ROLE OF LUNGS AND GILLS IN AN AFRICAN FRESH-WATER CRAB,
POTAMONAUTES WARRENI (DECAPODA: POTAMOIDEA), IN GAS
EXCHANGE WITH WATER, WITH AIR, AND DURING EXERCISE
A. M. Adamczewska, W. J. van Aardt, a n d S. Morris
A B S T R A C T
Respiratory gas transport and acid-base state were investigated in the crab Potamonautes warreni breathing air or water for at least 24 h to assess the role of the lungs and gills. The efferent
pulmonary and arterial samples had similar O2 content and thus either the lungs received most of
the hemolymph flow or the lungs and gills were of equal importance during air-breathing. The gills
were more important in immersed crabs. The O2 partial pressure in arterial hemolymph decreased
from 8.6 kPa in air-breathing crabs to 4.5 kPa in water-breathing crabs. Despite an increase in O2
diffusion limitation during water-breathing (Ldiff = 0.78) compared to air-breathing crabs (Ldiff = 0.57)
the arterial-venous O2 content difference did not change. In immersed crabs, the C O , content o f the
hemolymph (6.1 mmol�L�1) was half that in air-breathing crabs, but the pH remained unchanged at
pH 7.4. Potamonautes warreni showed no specific adaptations to the �1400-m altitude of its habitat other than apparently relatively improved O2 diffusion.
Polamonautes warreni performed well during exercise in air. Crabs exercised at slow speed (1.8
m�min�1) experienced smaller hemolymph acid-base perturbations (ApH = 0.18) than crabs exercised
at fast speed (ApH = 0.36; 3 m�min�1). While the partial pressure of 0 , in the arterial and efferent
pulmonary hemolymph during 20 min of fast exercise decreased from near 9 to 6 kPa or less, the
hemocyanin 0 , saturation was maintained near 80% and the arterial-venous O2 difference doubled.
An increase in Hc-O2 affinity in exercising animals partially offset the Bohr effect and assisted in
0 , uptake at the gas-exchange surfaces. The relative importance of lungs and gills in gas exchange
after exercise in air was apparently similar to that in resting crabs. There was little requirement for
anaerobiosis during submaximal exercise. Hemolymph L-lactate levels peaked at 3.6 mmol�L�1 after 20 min of fast exercise and were similar to L-lactate levels in the muscle.
The evolutionary transition from water to
air-breathing requires major physiological
and morphological adjustments. Air-breathing crabs have both gills and lungs (Taylor
and Greenaway, 1979; McMahon and Burggren, 1988; Farrelly and Greenaway, 1993) and
hemolymph may be preferentially directed to
gills or lungs depending on the medium that
the crabs were required to breathe (Taylor and
Greenaway, 1984; Al-Wassia et al., 1989).
While respiratory and acid-base changes in
crustaceans that breathe air and water have
been examined in the laboratory (e.g., Zoond
and Charles, 1931; Cameron, 1981; O ' M a honey and Full, 1984; Al-Wassia et al., 1989;
van Aardt, 1990a, b; Morris and Butler, 1996;
Butler and Morris, 1996), our understanding
of amphibious crustaceans is far from complete. For example, Cardisoma spp. were previously thought of as amphibious, but recent
field studies have shown that during voluntary immersion these crabs were reluctant to
ventilate water, which promoted internal hypoxia and anaerobiosis (Adamczewska and
Morris, 1996; Morris and Adamczewska,
1996). Furthermore, surprisingly few studies
have examined exercise performance of amphibious crabs in air and water (e.g., Houlihan and Innes, 1984; Innes et al., 1986; van
Aardt, 1990b).
Locomotion requires a significant energy
expenditure and is inextricably linked to all
activities from foraging and predator avoidance to breeding. Some aspects of locomotion in crustaceans have been relatively well
studied. These include the relationship between speed and endurance (Full et al., 1985;
review, Herreid and Full, 1988; van Aardt,
1990b), the costs of locomotion, heart and
ventilation rates, and the hemolymph gas and
acid-base status after exercise in terrestrial
and aquatic crabs (e.g., Wilkens, 1981; Herreid, 1981; Wood and Randall, 1981 a, b;
Booth et al., 1982, 1984; Greenaway et al.,
1988; Herreid and Full, 1988), providing
valuable information about respiratory and
circulatory physiology. The role of hemocyanin (He) in oxygen transport and the importance of modulation of Hc-02 affinity has
also been studied extensively in many species
of crustaceans (see Mangum, 1983; Morris,
1991; Truchot, 1992; Morris and Bridges,
1994, for reviews). While there are extensive
data on the limits and costs of locomotion in
crustaceans, there are considerably fewer for
more ecologically relevant locomotory behaviors (e.g., Wood and Randall, 1981 a;
Wheatly et al., 1985, 1986; Weinstein and
Full, 1992, 1994; Weinstein et al., 1994), especially for amphibious species, including the
fresh-water crabs.
The superfamily Potamoidea have a wide
global distribution, and, while referred to as
"fresh-water crabs," they are commonly seen
foraging on land (Bishop, 1963; Greenaway
et al., 1983a; Burggren and McMahon, 1988;
Gherardi and Vannini, 1989; Charmantier,
1992). Egg-laden Potamonaute,s warreni
(Caiman) have been observed during daylight
several 100 m distant from water (van Aardt,
personal observation). Limited data exist for
lung structure in P.seudothelphusia sp. (Diaz
and Rodriguez, 1977; Innes et al., 1987) and
more for Holthuisnnn transversa (Martens)
(see Taylor and Greenaway, 1979; Farrelly and
Greenaway, 1993), but there is no comparable information for lung structure in Potamonautes spp. Morphological examination of the
gills of P. warreni suggests that the surface
area of the gills is smaller than in obligate water-breathing crabs (van Aardt, 1990a).
The few respiratory studies on the amphibious habits of this superfamily o f crabs
suggest that the Potamoidea are equally adept
at breathing air and water (Zoond and Charles,
1931; Greenaway et al., 1983a, b; van Aardt,
1990a). Preliminary investigations of hemolymph respiratory gas and metabolic state
of P. warreni exercised to exhaustion in either air or water concluded that these crabs
performed similarly in the two media (van
Aardt, 1990b). Different values exist for the
O2 binding affinity of P. warreni Hc (van
Aardt, 1990b, 1993), and to date there have
been no measurements of respiratory gas state
in postpulmonary hemolymph, which are required to assess lung function. There are some
postexercise physiology data for P. warreni
(see van Aardt, 1990b), but information as
to respiratory gas transport and Hc function
during realistic submaximal exercise are still
required. It was considered crucial to integrate whole animal studies with in vitro measurements to assess the respiratory physiology of P. warreni with respect to its habitat
a n d a m p h i b i o u s h a b i t . P. w a r r e n i o c c u r s i n
t h e h i g h v e l d o f S o u t h A f r i c a , a n d t h u s O2 u p t a k e o c c u r s at a l o w e r o x y g e n p a r t i a l p r e s s u r e
( P o 2 � 18 k P a ) a n d r e l a t i v e l y l o w h u m i d i t y
i n air. R e d u c e d a m b i e n t P o , w o u l d r e q u i r e e i ther an increase in the H c - 0 , affinity to acc o m m o d a t e lowered arterial Po2 or a m a r k e d
i m p r o v e m e n t i n t h e i n w a r d d i f f u s i o n o f O2
to m a i n t a i n arterial P o , . Further, e c o l o g i c a l l y
relevant studies of air-breathing by fresh water crabs should include m o r e c o m p l e t e hem o l y m p h sampling and relate l o c o m o t o r perf o r m a n c e to their use o f terrestrial habitats.
This study e x a m i n e s the role o f gills and
l u n g s o f P. w a r r e n i i n g a s e x c h a n g e w i t h a i r
and also while crabs are s u b m e r g e d by m a k i n g n o v e l m e a s u r e m e n t s in p o s t p u l m o n a r y ,
arterial and venous h e m o l y m p h . T h e import a n c e o f t h e g i l l s a n d l u n g s o f P. w a r r e n i i n
gas e x c h a n g e with air w a s assessed by subj e c t i n g crabs to n o n e x h a u s t i v e e x e r c i s e and
determining the respiratory gas and acid-base
status, as w e l l as m e t a b o l i t e
changes.
The
o x y g e n - b i n d i n g p r o p e r t i e s o f H c o f P. w a r reni were reinvestigated using physiological
salines. In this way, t h e s e in vitro d a t a c o u l d
b e u s e d t o g e t h e r w i t h t h e in v i v o m e a s u r e ments
to p r o v i d e
a more
complete
under-
standing of respiratory gas transport during
a c h a n g e f r o m w a t e r to a i r - b r e a t h i n g a n d in
r e s p o n s e t o e x e r c i s e i n air.
MATERIALS AND M E T H O D S
P o t a n n n a u t e s warreni (mass 78.5 ± 5.3 g, range
5 8 . 6 - 1 0 5 ) were captured during May 1995 in the Mooi
River running through Potchefstroom, South Africa. The
crabs were maintained at Potchefstroom University (altitude 1,443 m) in the laboratory at 23°C in plastic boxes
(dimensions 13 x 30 x 14 cm) with 3 cm of Mooi river
water in the bottom, enabling the crabs to immerse themselves completely. Each box also contained a PVC tube
providing both a hiding place simulating a burrow and a
perch above the water line. The river water was collected
from the Mooi river on a regular basis. The crab boxes
were cleaned twice a week, the crabs were fed cat-food
biscuits (Catmore, S.A. Oil Mills) and were maintained
on a 12:12 h light: dark regime.
Potamonautes warreni were prepared for experiments
at least 24 h prior to sampling. For "air-breathing" treatments the water level was lowered to a thin film at the
bottom of the box to maintain humidity in the boxes, but
to ensure that the crabs were breathing air. Crabs were
exposed to 1 of 4 different treatments while breathing
air or were required to breathe water: (a) resting c r a b s crabs sampled after breathing air for a minimum o f 24 h
(Air group, N = 6); (b) 5-min slow e x e r c i s e - c r a b s
breathing air for a minimum of 24 h and then exercised
on a treadmill for 5 min at constant speed of 1.8 m-min-I
(Slow group, N = 6); (c) 20-min slow e x e r c i s e - c r a b s
breathing air for a minimum of 24 h and then exercised
for 20 min on a treadmill at constant speed of 1.8 m-min 'I
(20SIow group, N = 6); and (d) 20-min fast e x e r c i s e crabs breathing air for a minimum of 24 h and then exercised for 20 min on a treadmill at constant speed of 3
m-min-I (20Fast group N = 6).
Air-breathing crabs were exercised on a custom-made
treadmill (University of Potchefstroom, van Aardt, 1990b)
supplied with a constant flow of humidified air. One half
of the treadmill was covered with dark plastic, while the
other half was exposed to the light. The crabs continued
running in the darkened side in all experiments and thus
were judged never to be exercised to exhaustion.
The water-breathing treatment comprised crabs totally
immersed in water for 24 h before hemolymph sampling.
The crabs were housed in boxes filled with water up to
the lid and supplied with air stones to maintain oxygenation of the water. The water-breathing crabs were
sampled in situ, that is, hemolymph was withdrawn from
the crabs while they were held submerged (water group,
N
=6).
Hemolymph S a m p l i n g . - T o facilitate hemolymph sampling, the carapace of the crabs was drilled with an OMNI
1000 battery-operated drill fitted with dental drills to provide access to the pericardial cavity and the pulmonary
vein. The crabs were drilled a minimum of 24 h prior to
h e m o l y m p h sampling. The position of the pericardium
and the efferent pulmonary vein were clearly distinguishable on the dorsal carapace of P. warreni, as previously described for other air-breathing crabs with lungs
(Greenaway and Farrelly, 1990; Farrelly and Greenaway,
1993). From each crab a hemolymph sample (200 ilL)
was taken from the efferent pulmonary vein (pulmonary
hemolymph). A second (400 ttL) sample was taken from
the pericardial cavity (arterial hemolymph) and a third directly from the venous sinus (venous hemolymph, 600
pL) by puncturing the arthrodial membrane at the base
of the last walking leg (handling time during hemolymph
sampling was approximately 1 min). Hemolymph samples were taken with chilled 1 mL plastic syringes and
kept on ice for the duration of the hemolymph respiratory
gas and pH analysis.
For recovery measurements of L-lactate, exercised animals were placed in their boxes and supplied with humidified air. Hemolymph samples were taken at 0.5, 1, 2,
5, and 24 h postexercise for both 20 min slow and 20 min
fast exercise treatments. The hemolymph was deproteinized and analyzed for L-lactate as described below.
Hemolymph Respiratory Gas a n d p H Analysis. — Hemolymph samples were immediately analyzed for respiratory gas and pH. The P o , was determined at 23°C with
a flow-through micro-oxygen probe (Activon, M I 1 6 - 7 3 0 )
which held a 15-pL sample, connected to a P H M 7 3
pH/blood gas monitor (Radiometer, Copenhagen, Denmark). The outlet of the electrode chamber was connected
to 5 cm PE90 catheter tubing to prevent inward diffusion of 0 , during the measurement. Hemolymph oxygen
content (Co,) was measured with a Tucker chamber thermostatted at 30°C (Tucker, 1967; Bridges et al., 1979)
connected to a PHM73 and monitored on a pen recorder
(REA112 Radiometer, Copenhagen). The m a x i m u m O2
binding capacity was determined by measuring the absorbance at 340 nm of I O-pL hemolymph sample diluted
in I mL of water. The absorbance was measured with a
Pye Unicam PU8800 UV/vis spectrophotometer and used
to calculate the He concentrations using the extinction coefficient 2.7 £ " , „ (Nickerson and Van Holde, 1971). Rel-
ative 0 , saturation of He was then calculated for each
individual sample, assuming He mass of 72kD. This
method proved quicker and just as consistent as measuring [ 0 , ] of air-equilibrated hemolymph samples.
Hemolymph CO, content (Cco,) was measured with
a P c o , electrode (model E5037/SI) connected to a PHM73
pH/blood gas monitor (Radiometer, Copenhagen, Denmark) using a Cameron chamber thermostatted at 30°C
(Cameron, 1971), calibrated with fresh 15 m m o l L -~''
N a H C O , standards in 300 mmol L ' NaCl. Changes in
P c o , were timed with a stopwatch until a linear rate of
change was recorded and the P c o , then interpolated to injection time (i.e., time 0). Hemolymph pH was measured
with a How-through micro pH-probe at 23°C (Activon,
M 1 1 6 - 7 0 5 ) , arranged as for the 0 , electrode and connected to the PHM73.
Hemolymph Analysis f o r Metabolites a n d l o n s . - H e m o l y m p h calcium was determined with an atomic absorption spectrophotometer (model GBC 900). A portion
of hemolymph was deproteinized with an equal volume
of HNO, (0.1 mol-L-1), centrifuged, and a sample of the
supernatant diluted with 7.1 mmol L ' LaCl3.7H,O (ratio
1:50). Hemolymph and leg muscle (see below) L-lactate
were measured with a test kit (number 139 084,
Boehringer, Mannheim), and hemolymph and leg muscle glucose with Sigma Diagnostics test kit number 510.
The hemolymph was prepared by mixing with an equal
volume of 0.6 mol-L"1 HCIO, and then neutralized with
2.5 mol-L-1 K,CO, (ratio 5:1, respectively).
Tissue Acquisition a n d P r o c e s s i n g . - L e g - m u s c l e samples
were obtained from crabs in each of the 4 air-breathing
treatments (see above). Crabs were encouraged to auautomize the second walking leg (the largest leg) from
which the complete muscle was immediately excised and
denatured in 2 mL of ice-cold HC1O4 acid (0.6 mol�L-').
The muscle tissue was homogenized (Wheaton type glass
homogenizer), centrifuged for 10 min at 10,000 g and the
supernatant removed. The pellet was resuspended in 0.7
ml of 0.4 mol�L-' HCIO, and centrifuged again. The second supernatant was removed, pooled with the first and
the whole neutralized with 0.8 ml of K=CO, (3.75
mol-L '). The resultant solution was again centrifuged and
the final supernatant used for lactate and glucose analysis. For glycogen analysis, a known amount of tissue extract was hydrolyzed with glucoamylase as described in
Bergmeyer (1985) and then analyzed for total glucose
content (Sigma test kit number 510).
Construction o f 0 , Eguilibrium C u r v e . s . - P o o l e d hemolymph from 6 P. warreni in each of the 4 treatment
groups, (a) rest in air, (b) rest in water, (c) 20Slow. and
(d) 20Fast, was used for the construction of oxygen equilibrium curves (OEC). The OEC were made at 23°C using a thin-layer optical cell method (Dolman and Gill,
1978; van Aardt, 1992). In this method, a 75-llm thick
film of hemolymph was placed in a small chamber where
the composition of gas could be controlled in a stepwise
dilution series. The absorbance of a deoxygenated hemolymph sample (He absorbance) was measured at 340
nm and the change in absorbance was monitored during
stepwise addition o f f , until full oxygenation. Five equilibrium curves spanning the normal physiological pH
range were produced for hemolymph from each experimental group by varying the C O , in the gas mixture
( 0 . 0 - 2 . 9 % CO,). The required equilibration gas mixtures
Fig. 1. The h e m o l y m p h oxygen content in Potamonautes warreni at rest breathing either air or water, and
in crabs exercised in air at either slow (1.8 m - m i r 1 ) or
fast speed (3 m-min 1). The 0 in the legend indicates the
water-breathing group was significantly lower than all
other groups. The "p" and "a" indicate a difference with
respect to pulmonary or arterial hemolymph, respectively.
of N" O2, and C O , were prepared using a gas mixing pump
(W6sth�ff, Bochum, Germany) and medical grade gases.
For each curve an 80-pL h e m o l y m p h sample was
equilibrated to the required gas mixture in a BMS 2 (Radiometer, Copenhagen) and the pH of the sample was
measured near the P5o using a micro-pH probe (Activon,
M I 1 6 - 7 0 5 ) at 23°C. The curves were analyzed for 0 ,
affinity by calculating the log P5U and cooperativity of
0 , affinity (n50) according to the Hill equation, using values between 25 and 75% saturation.
The effect o f L-lactate on the affinity o f the hemolymph of P. warreni was tested by enriching a pooled
hemolymph sample from air-breathing crabs with L-lactate. Briefly, a h e m o l y m p h sample was centrifuged at
161,000 g for 20 min to sediment the He and 10% of the
supernatant was replaced with either Potamon Ringer solution (NaCI 240, KCl 5.85, CaC], 15.8, and M g S 0 4 3.21
mmol�L-') or with Ringer solution containing 100
mmol�L-' L-lactate. The O2 equilibrium curves were constructed as described above.
Nonbicarbonate Buffering Capacity of the H e m o l y m p h . The buffering capacity of the h e m o l y m p h was investigated by equilibrating hemolymph for 20 min at 23°C
with gas mixtures of different P c o , and determining the
C e o , and pH as described above. The buffer capacity was
determined for a pooled hemolymph sample from crabs
in each of the 3 treatment groups: (a) air, (b) 20Fast, and
(c) 20Slow. The O=, C O " and N, mixtures were prepared
with a gas-mixing pump and then humidified before flowing over the hemolymph in the tonometers of a BMS2
blood/gas analyzer (Radiometer). The proportion of CO,
in the equilibration gas ranged from 0 . 0 1 - 4 % to provide
pH values which spanned in vivo pH measurements (pH
7.06-8.24).
Analysis of Results.-Bartletts x2 was used to test for homogeneity of variance in the data. Statistical analyses on
hemolymph gas parameters were carried out with two-way
analysis of variance (ANOVA), and one-way ANOVA was
used for metabolite and ion comparisons, using Systat 5.03
statistical package. Post hoc testing was carried out with
Fig. 2. The hemolymph oxygen partial pressure in Potamonautes warreni at rest in either air or water, and in
crabs exercised in air at either slow (1.8 m-min-1) or fast
speed (3 m-min'). The * in the legend indicates the water-breathing group was significantly different to resting
crabs in air. The "p" and "a" indicate a difference with
respect to pulmonary or arterial hemolymph, respectively,
and "r" a difference with respect to resting crabs.
Tukey HSD multiple means comparison or contrast analysis. Oxygen equilibrium curves were analyzed by analysis of covariance after testing for heterogeneity of slopes.
Significance level was taken as P = 0.05 and all data are
presented as mean ± SEM, unless stated otherwise.
RESULTS
Hemolymph Gas Transport
The oxygen contents (Coz) of pulmonary
and arterial hemolymph were very similar in
P. warreni breathing air (approximately 0.7
mmol-L-') regardless of treatment (Fig. 1), but
the C02 was significantly reduced in the venous hemolymph (P � 0.001). The arterialvenous 0 , content difference was 0.15
mmol-L"1 in air-breathing crabs at rest, but increased to 0.27 mmol-L-1 after 20 min at the
fast walking speed (3 m-min-1). In P. warreni
breathing water, the hemolymph O2 content
was significantly lower than in crabs breathing air. In crabs breathing water the arterial
hemolymph had a greater Co� (0.5 ± 0.13
mmol-L-1) than the pulmonary and venous
hemolymph, but the high and heterogenous
variance in these data made statistical confirmation difficult. While the venous Co2 of
crabs in water (0.31 ± 0.07 mmol-L-1) was
significantly lower than in the crabs breathing air (0.5 ± 0.04 mmol-L-1), the mean arterial-venous difference was maintained at 0.19
mmol-L"1 (Fig. 1).
Within each of the four air-breathing treatment groups, the arterial and efferent pulmonary oxygen partial pressures (Po2) were
Fig. 3. The hemolymph pH in Potamonautes warreni at
rest in either air- or water-breathing, and in crabs exercised in air at either slow (1.8 m m i n or fast speed (3
m-min-1). The 0 in the legend indicates the treatment was
significantly different from all other groups.
similar, but decreased significantly in the venous hemolymph (Fig. 2). Both postpulmonary and arterial Po2, however, decreased
with increase in exercise regime, with the result that the PPo2 = 6.34 ± 0.92 kPa ( l k P a =
7.5 torr) measured in crabs exercised for 20
min fast (Fig. 2) was significantly lower than
the P o, of resting crabs (9.19 ± 1.13 kPa),
(P �z.001).
In water-breathing P. warreni, the hemolymph Po2 was generally significantly lower
than in air-breathing crabs (P �0.001). Furthermore, the P.,oz (4.46 ± 1.62 kPa), but not
the Ppo, (2.51 ± 1.26 kPa), was significantly
greater than the Pvo, (0.71 ± 0.07 kPa) (Fig.
2). The O2 saturation of hemocyanin (He) in
pulmonary vein samples was between 80 and
90% in crabs breathing air, but significantly
less in venous hemolymph. In contrast, the
He of efferent pulmonary hemolymph from
crabs breathing water was lower and only
52% saturated (P �0.01). There were no differences in the He saturation of postpulmonary, arterial, and venous samples taken
from crabs breathing water.
The pH values of the hemolymph were
similar in the arterial, postpulmonary, and venous samples within any treatment group
(Fig. 3). A pH of 7.4 was maintained in P.
warreni at rest in either air or water, but the
hemolymph became significantly acidotic
during 20 min of slow exercise (pH 7.33 ±
0.02) and more so after 20 min of fast exercise (pH 7.08 ± 0.02) (P � 0.001) (Fig. 3).
Within any treatment, the carbon dioxide
Fig. 4. The hemolymph CO, content in Potamonautes
warreni at rest in either air or water, and in crabs exercised in air at either slow (1.8 m min ') or fast speed (3
m-min-'). The if in the legend indicates the treatment was
significantly different from all other groups.
content of the hemolymph did not differ between efferent pulmonary, arterial, and venous samples (Fig. 4). The mean Ccoz of pulmonary, arterial, and venous samples decreased from 13 ± 0.6 mmol-L-1 in crabs at
rest in air to 10.6 ± 0.5 mmol-L"1 after 20 min
of fast exercise (P � 0.001). The Cco2 of
crabs in water (6.9 ± 1 . 1 mmol-L"1) was only
half that of crabs in air (Fig. 4).
Hemolymph Metabolites and Ion Status
The hemolymph Ca concentration increased steadily with increased intensity of
exercise in air from 10.7 mmol-L-1 in crabs
at rest to 15.0 mmol-L-1 in crabs exercised for
20 min at fast speed (Table 1). The concentration of hemolymph glucose was similar in
all groups sampled and was normally similar
to the concentrations measured in leg muscle (Table 1). However, there was a significant increase in muscle glucose after 20 min
of fast exercise (P �0.001) (Table 1). Muscle glycogen levels did not change significantly and averaged 165 mmol.kg-' glucose
units (Table 1).
In crabs resting in either air or water the
hemolymph L-lactate levels were below 0.1
mmol-L-1. L-lactate levels increased significantly after 5 min of slow exercise (1.8
m-mirr1) to 0.8 rnmol-L"' (Table 1) and again
after 20 min of fast exercise (3 m-min-') to
3.6 mmol-L-' (P �0.001). L-lactate levels in
the leg muscle of resting crabs and the two
Table 1. Hemolymph calcium and hemolymph and leg muscle tissue metabolites in air-breathing, exercised, and
water-breathing Potamonautes warreni. Glycogen is expressed as glucose units. The * indicates that the treatment is
significantly different from the resting group a n d * a significant difference between muscle and hemolymph concentration.
groups subject to slow exercise were normally similar to the hemolymph values. Tissue L-lactate levels in the 20-min fast exercise group were lower than those in the hemolymph (Table 1) (P = 0.049). Removal of
L-lactate from the hemolymph required 2 h
in P. warreni exercised for 20 min at a slow
speed, but after 20 min of fast exercise required up to 5 h (Fig. 5).
In Vitro Studies
Oxygen equilibrium curves were constructed using hemolymph collected from P.
warreni at rest in either air or water, as well
as from crabs exercised for 20 min at either
the slow (1.8 m-min1) or fast (3 m-min1)
speeds. The Bohr shift (Alog P5./ApH) was
large and ranged from -1.45 to -1.80 for the
4 treatment groups. Analysis of the data
Fig. 5. Hemolymph L-lactate concentration during 24-h
recovery from 20-min exercise in air at slow speed (1.8
m-min-1) and fast speed (3 m ' m i n in Potamonautes warreni. Time 0 is immediately after 20 min of exercise. The
"*" indicates significantly elevated above resting (Table
1) and "#" a significant difference between fast and slow
treatment groups. For this analysis only the critical P
value was set at a more stringent 0.01.
showed that the O2 affinities of the Hc were
very similar in resting and 20-min slow exercise crabs (P50 at pH 7.4 was 4.9 and 4.8
kPa, respectively) (Fig. 6a). The H c - 0 , affinity increased to a similar extent in 20-min fast
exercise and water-breathing crabs, P5" = 2.5
kPa at pH 7.4. The O2 binding cooperativity
(n5o = 3.5) was similar in the four treatment
groups (Fig. 6b).
In vitro measurements using whole hemolymph showed that L-lactate had no effect on H c - 0 , affinity nor cooperativity o f
Fig. 6. (a) H c - 0 , affinity as log P50 of Potamonautes
warreni in hemolymph taken from crabs at rest in either
air or water, and from crabs exercised in air at either slow
(1.8 m-mirr1) or fast (3 m ' m i n speed. Note: log P50
provided as log (kPa-10"1) to avoid negative values and
since the product is very similar to that produced by P5o
50
as torr. (b) The HC-02 binding cooperativity (n5o) for the
same equilibrium curves.
DISCUSSION
Air and Water-breathing
Potamonautes warreni was considerably
better at breathing in air than in water. Since
this crab has similar oxygen uptake (MO,) in
air a n d w a t e r ( v a n A a r d t , 1 9 9 0 b ) a n d t h e O2
Fig. 7. (a) Hc-0, affinity as log P5(l of Potamnrrautes
warreni in hemolymph taken from air-breathing crabs at
rest (low L-lactate) and with increased L-lactate content.
Note: log PS11 is provided as log (kPa.)O '). (b) The HcO2 binding cooperativity (n5") for the same equilibrium
curves.
O, binding at the concentration of 11.4
mmol-L"1. The Alog P50/ApH relationship
could thus be described by a common equat i o n : l o g P , o ( k P a - 1 0 1 ) = 1 1 . 0 5 - 1 . 2 8 - p H , r2
= 0.93 (Fig. 7a, b).
The Hc content in the hemolymph used to
construct the nonbicarbonate buffer lines was
similar for all three treatments, but the buffering capacity (A[HCO3~]/ApH) from the two
exercise groups was significantly greater than
for the resting group (Table 2). The regression equations for nonbicarbonate buffer lines
are given in Table 2. While heterogeneity of
the variance in the data (different slopes) rendered the data unsuitable for further analysis
of covariance, the nonbicarbonate buffer lines
from the two exercise groups appeared elevated from air-breathing resting crabs.
Table 2.
requirement thus remains constant, the greater
hemolymph O2 in air-breathing crabs is indicative of easier gas exchange compared
with water-breathing P. warreni. Similar postpulmonary and arterial oxygenation in crabs
breathing air shows that either the gills and
lungs of P. warreni function similarly in gas
exchange or that there is preferential direction of hemolymph to the lungs (Taylor and
Greenaway, 1984; AI-Wassia et al., 1989).
The P�o2 of air-breathing crabs at rest was
comparable to previous values for this species (van Aardt, 1990a, 1993). The P,,02 of
P. warreni in the present study was within the
range measured by van Aardt (1993) and similar to the Po, of 7.48 kPa in Holthuisana (see
Greenaway et al., 1983b) and in other amphibious (e.g., Wood and Randall, 1981b) as
well as terrestrial crustaceans (e.g., Greenaway et al., 1988; McMahon and Burggren,
1988; Farrelly and Greenaway, 1994; Adamczewska and Morris, 1994a).
In P. warreni, breathing water, the postpulmonary Po2, C o " and He saturation values were consistently lower than those for arterial hemolymph, significantly so for 0 , content (paired t-test). Thus, the gills appear
more important than lungs in O2 uptake from
water. Nonetheless, the mean arterial-venous
Co, difference of air-breathing P. warreni was
maintained by crabs breathing water and
L-lactate levels remained low. Thus, the crabs
maintain aerobic metabolism while at rest regardless of the respiratory medium. The constant a-v Co2 difference together with the similar MOZ of this species in air and water (van
Aardt, 1990b) means that cardiac output is
constant. The relative internal hypoxia of P.
warreni in water is consistent with impaired
O, exchange compared to crabs in air and
Nonbicarbonate buffering capacity of the hemolymph of Poramonautes warreni in air.
Fig. 8. Model 0, equilibrium curves at 23°C for Potamonautes warreni based on log P,,, and n50 determined
from in vitro measurements. The model curves were constructed for pH 7.4.
thus O2 uptake can only be facilitated with a
greater APo2 across the gas exchange organs.
The general reduction in hemolymph oxygenation will severely reduce the scope for
activity in water-breathing P. warreni. An increase in H c - 0 , affinity in water (Fig. 8)
would further assist O2 extraction from the
water, reducing the need for hyperventilation.
The diffusion limitation for O2 uptake across the
gas exchange organs can be defined as Dill (Piiper, 1982; Innes et al., 1987) and values are
provided by Taylor and Taylor (1992) for a
range of species. The majority o f terrestrial
crabs show Ldifr 0 . 4 - 0 . 6 , where 0 indicates
perfect equilibrium and 1 severe diffusion
limitation. The Ld;ff for resting P. warreni in
air was typical of land crabs at 0.57, but increased to 0.78 when P. warreni was required
to breathe water. Thus, P. warreni is clearly a
facile air-breather and is more diffusion-limited
in water than are aquatic crabs (Taylor and Taylor, 1992).
The general model for amphibious animals
predicts a respiratory alkalosis during immersion, causing an increase in hemolymph
pH (Taylor and Butler, 1978; review in
McMahon and Burggren, 1988). Analysis using a Henderson-Hasselbalch diagram (Fig.
9) revealed that during immersion P. warreni
experienced a metabolic acidosis which
masked the usual respiratory alkalosis, thereby
maintaining pH at 7.4 as in air-breathing
crabs. The source of the metabolic acidosis
was not, however, L-lactate production (see
Table 1). Constant pH in this species during
Fig. 9. The pH/HCO-, relationship for Potamonautes
warreni hemolymph at 23°C showing the response to exercise and immersion. The values shown are the mean venous hemolymph pH and calculated HCO 3 to illustrate
major acid-base changes. The fco, (kPa) isopleths were
derived from measurements in vitro and the HendersonHasselbalch relationship using aCO� = 0.043 and pK =
5.986. The three appropriate buffer lines determined from
hemolymph from separate groups of crabs at rest in air,
and exercised in air at slow or fast speed are also provided ( - , rest; - - , slow; - - - , fast).
air- and water-breathing has been described
previously (van Aardt, 1990b), but this is also
maintained in the amphibious Leptograpsus
variegatus (Fabricius) (see Morris and Butler, 1996), Cardisoma carnifex (Herbst) (see
Cameron, 1981), Cyclograpsus lavauxi H.
Milne Edwards (see Innes et al., 1986), and
Herrtigrapsus nudus (Dana) (see Morris et al.,
1996). The increased metabolic acid load in
the hemolymph of immersed P. warreni could
have originated either from the tissues or by
ion exchange through the gills during immersion, leading to a net loss of nonrespiratory
base (Wood and Cameron, 1985; Cameron,
1985). Potamonautes warreni can be considered truly amphibious, since the crabs maintained acid-base status and a similar arterialvenous O� content difference in both air and
water despite fluctuations in the hemolymph
respiratory gas variables.
Exercise in Air
In P. warreni exercised in air, the hemolymph remained well oxygenated, indicating that the gas exchange surfaces did not
limit oxygen uptake. At slow exercise speed
(1.8 mmin"1) hemolymph acid-base and respiratory gas perturbations were much smaller
Table 3.
taceans.
Measured changes in hemolymph pH and L-lactate, before and after exercise, in various species of crus-
than at the fast speed (3 m-min-'). Sustained
locomotion at the slow speed is equivalent
to 0.4 body lengths-s"1 (average carapace
width 65 mm). Thus, the exercise capacity
of P. warreni on land is comparable to some
other amphibious crabs (e.g., Wood and Randall, 1981b; Full and Herreid, 1984; Full et
al., 1985). The arterial-venous O2 difference
almost doubled during exercise, coinciding
with a doubling of Mo2 after exhaustive
exercise (van Aardt, 1990b). Thus, the He
was important in O2 transport and minimal
changes in cardiac output were required.
However, decreased postpulmonary and arterial P02 in the 20Fast group implies that hemolymph is resident at the respiratory exchange surfaces for a shorter time than is necessary for the P02 to equilibrate to the same
extent as in the resting crabs. This is exemplified by the increase in L,., from 0.57-0.77,
similar to the L diff in water. While postpulmonary and arterial P02 decreased, the He
saturation nonetheless remained high during
all exercise treatments, indicating that the Hc
maintained its role in oxygen transport.
The in vitro analysis of Hc-O� affinity characterized the pigment as having a high Hc
subunit cooperativity (n5o = 3.5) and a high
sensitivity to pH (41ogP5o/OpH = -1.55).
While the n50 = 3.5 was similar to previous
values for this species, the Bohr shift was
greater and the O, affinity lower than previous values (van Aardt, 1990b, 1993). Special
care was taken in the present study to use
fresh hemolymph (never frozen), the appropriate physiological saline solutions, and a realistic Pco� range for making the OEC, so
that the data could be used for interpreting the
in vivo information. The decrease in pH from
7.4 (rest) to 7.08 in the 20Fast group should
have resulted in a substantial decrease in HcO, affinity (factor of 2.9) according to the
Bohr shift. However, in vitro analysis of HcO, on the 20Fast group hemolymph showed
an increase in intrinsic Hc affinity when compared at pH 7.4 to that of the resting crabs
(Fig. 8). This increase in affinity would be advantageous by partially offsetting the effects
of the Bohr shift and assisting in O, uptake.
While both organic and inorganic factors are
known to affect H c - 0 , affinity in crustaceans
(Morris et al., 1985; Morris, 1990; Truchot,
1992), the Hc of P. warreni was insensitive
to L-lactate. Since hemocyanins of terrestrial
crustaceans, including some Potamoidea, appear to be characterized by low pH sensitivity and low or no sensitivity to L-lactate
(Morris et al., 1988; Morris, 1991), the Hc
of P. warreni shows characteristics of Hc of
both air- and water-breathing crustaceans.
The decrease in hemolymph pH of 0.36 pH
units (venous) following 20-min fast exercise
was similar to the ApH for other crustaceans
exercised for 2 0 - 2 5 min (Table 3). The acidosis was mixed (Fig. 9), metabolic/respiratory, as is common for other crustaceans
(Smatresk et al., 1979; Greenaway et al.,
1988; Forster et al., 1989, Adamczewska and
Morris, 1994a), but in P. warreni the respiratory component was predominant. While
CaCO, mobilization from the carapace is
thought to contribute to buffering hemolymph
acidosis (deFur et al., 1980), the increase in
hemolymph Ca in 20Fast group occurred over
an unusually short time period (Wood and
Randall, 1981b; Cameron, 1985; van Aardt,
1988). The range of Ca concentrations measured are within the variation found in rest-
ing P. warreni (11-16 mmol-1-1; see van Aardt,
1988, 1990a). A rapid exercise-induced elevation of hemolymph calcium concentration
occurs in other air-breathing crabs, Gecarcoidea natalis (Pocock) and Cardisoma hirtipes (Dana) (Adamczewska, unpublished),
and is most likely a movement of calcium from
an intracellular pool into the hemolymph.
A significant increase in muscle glucose in
the 20Fast group but not in the hemolymph
implicates mobilization from the endogenous
muscle glycogen stores. Glycogen levels were
high in the leg muscle of P. warreni ( - 1 3 5
mmol.kg-' glucose) and approached those of
laboratory-fed G. natalis, 2 3 0 - 2 8 0 mmol.kg-'
glucose (Adamczewska and Morris, 1994b).
Thus, glucose mobilization during 20-min exercise makes no appreciable difference to the
glycogen store. Hyperglycemia occurs in
crustaceans during stress or oxygen shortage
(Telford, 1968; Taylor and Spicer, 1987; van
Aardt, 1988). In P. warreni, the increase in
hemolymph L-lactate (3.6 m m o l l 1 ) after exercise was low compared to values previously
found for P. warreni and other species with
a similar decrease in pH (Table 3; van Aardt,
1990b). Furthermore, in contrast to the present study, exercised G. natalis showed a 5fold increase in glucose coupled to a large
anaerobiosis (Adamczewska and Morris,
1994b). It seems likely that the ecologically
realistic, submaximal exercise regime used in
the current study promoted a different efflux
of L-lactate and H+ from the muscle compared to these previous studies. Employing
the analysis used by Greenaway et al. ( 1988),
the 20Fast group experienced a primarily respiratory acidosis, whereas exhausted P. warreni (see Van Aardt, 1990b) apparently had
a relatively larger metabolic component to the
acidosis. Comparisons of crustacean species
having varied air-breathing ability revealed
that a pH decrease of 0 . 2 - 0 . 5 pH units during exercise can be accompanied by quite different hemolymph [L-lactate] changes (Table
3). The differences are most likely the result
o f varied exercise regimes (Adamczewska
and Morris, 1994b; Reidy et al., 1995) and
variable rates of H ' and lactate efflux from
muscle tissue (Benade and Heisler, 1978).
The maximal recovery rate for hemolymph
L-lactate in P. warreni is unexceptional compared to other crabs (Wood and Randall,
1981b; Henry et al., 1994). The low hemolymph and muscle lactate levels after ex-
ercise show that P. warreni are well able to
aerobically support locomotion on land.
The gills become relatively more important
than the lungs in gas exchange by waterbreathing P. warreni. During air breathing, either the lungs and gills have similar gas-exchange properties or most of the hemolymph
passes through the pulmonary rather than the
branchial circuit. Detailed studies of hemolymph flow and distribution are required
to determine the precise functioning of the
twin gas-exchange organs. While oxygen uptake in exercised P. warreni became progressively diffusion-limited and P�,o2 was
lowered as a consequence, the increase in
HcO, affinity partially offset the Bohr shift
and
maintained
Hc-02
saturation
and
O2
transport. Potamonautes warreni lives at altitudes approaching 1,800 m and ambient P02
may fall to 75% of sea-level values, �80%
in the present study. However, the efferent
pulmonary and arterial P02 are comparable to
or better than those in many air-breathing
crabs. Thus, there must be a relative decrease
in diffusion resistance to aid 0 , uptake, especially since this species shows no special
adaptation of the Hc to breathing air with a
lowered Po,. Further studies are necessary to
determine the structure and degree of development of the lung, as well as further studies to compare exercise performance in water and air.
ACKNOWLEDGEMENTS
AMA and SM thank Prof. G. C. Loots and all members of the Zoology Department, Potchefstroom University for CHE, especially Ryno Erdmann, for their assistance and generous hospitality. This work was supported
by ARC grants, a Sydney University Special Studies Programme Grant and PU for CHE funds to SM, and by FRD
grants to WJvA. This work was carried out while AMA
was in receipt of an Australian Post Graduate Award.
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RECEIVED: 8 October 1996.
ACCEPTED: 11 March 1997.
Addresses: (AMA, SM) School of Biological Sciences
(A08), University of Sydney, Sydney, N.S.W., Australia
2006. ([email protected]) (e-mail: stevems@bio.
usyd.edu.au); (WJA) Zoology Department, Potshefstroom
University for CHE. Potchefstroom 2520, South Africa.
([email protected])
ANNOUNCEMENT
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