Respiratory and acidbase responses during migration and to exercise by the terrestrial crab Discoplax (Cardisoma) hirtipes, with regard to season, humidity and behaviour
Integrative and Environmental Physiology, School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK
e-mail: steve.morris{at}bristol.ac.uk
Accepted 3 October 2005
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Summary |
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Key words: land crab, Cardisoma, Discoplax, exercise, migration, respiration, acidbase, behaviour
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Introduction |
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D. hirtipes have well-developed lungs (Farrelly and Greenaway,
1992,
1993
) and are competent air
breathers (Adamczewska and Morris,
1996
; Morris and Dela-Cruz,
1998
) but require periodic immersion to facilitate nitrogen
excretion (surface water for drinking is inadequate;
Dela-Cruz and Morris, 1997b
).
Thus, the necessity for immersion apparently limits the dry-season range of
D. hirtipes. A reduced locomotor capacity in land crabs during the
dry season would further constrain their distribution, foraging ranges and
ability to escape from predators.
There are several studies of exercise capacity in a variety of decapod
crustaceans (for reviews, see McMahon,
1981; Herreid and Full,
1988
; Full and Weinstein,
1992
; Adamczewska and Morris,
2000b
; Morris,
2002
) but very few of migration physiology, or of seasonality, or
under field conditions (e.g. Adamczewska and Morris,
2001a
,b
).
Increasing the walking speed of terrestrial crabs induces fatigue and reduces
their distance capacity (e.g. Wood and
Randall, 1981a
; Full and
Herreid, 1984
; van Aardt,
1990
; Weinstein and Full,
1992
; Adamczewska and Morris, 1998a). In most crustaceans, a
functional shortage of O2 during locomotion is associated with some
degree of anaerobiosis, leading to a lactacidosis that is often exacerbated by
a respiratory component (e.g. Wood and
Randall, 1981b
; Booth et al.,
1984
; Greenaway et al.,
1988
; Forster et al.,
1989
; Henry et al.,
1994
; Adamczewska and Morris,
1998
).
Seasonally variable rainfall and/or ambient humidity has received
relatively little consideration as a limiting factor in exercise physiology of
crabs, although dehydration is known to interfere with O2 transport
in land crabs (Burggren and McMahon,
1981). In ghost crabs, dehydration markedly decreased the maximum
aerobic scope (Weinstein et al.,
1994
). However, the terrestrial G. natalis exhibited a
seasonal respiratory acidosis that correlated with increased activity and
respiration rate during the wet season rather than with hydration state,
although hydration state may facilitate the altered behaviour
(Adamczewska and Morris,
2000a
). The role of ambient humidity in limiting locomotor
capacity and activity is important in understanding the migration physiology
of D. hirtipes.
The majority of studies of locomotion in crustaceans have used exhaustive
exercise regimens under laboratory conditions, resulting, unsurprisingly, in a
lactacidosis (e.g. McMahon et
al., 1979; Greenaway
et al., 1988
; Forster et al.,
1989
; Adamczewska and Morris,
1994a
,b
;
Morris and Adamczewska, 2002
).
However, field studies of G. natalis on Christmas Island revealed
that these crabs migrated over more than 5 km without becoming anaerobic.
Generally, crustaceans, including land crabs, are not adept at re-oxidising
lactate and recover relatively slowly from an O2 debt (e.g.
Henry et al., 1994
;
Adamczewska and Morris, 1998
;
Morris and Adamczewska, 2002
).
Slow lactate oxidation is not necessarily problematic if the lactacidosis is a
consequence of a `one-off' sprinting event (e.g. predator avoidance) with a
protracted recovery period. However, the blue crab migration is an endurance
activity and, if the crabs become anaerobic, low rates of oxidation would lead
to both more rapid and more persistent lactacidosis, which will compromise the
migration. Thus, it is important to determine the extent of any such acidosis
and the recovery capacity of D. hirtipes or whether they limit,
behaviourally, migration exercise to within their maximum aerobic scope (MAS).
Furthermore, it should be determined whether low humidity and dry-season
conditions increase the likelihood of anaerobiosis. Is there a respiratory or
energetic penalty for exercising under dry conditions or is the risk limited
to problems of ion and water homeostasis (e.g.
Harris and Kormanik, 1981
;
Dela-Cruz and Morris, 1997b
),
which constrain the commitment to exercise?
The migration of blue crabs on Christmas Island requires the establishment
of the wet season and this is clearly a general determinant of seasonal
behaviour (Hicks et al., 1990)
and, thereby, of the energetic demands of locomotion. Preparation for the
migration requires a complex of physiological changes (e.g. maturation of
reproductive structures and gametes;
Linton and Greenaway, 2000
)
but these are concomitant with a highly dichotomous activity level. However,
it is unclear to what extent increased humidity may release constraints on
behaviour and thus allow an increased locomotion and therewith the migration.
Alternatively, seasonal changes in physiology may equip the crabs for
migration in anticipation of the rigours of the wet season. The energetic
investment in walking is seasonal and indicates a proclivity, and a greatly
increased commitment, for walking during the wet season. Thus, behaviour may
be modulated by ambient humidity but, at the same time, changes in underlying
physiological state may also be required in order to facilitate increased
levels of exercise.
To establish direct (physiological) and possible indirect (behaviourally mediated) influences of a seasonal rainfall pattern and humidity on exercise and migration in D. hirtipes, both field and laboratory studies were conducted in which respiratory, acidbase and metabolite status was determined. On Christmas Island, the respiratory and energetic status of D. hirtipes during the wet season (migration) was compared with that of crabs in the dry season (quiescent), both in their natural state in situ and with an additional load of 5 min enforced walking. In the laboratory, the influence of humidity was probed by comparing crabs exercised for 5 min at either 40% or 90% relative humidity. Recovery rates and duration following an exercise-induced lactacidosis were determined. The data are evaluated in regards to the seasonal ecology and breeding migration of D. hirtipes and land crabs generally.
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Materials and methods |
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Exercise in the laboratory simulations
Male and female blue crabs were used in equal numbers for experiments with
body mass ranging from 331 to 557 g (mean 388±43 g). The crabs were
deprived of food and water 1 day prior to experimentation. The effects of
exercise at either low (40%) or high (90%) relative humidity (RH) on the
respiratory gas exchange and metabolic status were examined. Blue crabs were
sampled either while resting in their terrarium (N=6) or after 5 min
of exercise in air at 40% RH (N=6) or at least 90% RH (N=6).
The crabs that were exercised walked individually in an arena 1.5 mx3 m
at a pace of their choosing, but gentle tactile stimulation was used to
encourage crabs to continue walking (speed 2.48 and 1.80 m
min1 at 90% and 40% RH, respectively). Humidity was
maintained using an electric steam humidifier activated by a relay attached to
a micro switch (RS Components Pty Ltd, Smithfield, NSW, Australia) mounted on
a Lambrecht 194 hygrometer (Lambrecht, Göttingen, Germany) accurate to
±2.5% RH.
Sampling and analysis in the laboratory
Each blue crab was sampled for pulmonary, arterial and venous haemolymph,
which was immediately analysed at 25°C for partial pressure and content of
O2 and CO2, as well as haemolymph pH. A sample of
haemolymph was frozen for measurement of osmolality, calcium and metabolites.
The crabs were kept in ventilated individual terrariums with fresh drinking
water for 24 h prior to experimentation. The carapace of the crabs was drilled
at least 24 h prior to experiments to facilitate sampling of haemolymph from
the pericardial cavity (700 µl; arterial haemolymph), from the efferent
pulmonary vessel (300 µl; for vascular anatomy; see
Farrelly and Greenaway, 1993)
and directly from the venous sinus (800 µl; venous haemolymph). The entire
sampling process required less than 30 s. Samples were taken in chilled 1 ml
syringes with 21-gauge hypodermic needles and held on ice for the duration of
haemolymph gas and acidbase analysis.
The partial pressure of O2 (PO2)
and CO2 (PCO2), as well as the pH of
the haemolymph, was measured using a BMS 3 MK II Blood Micro System
(Copenhagen, Denmark) thermostatically controlled at 25±0.2°C and
connected to a PHM73 pH/blood gas monitor (Radiometer, Copenhagen, Denmark).
The electrodes were calibrated with humidified gases each day before use. The
O2 electrode was calibrated using O2-free gas and with
humidified air and the CO2 electrode using 0.5% and 2.5%
CO2. The pH electrode was calibrated with Radiometer precision
buffers of pH 7.410 (S1510) and 6.865 (S1500), accurate to ±0.005 at
25°C. Haemolymph oxygen contents ([O2]) were measured using the
modified Tucker chamber method (Tucker,
1967) as outlined by Bridges et al.
(1979
). The O2
electrode was maintained at 32°C and connected to an oxygen meter
(Strathkelvin model 781, Glasgow, Scotland). The changes in
Po2 were recorded on a pen recorder (model BD111; Kipp and
Zonen, Delft, The Netherlands). The haemolymph CO2 content was
measured using a Corning 965 CO2 analyser (Medfield, MA, USA;
calibrated with HCO3 standard, 15 mmol
l1).
The haemocyanin content of the haemolymph was measured by
spectrophotometric scanning of a 10 µl haemolymph sample in 1 ml of 1% EDTA
in Milli-Q water (Sydney, NSW, Australia). The peak absorbance near 338 nm was
used to calculate the haemocyanin concentrations using the extinction
coefficient 2.69
(Nickerson and Van Holde,
1971
). The haemocyanin concentration was used to derive the
maximum capacity for haemocyanin-bound O2 of each sample and
thereby the relative haemocyanin O2 saturation.
An aliquot of the remaining haemolymph samples was mixed (ratio 1:1) with ice-cold 0.6 mol l1 HClO4 to denature proteins and was neutralised with 2.5 mol l1 K2CO3. The denatured sample was centrifuged at 10 000 g at 4°C for 10 min and the supernatant frozen for subsequent L-lactate analysis (test kit No. 138 084; Boehringer Mannheim, Mannheim, Germany). Whole haemolymph samples were maintained at 4°C for a maximum of 15 min before freezing for later analysis for glucose (test-kit No. 510; Sigma Diagnostics, Sydney, NSW, Australia) and urate concentrations (Sigma Diagnostics test kit No. 685).
Haemolymph osmolality was measured using a vapour pressure osmometer (Wescor 5100C, Logan, UT, USA) calibrated with two precision standards, 290 and 1000 mOsm. The concentration of Ca2+ in the haemolymph was measured using an atomic absorption spectrophotometer (GBC 906, Melbourne, Australia) with a sample of haemolymph deproteinised with HNO3 (0.1 mol l1; ratio 1:1). To suppress interference during measurements, samples and standards were diluted with 7.2 mmol l1 LaCl3.
The concentration of L-lactate from the haemolymph and the
changes in the concentration of circulating glucose after 5 min of exercise
were monitored at intervals for 24 h in crabs exercised at 90% RH by
repeated sampling (50 µl) using an ice-cold 100 µl Hamilton syringe with
a 26-gauge needle inserted through the arthrodial membrane at the base of the
walking legs. During recovery, blue crabs were maintained individually and
supplied with a continuous flow of humidified air.
Exercise in the field in situ
D. hirtipes were sampled on Christmas Island during two seasons:
June (dry season), when the crabs were quiescent, and during the following
February (wet season), when the blue crabs were engaged in the seaward
breeding migration. In June, sampling was carried out at Ross Hill Gardens
(10°29'11''S, 105°40'41''E), where
the blue crabs were congregated around freshwater seepages. During the
February wet season, the crabs were migrating from Ross Hill Gardens to the
coast, and sampling was carried out at a lower forest terrace, approximately
500 m from Ross Hill Gardens (10°29'29''S,
105°40'43''E). During the dry season study period
(June), RH was as low as 63% whereas in the wet season period (February) it
never fell below 100% and quite often the air was supersaturated with water
vapour (mist).
Two groups of crabs were sampled during each of the study seasons (N>6 for each group). The first group was the free-ranging crabs (FR); this group comprised crabs that were above ground outside of their burrows. Each crab was captured for haemolymph sampling, but any crabs that attempted to escape prior to capture were marked and excluded from the experiment. A second group of crabs was exercised for 5 min. A blue crab was selected at random and an observer approached the crab until the crab began to move away; when the crab stopped walking, the observer approached the crab again. After 5 min of this exercise the crab was captured for haemolymph sampling.
Samples were taken and analysed as described for the samples from crabs
exercised in the laboratory (above) except that the samples were transported
in sealed syringes on ice for the 14 min drive to the Research Station on
Christmas Island and different electrodes were used.
PO2 was determined with a flow-through micro
oxygen probe (Microelectrodes, MI16-730, Bedford, NH, USA) connected to a
PHM73 pH/blood gas monitor (Radiometer) calibrated at ambient temperature.
Haemolymph pH was measured with a flow-through micro pH-probe
(Microelectrodes, MI16-705), connected to the PHM73 also at ambient
temperature, which remained effectively constant throughout
(25±2°C). Changes in PO2 of the
Tucker chamber were timed with a stopwatch until a linear rate of change was
recorded and the PO2 then interpolated to
injection time (i.e. time 0). Haemolymph CO2 content
([CO2]) was measured with a PCO2
electrode (model E5037/SI) connected to a PHM73 pH/blood gas monitor using a
Cameron chamber also at 32°C (Cameron,
1971) and calibrated with fresh 15 mmol l1
NaHCO3 standards. The changes in
PCO2 were recorded until a linear rate change
was achieved and interpolated to injection time. A sample of the haemolymph
was frozen and saved for analysis of metabolites and selected ions. Samples
for Mg2+ analysis were prepared as for Ca2+ (above), as
were samples for Na+ and K+ analysis, except they were
diluted in CsCl. The Cl- concentration was measured using a CMT10
titrator (Radiometer) calibrated with 100 mmol l1 NaCl.
Muscle samples were also obtained for measurement of metabolites. A
different group of blue crabs was used for tissue sampling. The crabs were
encouraged to autotomise the penultimate walking leg, and the muscle tissue
(0.3 g from the merus) was extracted from either FR crabs (N=8) or
crabs exercised for 5 min (N=8). The muscle tissue was immediately
deposited into a pre-weighed tube with 2 ml of ice-cold HClO4 (0.6
mol 11) to deproteinise the sample. The vials with the
muscle tissue were weighed and then homogenised with an OMNI 1000 homogeniser
(Marietta, GA, USA) and frozen until further processing as described
previously (Adamczewska and Morris,
1996, 1998a,
2001a
). The muscle tissue was
analysed for L-lactate (using Boehringer test kit no. 138 084) as
well as glucose by methods described by Bergmeyer
(1985
). The concentrations of
metabolites in tissues were expressed in mmol kg1 wet tissue
mass. The muscle and haemolymph samples were air-freighted to the laboratory
at 40°C in dry ice.
Data analysis
All data, except the determinations of L-lactate and glucose in
the haemolymph during post-exercise recovery, were independent with regard to
treatment (e.g. exercise vs resting) and were analysed for treatment
effects by analysis of variance (ANOVA). Data sets containing means with
heterogenous variance (Bartletts' and Levene's tests) were log or square-root
transformed before analysis. Post-hoc testing was by Tukey's HSD
test. The changes in L-lactate and glucose concentration of the
haemolymph following exercise were analysed using a one-way repeated measures
design. Pulmonary, arterial and venous haemolymph samples were not completely
independent within treatments since they were taken from each crab, and thus
comparisons between these values were made using serial Friedman's two-factor
ANOVA for ranked related samples (non-independent). This is comparable to a
one-factor ANOVA and was verified using Minitab 14 in addition to Systat,
which was employed for the other analyses. In all cases, P<0.05
was taken as significant. Values are presented as means ±
S.E.M.
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Results |
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The effects of exercise at 90% RH on haemolymph CO2 and pH were more extreme than those at 40% RH (ANOVA; Table 1). [CO2] in the haemolymph was similar in the pulmonary, arterial and venous haemolymph within each exercise regimen (Table 1). The haemolymph [CO2] of crabs after 5 min of exercise (e.g. [CO2]a 12.51 and 12.79 mmol l1 at 40% and 90% RH, respectively) was significantly lower than in the crabs at rest (e.g. [CO2]a 17.49 mmol l1). By contrast, the haemolymph PCO2 of exercised crabs (e.g. PvCO2 2.24 and 2.69 kPa at 40% and 90% RH, respectively) was higher than that of crabs at rest (e.g. PvCO2=1.56 kPa; Table 1). The pH of the haemolymph was similar in pulmonary, arterial and venous haemolymph of crabs within each exercise regimen (Table 1). However, the pH of the haemolymph in crabs exercised for 5 min was significantly lower in exercised crabs (pHv=7.35) than in those at rest (pHv=7.59). This relative acidosis was more pronounced in crabs exercised in 90% RH (pHa=7.40 vs pHa=7.49; Table 1).
Osmolality and metabolites in the haemolymph
Osmolality (OP) was approximately 70 mOsm greater in the laboratory crabs
compared with the free-ranging D. hirtipes on Christmas Island
(below), although haemolymph Ca2+ levels were lower. Exercise
promoted an increase in OP from 574 to 677 mOsm after 5 min exercise at 90%
RH, while the increase in OP after exercise at 40% RH was considerably smaller
(Table 2). The increase in OP
was reflected in a 5.1-fold increase in Ca2+ concentration in the
haemolymph of blue crabs after 5 min exercise at 90% RH but there was no
significant increase during exercise at 40% RH
(Table 2). There was no
detectable change in haemolymph urate concentration (0.040.10 mmol
l1), which was within the range for field data (below),
consequent to exercise in blue crabs, nor in the concentration of glucose
(0.350.60 mmol l1). Exercise had a pronounced effect
on haemolymph L-lactate concentration, which was 0.24 mmol
l1 in resting laboratory animals but increased to 3.68 and
5.41 mmol l1 after 5 min exercise at 90% and 40% RH,
respectively.
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Recovery from exercise at 90% RH
Subsequent to 5 min exercise at 90% RH, L-lactate in the
haemolymph continued to accumulate for at least the next 30 min and exceeded 4
mmol l1 (Fig.
1). Haemolymph L-lactate was then progressively cleared
and although after 5 h recovery the mean L-lactate concentration
was still 1.9 mmol l1, this was no longer statistically
elevated compared with values from resting crabs. L-lactate
declined to 0.19 mmol l1 after 24 h recovery. Haemolymph
glucose varied in the haemolymph of post-exercised D. hirtipes
differently to L-lactate (Fig.
1). The haemolymph glucose increased from 0.35 mmol
l1 in resting crabs for at least 2 h after exercise to 1.73
mmol l1, and this hyperglycaemia persisted even after 24 h
recovery, at which time the concentration was still 0.74 mmol
l1 (Fig.
1).
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In situ assessment of exercise in D. hirtipes on Christmas Island
In each of the sample groups, the PO2 in the
arterial and pulmonary haemolymph was similar but decreased significantly in
the venous samples (Table 3).
In June (dry), the PpO2 of exercised crabs
(6.5±1.8 kPa) was significantly lower than in the FR crabs
(12.0±2.8 kPa). By contrast, during February (wet), the mean
PpO2 of FR and the exercised crabs was similar
(combined mean 5.5±1.2 kPa; Table
3).
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Like the PO2, [O2] in the haemolymph was similar in pulmonary and arterial haemolymph (Table 3). However, while the [O2]p of FR and exercised crabs was similar in June, in February (wet) the [O2]p of exercised crabs (0.66±0.04 mmol l1) was lower than in the FR (migrating) crabs (1.00±0.15 mmol l1). The venous [O2] ranged from 0.26 to 0.36 mmol l1 and was not significantly different among any of the groups (Table 3).
Despite the differences in PO2 and [O2] between some treatments, the Hc was always well saturated at the gas exchange surfaces in all sample groups (pulmonary Hc O2>95%) but decreased to below 30% in the venous haemolymph of crabs exercised in the dry season (Table 3).
There was no difference in [CO2] between the pulmonary, arterial and venous haemolymph within any of the sample groups (Table 3). While the mean haemolymph [CO2] in crabs sampled in June was similar in FR and exercised crabs (e.g. [CO2]v=16.70±0.90 and 16.60±1.02 mmol l1 respectively), during the February wet season the [CO2] of exercised crabs (e.g. [CO2]v=11.38±0.57 mmol l1) was significantly lower than in FR crabs ([CO2]v=17.28±1.86 mmol l1).
The pH of pulmonary, arterial and venous haemolymph was similar within each of the sample groups, but exercise induced a haemolymph acidosis (Table 3). The haemolymph of crabs sampled in June (dry) had a pHv of 7.60±0.03, but after 5 min of exercise this decreased to pHv 7.26±0.08. Similarly, in crabs sampled in February (wet), the haemolymph pHv of 7.45±0.03, for example, decreased to an even lower value of 6.99±0.04. While the relative acidosis induced by 5 min enforced walking was similar in both seasons, the initial haemolymph pH of FR crabs sampled in February was significantly lower than that of the FR crabs sampled in June (Table 3).
Haemolymph and tissue metabolites
The concentration of glucose in the haemolymph of FR crabs sampled in
February (wet season; 0.22±0.03 mmol l1) was almost
twice that of FR crabs sampled in June (dry; 0.13±0.02 mmol
l1; Table 4).
Likewise, the increase in haemolymph glucose after 5 min of exercise in
February (0.14 mmol l1) was twice that measured in June
(Table 4). While the glucose
concentration in the muscle of FR crabs was comparable with the concentrations
in the haemolymph, after 5 min of exercise the increase in muscle [glucose]
was much greater at 1.1 mmol kg1 in June and 0.7 mmol
kg1 in February (Table
4).
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The concentration of urate in the haemolymph was similar in both seasons (0.0750.121 mmol l1) and did not change after 5 min of exercise (Table 5). Haemolymph L-lactate was similar in the two sampling seasons and increased from a mean of 0.76±0.17 mmol l1 in FR crabs to a mean of 9.2±1.2 mmol l1 after 5 min of exercise (Table 4). The concentration of L-lactate in the muscle tissue was consistently higher than in the haemolymph and increased from a mean (combining both dry and wet season data) of 2.3±0.81 mmol kg1 in resting crabs to a mean of 13.9±2.5 mmol kg1 after 5 min of exercise (Table 4).
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Osmotic and salt balance
There was no difference between the haemolymph OP of D. hirtipes
sampled in the dry season and those sampled in the wet season
(Table 5). Exercising blue
crabs for 5 min promoted an increase in haemolymph osmolality of 73.1 mOsm in
June and 58.8 mOsm in February (Table
5). Similarly, there was no seasonal variation in haemolymph
Ca2+ concentration, but the 5 min exercise promoted a 4045%
increase in circulating Ca2+ levels
(Table 5). During the June
sampling season, exercised D. hirtipes, compared with FR crabs,
showed no change in Na+ and Cl-, but Mg2+ and
K+ were increased by 31% and 22%, respectively
(Table 5).
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Discussion |
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Respiratory competence of the lungs in supporting exercise
Afferent systemic haemolymph O2 saturation was not a simple
limitation to exercise in D. hirtipes. In the laboratory, the
PpO2 and PaO2
values in the haemolymph in resting D. hirtipes were similar to
previous in situ values
(Adamczewska and Morris, 1996)
and to those in quiescent crabs in the field during the dry season. The
haemolymph of D. hirtipes has a high O2-carrying capacity
(
1 mmol l1) and is normally well saturated leaving the
lungs (Farrelly and Greenaway,
1994
; Adamczewska and Morris,
1996
). The Hc in D. hirtipes has a very high affinity for
O2 (P50=0.62 kPa at pH 7.8 and 25°C;
Dela-Cruz and Morris, 1997a
)
and saturates at PO2 values well below those in
resting crabs.
The index Ldiff
(Piiper, 1982;
Taylor and Taylor, 1992
)
assesses diffusion limitation to gas exchange and, for terrestrial crabs, is
generally between 0.4 and 0.5 (Innes and
Taylor, 1986
, Taylor and
Taylor, 1992
; Adamczewska and
Morris, 1998
). D. hirtipes resting in the laboratory or
quiescent in the field showed typical Ldiff values of
0.41±0.03 and 0.44±0.14, respectively, characteristic of an
air-breather (Morris and Dela-Cruz,
1998
).
The relative haemolymph flow through the lungs of resting D.
hirtipes was 82.1%, with only 17.9% through the gills (S.M., unpublished
observation) as determined using an injected radiolabelled micro-sphere method
(e.g. Taylor and Greenaway,
1984). The gills may be important in the excretion of
CO2 (Farrelly and Greenaway,
1994
) but this could not be substantiated (see also
Dela-Cruz and Morris, 1997a
).
The only significant (Friedmans test) decrease in haemolymph [CO2]
was during lung transit (0.87±0.36 mmol l1),
which supports the contrary suggestion that CO2 excretion is over
the lungs. The [CO2] in the haemolymph of resting D.
hirtipes was between 16.5 and 17.3 mmol l1 and was very
similar to both previous laboratory
(Farrelly and Greenaway, 1994
)
and field values (Adamczewska and Morris,
1996
). Similarly, the PCO2 and pH
values of the two resting groups of D. hirtipes (laboratory and dry
season field crabs) were similar to those in previous in situ studies
(Adamczewska and Morris,
1996
).
The influence of humidity on exercise in the laboratory
Rather than low humidity exacerbating the demands of exercise on
respiration, increasing the humidity encouraged the crabs to a greater
commitment to walking. In the laboratory, exercised D. hirtipes
exhibited pronounced reductions in the haemolymph
PO2, especially in the
PpO2, which was reduced by 59% and 80% in crabs
exercised at 40% RH and 90% RH, respectively. These decreases are consistent
with a partial failure of the lungs to oxygenate the haemolymph, especially in
crabs exercising in air at 90% RH. The Ldiff for animals
exercised at 40% RH increased to 0.77±0.07, and at 90% RH to
0.93±0.02, indicating severe diffusion limitation in the crabs
exercising under humid conditions. In exercising G. natalis,
Ldiff increased from 0.53 to 0.77
(Adamczewska and Morris, 1998)
and in exercised Potamonautes warreni from 0.57 to 0.77
(Adamczewska et al., 1997
). If
Ldiff increases in D. hirtipes during severe
exercise then this correlates with a 38% faster walking speed in crabs under
90% compared with 40% RH. This large effect of laboratory exercise in lowering
haemolymph PO2 was not reflected in the oxygen
content of D. hirtipes haemolymph, which remained generally high (the
increase in [O2] in crabs exercised at 90% RH was due to unusually
high [Hc]). The venous Hc O2 saturation was significantly lower in
the crabs exercised at 90% RH compared with both controls and those exercised
at 40% RH. This relative lowering of venous Hc oxygenation is consistent with
comparatively greater respiratory demand in that group, consequent on their
faster locomotion.
The increased haemolymph calcium in both laboratory and field crabs may
explain how Hc-O2 saturation remained high despite the internal
hypoxia. The O2 affinity of D. hirtipes Hc is
significantly improved by increased haemolymph Ca
(logP50/
log[Ca2+]=0.45 at
pH 7.4) but is not sensitive to L-lactate
(Dela-Cruz and Morris, 1997a
).
By employing the Bohr coefficient for the pH sensitivity of O2
binding by D. hirtipes Hc (
=0.57;
Dela-Cruz and Morris, 1997a
),
the P50 of D. hirtipes Hc in laboratory exercised
crabs at 90% RH would be 1.06 kPa. Incorporating the effect of increased
Ca2+ reduced the P50 to 0.51 kPa in D.
hirtipes exercised at 90% RH, which is an improvement of the Hc affinity
for O2 by 52%. In crabs exercised at 40% RH the Ca2+
increase was less, thus the potentiation of Hc O2 affinity was
proportionately less and correlated with the less severe internal hypoxia.
In Cardisoma guanhumi, fatigue set in at a walking speed of 3 m
min1 (Herreid et al.,
1979), which was only slightly faster than 2.48 m
min1 by D. hirtipes at 90% RH and was thus likely
close to the maximum speed for D. hirtipes. While haemolymph
L-lactate increased after 5 min exercise in the laboratory,
35 mmol l1 is not high for land crabs (e.g.
Greenaway et al., 1988
;
Adamczewska and Morris, 1994,
1998
). Short-term, vigorous
exercise affected urate oxidase and increased haemolymph urate in G.
natalis (Adamczewska and Morris,
1998
) but in D. hirtipes 5 min laboratory exercise was
insufficient to alter [urate]. Furthermore, there was no
significant hypergylcaemia until after the exercise demand had ceased.
In crustaceans, low rates of lactate reoxidation (0.82.6 mmol
l1 h1) are the norm
(Wood and Randall, 1981b;
Forster et al., 1989
;
Henry et al., 1994
;
Adamczewska and Morris, 1998
)
but the rate of 0.49 mmol l1 h1 in D.
hirtipes exercised in the laboratory was unusually slow, although Henry
et al. (1994
) reached a
similar conclusion for C. guanhumi. Thus, in the long term, migrating
D. hirtipes would be severely constrained to within the MAS. In
D. hirtipes, mobilised glucose accumulates in the haemolymph while
lactate is being re-oxidised. The primary site for glucose mobilisation in
land crabs appears to be the leg muscles
(Morris and Adamczewska,
2002
). The simplest explanation is that, subsequent to short-term
exercise, glucose continues to flux from the muscle of D. hirtipes
under conditions where O2 supply is restored and
L-lactate can slowly re-enter into the respiratory pathway,
obviating the need for elevated glycolysis.
|
These data are also consistent with different work loads (walking speeds)
between the groups and/or differential lactate and H+ efflux rates.
Assuming that the L-lactate efflux into the haemolymph was
accompanied by a stoichiometric efflux of H+, the haemolymph pH due
to metabolic acid [pHa(m)]could be predicted for each crab (for
method, see Greenaway et al.,
1988; as derived from Wood et
al., 1977
). The haemolymph of crabs exercised at 90% RH was
pHa=7.40, which was not different to the pHa=7.45 in
those crabs exercised at 40% RH (Table
1; Fig. 2). The
corresponding increases in haemolymph lactate were 3.44 and 5.17 mmol
l1, respectively, which provided a statistically unchanged
pHa=7.48 for the crabs in 40% RH but a higher
pHa=7.51±0.02 in those crabs exercised in 90% RH (paired
t-test, T=4.63, P=0.002). Thus, in D.
hirtipes exercised at 90% RH, there appears to be a preferential
retention of L-lactate over H+ by the tissues, perhaps
as a consequence of the severity and brevity of the exercise period (see table
2 in Adamczewska et al., 1997
).
When land crabs were exercised to exhaustion, the L-lactate efflux
exceeded that of H+ (e.g.
Greenaway et al., 1988
;
Adamczewska and Morris, 1994b
)
whereas G. natalis under non-exhausting conditions
(Adamczewska and Morris, 1998
)
exhibit values similar to those from D. hirtipes.
Behavioural responses to humidity are paramount since the differences between the D. hirtipes exercised at 40% and 90% RH were not directly debilitating effects of low humidity but rather are due to an increased proclivity to exercise at higher humidity. The increased commitment to walking in 90% RH accounts for the relative internal hypoxia as well for the deeper hypercapnic lactacidosis.
Exercise in the field and relevance to the seasonal migration
The free-ranging D. hirtipes during the dry season were
constrained in their distribution around freshwater seepages, were relatively
inactive and moved only short distances
(Hicks et al., 1990;
Greenaway and Raghaven, 1998
;
S.M., unpublished radio-tracking data). Free-ranging D. hirtipes in
the wet season were migrating and walked persistently but not continuously
during the daylight hours. The establishment of the wet season is a
requirement for the migration (Hicks et
al., 1990
; S.M., personal observations), and the resultant
seasonal differences in the behaviour of free-ranging crabs correlate with
changes in the haemolymph respiratory gas status. Thus, the relative humidity
does not affect O2 transport directly but instead determines the
behaviour of the animal and thereby the demands that the crabs then make on
their oxygen uptake and transport systems.
The haemolymph PO2 of free-ranging crabs in June was not different from that measured in crabs at rest in the laboratory, implying that the D. hirtipes were indeed quiescent in the field during the dry season. However, in the wet season, the PO2 values of migrating crabs declined markedly and were similar to those of exercised crabs in the laboratory. Thus, while Ldiff was 0.44 in quiescent dry season crabs, the uptake of O2 was more severely diffusion limited in the migrating D. hirtipes (Ldiff=0.75). Superimposing 5 min exercise on the quiescent dry season animals further reduced their haemolymph PO2 and increased Ldiff to 0.95, as in the laboratory exercised crabs. In the wet season animals that were migrating, the extra 5 min exercise load failed to elicit any further reduction in PO2, and Ldiff remained at 0.78.
The low PO2 of the haemolymph in naturally
migrating crabs was again not manifested in low O2 content because
of the high affinity of the Hc for O2. The apparent decreased
O2 content in crabs exercised during February was due to relatively
low [Hc] since the % saturation was unaffected
(Table 3). In June (dry
season), subsequent to the 5 min exercise, the increase in Ca2+
content would have decreased the P50 (increased
O2 affinity) from 1.21 to 0.91 kPa, and in the migrating wet season
crabs from 1.60 to 1.18 kPa. Actively foraging G. natalis on
Christmas Island in the wet season exhibited a relative haemolymph hypoxia
(2.9 kPa) compared with the relatively inactive crabs in the dry season, which
correlated with activity (Adamczewska and
Morris, 2000a).
Migrating crabs in the wet season maintained low L-lactate
levels, indistinguishable from those in June during the dry season. Therefore,
the breeding migration of blue crabs is completely aerobic, and within the
MAS. Migrating G. natalis also exhibited low and constant haemolymph
lactate levels (Adamczewska and Morris,
2001b) and this may be a feature of gecarcinid land crabs. The
elevated O2 demand of migrating crabs (Feb) compared with those
confined to around the freshwater springs in June is reflected in a relative
hyperglycaemia typical of exercised crustaceans
(England and Baldwin, 1983
;
Morris and Adamczewska, 1994b
,
2002
). In both June and
February, the 5 min imposed exercise elevated haemolymph lactate to a similar
extent, at least 9 mmol l1, and tissue [lactate] also
increased similarly in both groups despite the obvious differences in the
haemolymph PO2 changes. The data are consistent
with migrating D. hirtipes walking close to their aerobic limit
during which time pulmonary PO2 is maximally
depressed, O2 uptake diffusion limited, and the
arterialvenous (av) PO2
difference minimised (albeit with little effect on the av Hc
O2 difference). Extra exercise imposed a requirement that exceeds
the aerobic scope and requires anaerobic supplementation.
As with the haemolymph oxygenation, the CO2 and acidbase status of relatively inactive crabs during the dry season was similar to that of resting crabs in the laboratory (Fig. 2). Red crabs, G. natalis, on Christmas Island showed a seasonal respiratory acidosis such that actively foraging crabs in the wet season accumulated an extra 3 mmol l1 of CO2 and a decrease in pH of 0.13 compared with the relatively inactive crabs in the dry season (Adamczewska and Morris, 2002a). The migrating D. hirtipes were similarly acidotic compared with the quiescent dry-season animals due to a respiratory acidosis with no evidence of any metabolic component (Fig. 2). Consequently, requiring the dry season (June) animals to exercise elicited both a respiratory component to the ensuing acidosis (as exhibited by migrating crabs) and a metabolic component, as evidenced by the accumulated lactate. The fundamentally different basic respiratory state of the wet-season animals, close to their aerobic limit, compared with dry-season crabs meant that when the additional exercise was imposed on migrating blue crabs the acidosis was primarily metabolic in origin.
Adamczewska and Morris
(1998) concluded that burst
locomotion has significant disadvantages if it exceeds the MAS and if the
crabs, like D. hirtipes, have slow rates of lactate reoxidation (e.g.
Henry et al., 1994
;
Morris and Adamczewska, 2002
).
Migrating D. hirtipes remain aerobic but exhibit a hypoxic
respiratory acidosis compared with those in the dry season. Any extra
energetic demand requires the recruitment of anaerobiosis and a debt that, in
D. hirtipes, would require many hours to repay and impair subsequent
exercise capacity. The best strategy for migrating blue crabs would appear
similar to that of G. natalis, to walk continuously but not to exceed
the MAS. During the dry season, extensive walking is resisted by either
species. In D. hirtipes, the seasonal activity is inextricably linked
to ion and water homeostasis, which modify the extent of activity which the
animals can engage in and, thereby, the seasonal metabolic and respiratory
demands. It is apparent from the laboratory trials and field observations that
D. hirtipes responds to ambient humidity and limits, even under
imposed locomotion, the commitment to exercise when at lower RH. This humidity
sensitivity is integral to the seasonal behaviour and thus to the resulting
routine metabolic demands in different seasons and to migration of the
crabs.
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Acknowledgments |
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References |
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