus maenas (L.) in sea water at 15°C
.
J. exp. mar. Biol. Ecol
.
74
,
221
230
.
Innes
,
A. J.
,
Forster
,
M. E.
,
Jones
,
M. B.
,
Marsden
,
I. D.
and
Taylor
,
H. H.
(
1986
).
Bimodal respiration, water balance and acid-base regulation in high-shore crab, Cyclograpsus lavauxiH. Milne Edwards
.
J. exp. mar. Biol. Ecol
.
100
,
127
145
.
Innes
,
A. J.
and
Taylor
,
E. W.
(
1986a
).
An analysis of lung function in the Trinidadian mountain crab
.
J. Physiol., Lond
.
372
,
43P
.
Innes
,
A. J.
and
Taylor
,
E. W.
(
1986b
).
.
Comp. Biochem. Physiol
.
85A
,
373
381
.
Innes
,
A. J.
and
Taylor
,
E. W.
(
1986C
).
The evolution of air-breathing in crustaceans: A functional analysis of branchial, cutaneous and pulmonary gas exchange
.
Comp. Biochem. Physiol
.
85A
,
621
637
.
Innes
,
A. J.
and
Taylor
,
E. W.
(
1987
).
Air-breathing in the trinidad mountain crab: a quantum leap in the evolution of the invertebrate lung?
Comp. Biochem. Physiol
.
87A
,
1
8
.
Johansen
,
K.
,
Lenfant
,
C.
and
Mecklenburg
,
T. A.
(
1970
).
Respiration in the crab, Cancer magister
.
Z. vergl. Physiol
.
70
,
1
19
.
Krough
,
A.
and
Weis-Fogh
,
T.
(
1951
).
The respiration exchange of the desert locust (Schistocerca gregaria) before, during and after flight
.
J. exp. Biol
.
28
,
344
357
.
Lallier
,
F.
,
Boitel
,
F.
and
Truchot
,
J. P.
(
1987
).
The effect of ambient oxygen and temperature on haemolymph L-lactate and urate concentrations in the shore crab Carcinus maenas
.
Comp. Biochem. Physiol
.
86A
,
255
260
.
Maitland
,
D. P.
(
1986
).
Crabs that breathe air with their legs - Scopimera and Dotilia
.
Nature
19
,
493
495
.
Mcdonald
,
D. G.
(
1977
).
Respiratory Physiology of the Crab Cancer magister
.
PhD thesis
.
University of Calgary, Calgary, Alberta, Canada
.
Mcdonald
,
D. G.
,
Mcmahon
,
B. R.
and
Wood
,
C. M.
(
1979
).
An analysis of acid-base disturbances in the haemolymph following strenuous activity in the dungeness crab, Cancer magister
.
J. exp. Biol
.
79
,
47
58
.
Mcmahon
,
B. R.
(
1981
).
Oxygen uptake and acid-base balance during activity in decapod crustaceans
.
In Locomotion and Energetics in Arthropods
(ed.
C. F.
Herreid
and
C. R.
Fourtner
), pp.
299
335
.
New York, London
:
Plenum Press
.
Mcmahon
,
B. R.
,
Mcdonald
,
D. G.
and
Wood
,
C. M.
(
1979
).
Ventilation, oxygen uptake and haemolymph oxygen transport, following enforced exhausting activity in the dungeness crab Cancer magister
.
J. exp. Biol
.
80
,
271
285
.
Mcmahon
,
B. R.
and
Wilkens
,
J. L.
(
1983
).
Ventilation, perfusion and oxygen uptake
.
In Internal Anatomy and Physiological Regulation. The Biology of Crustacea
, vol.
5
(ed.
D. E.
Bliss
and
L. H.
Mantel
), pp.
289
372
.
New York, London
:
.
Mercier
,
J. A.
and
Wilkens
,
J. L.
(
1984
).
Analysis of the scaphognathite ventilatory pump in the shore crab Carcinus maenas. I. Work and power
.
J. exp. Biol
.
113
,
55
68
.
Morris
,
S.
,
Bridges
,
C.R.
and
Grieshaber
,
M. K.
(
1985
).
A new role for uric acid: modulator of haemocyanin oxygen affinity in crustaceans
.
J. exp. Zool
.
235
,
135
139
.
Morton
,
R. H.
(
1985
).
Two-dimensional short-term model of oxygen uptake kinetics
.
J. appl. Physiol
.
58
,
1736
1740
.
Pasquis
,
A.
,
Lacaisse
,
A.
and
Dejours
,
P.
(
1970
).
Maximal oxygen uptake in four species of small animals
.
Respir. Physiol
.
9
,
298
309
.
Priede
,
I. G.
(
1985
).
Metabolic scope in fishes
.
In Fish Energetics: New Perspectives
(ed.
P.
Tytler
and
P.
Calow
), pp.
33
64
.
London, Sydney
:
Croom Helm
.
Schmidt-Nielsen
,
K.
(
1984
).
Scaling, Why is Animal Size so Important?
pp.
151
164
.
London
:
Cambridge University Press
.
Smatresk
,
N. J.
and
Cameron
,
J. N.
(
1981
).
Post-exercise acid-base balance and ventilatory control in Birgus latro, the coconut crab
.
J. exp. Zool
.
218
,
75
82
.
Smatresk
,
N. J.
,
Preslar
,
A. J.
and
Cameron
,
J. N.
(
1979
).
Post-exercise acid-base disturbance in Gecarcinus lateralis
.
J. exp. Zool
.
210
,
205
210
.
Snedecor
,
G. W.
and
Cochran
,
W. C.
(
1972
).
Statistical Methods
.
Iowa
:
Iowa State University Press
.
Sokal
,
R. R.
and
Rohlf
,
F. J.
(
1969
).
Biometry
.
San Francisco
:
Freeman Publications
.
Taylor
,
A. C.
(
1976
).
The respiratory responses of Carcinus maenas to declining oxygen tension
.
J. exp. Biol
.
65
,
309
322
.
Taylor
,
E. W.
(
1982
).
Control and co-ordination of ventilation and circulation in crustaceans: responses to hypoxia and exercise
.
J. exp. Biol
.
100
,
289
319
.
Taylor
,
E. W.
and
Butler
,
P. J.
(
1978
).
Aquatic and aerial respiration in the shore crab, Carcinus maenas (L.), acclimated to 15°C
.
J. comp. Physiol
.
127
,
315
323
.
Taylor
,
H. H.
and
Greenaway
,
P.
(
1984
).
The role of the gills and branchiostegites in gas exchange in the bimodally breathing crab, Holthuisana transversa: evidence for a facultative change in the distribution of the respiratory circulation
.
J. exp. Biol
.
111
,
103
121
.
Thomas
,
S. P.
(
1975
).
Metabolism during flight in two species of bats, Phyllostomus hastatus and Pteropus gouldii
.
J. exp. Biol
.
63
,
273
293
.
Truchot
,
J. P.
(
1976a
).
Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L.): CO2-dissociation curves and Haldane effect
.
J. comp. Physiol
.
112
,
283
293
.
Truchot
,
J. P.
(
1976b
).
Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L.): carbon dioxide solubility coefficient and carbonic acid dissociation constants
.
J. exp. Biol
.
64
,
45
57
.
Truchot
,
J. P.
(
1980
).
Lactate increases the oxygen affinity of crab haemocyanin
.
J. exp. Zool
.
214
,
205
208
.
Waldron
,
F. M.
,
Taylor
,
H. H.
and
Forster
,
M. E.
(
1986
).
Acid-base disturbances following exercise in a high-shore crab, Cyclograpsus lavauxi
.
N.Z. J. mar. freshwater Res
.
20
,
479
487
.
Wheatly
,
M. G.
,
Mcmahon
,
B. R.
,
Burggren
,
W. W.
and
Pinder
,
A. W.
(
1986
).
Haemolymph acid-base, electrolyte and gas status during sustained voluntary activity in the land hermit crab Coenobita compressas
.
J. exp. Biol
.
125
,
225
243
.
Wilkens
,
J. L.
,
Wilkes
,
P. R. H.
and
Evans
,
J.
(
1984
).
Analysis of the scaphognathite ventilatory pump in the short crab, Carcinus maenas. II. Pumping efficiency and metabolic cost
.
J. exp. Biol
.
113
,
69
81
.
Wood
,
C. M.
,
Mcmahon
,
B. R.
and
Mcdonald
,
D. G.
(
1979
).
Respiratory gas exchange in the resting flounder, Platichthys stellatus: a comparison with other teleosts
.
J. exp. Biol
.
78
,
167
179
.
Wood
,
C. M.
and
Randall
,
D. J.
(
1981a
).
Oxygen and carbon dioxide exchange during exercise in the land crab (Cardisoma carnifex)
.
J. exp. Zool
.
218
,
7
22
.
Wood
,
C. M.
and
Randall
,
D. J.
(
1981b
).
Haemolymph gas transport, acid-base regulation, and anaerobic metabolism during exercise in the land crab (Cardisoma carnifex)
.
J. exp. Zool
.
218
,
23
35
.