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The effects of temperature on aspects of respiratory physiology of the semi-terrestrial crabs, Uca inversa (Hoffmann) and Metopograpsus messor (Forskål) from the Red Sea
Comp. B&hem. Physiol. Vol. 114A, No. 4, pp. 297-304, Copyright 0 1996 Elsevier Science Inc.
1996
ISSN 0300-9629/96/$15.00 PI1 SO300.9629(96)00007-2
ELSEVIER
The Effects of Temperature on Aspects of Respiratory Physiology of the Semidterrestrial Crabs, Uca inversa (Hoffmann) and Metopogrupsus messor (Forsksl) from the Red Sea A. A. Eshky,’ A. C. Taylor,2 and R. _I. A. Atkinson3 ‘FACULTYOF MARINE SCIENCE,KING ABDULAZIZUNIVERSITY,JEDDAH,SAUDI ARABIA, *INSTITUTEOF BIOMEDICAL AND LIFESCIENCES,GRAHAM KERRBUILDING,UNIVERSITYOF GLASGOW,GLASGOW, G12 8QQ, UK, AND ‘UNIVERSITY MARINE BIOLOGICAL STATION, MILLPORT,ISLEOF CUMBRAE,KA28 OEG, UK
ABSTRACT. The effects of temperature on aspects of the respiratory physiology of two semi-terrestrial crabs, Uca inoersa and Metopoppsus messor have been studied. When exposed to increasing temperatures in the laboratory both species show a pronounced increase in both the rate of oxygen consumption (tiol) and in heart rate; Q10 values were approximately 2-3 over the temperature range 15-40°C for both Mo2 and heart rate. Temperature did not, however, affect the relationships between h;lo2 and fresh body weight and between heart rate and fresh body weight. The haemocyanin of both species was found to have a high oxygen affinity (PsO = 6.4 and 12.4 Torr for U. inversa and M. messor, respectively, pH = 7.9, temperature = 30°C) and moderately high Bohr values (- 1.07 and -0.96 for U. inversa and M. messor, respectively). An increase in temperature resulted in a significant decrease in the oxygen affinity of the haemocyanin of both species (AH = -51.7 and -57.4 kJ.mol-’ for U. inversa and M. messor, respectively). Changes in temperature did not have a significant effect on either the Bohr value or the cooperativity of the pigment. COMP BIOCHEMPHYSIOL114A;4:297-304, 1996. KEY WORDS. Crustacea, Decapoda, Uca, Metopogmpsus, temperature, cyanin
INTRODUCTION Crabs are important
members of mangrove
ecosystems
in
tropical regions (3 1,13). One of the major problems that they face in this environment is that they may be exposed to temperatures that are potentially lethal. In a previous study (7) we discussed aspects of the ecology and behaviour in Red Sea mangroves of the two crabs used in the present study, the ocypodid Uca (Amphiucu) inversa inversa (Hoffmann) and the grapsid, Metopograpsus messor (Forsksl). The ecology of these two species is quite distinct; LJ. inversa occupies burrows in the upper eulittoral zone whereas M. mesSOTis found amongst mangrove trees (Auicennia marina) although it may also occur on rocky shores (29). Eshky et al. (7) showed that U. inversa was more tolerant of thermal stress than M. messor and that both species used behavioural and physiological means to survive exposure to high temperatures. In the case of U. inuersa, the burrow provided an essential refuge from environmental extremes since measurements
of the microclimatic
conditions
within
Address reprint requests to: A.C. Taylor, Institute of Biomedical and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow, G12 8QQ UK. Received 14 September 1995; revised 24 November 1995; accepted 5 December 1995.
oxygen consumption,
heart rate, haemo-
the burrow showed that the temperature within the burrow was consistently less variable than that of the sediment surface or of the air just above the sediment. Similarly, the relative humidity inside the burrow was much greater than that of the air outside and the burrow characteristically gave access to standing water. In contrast, M. messor constructs burrows only infrequently.
Instead, this species makes stra-
tegic use of the mangrove vegetation
to minimize the effects
of thermal stress; M. messor was observed to utilize the shade provided by the Avicennia plants during the the hottest times of the day but then foraged more widely on the mud plain during cooler periods. Although this species was observed to spend brief periods in shallow water at the base of the mangrove bushes, our observations indicate that it tends to avoid periods of prolonged immersion. Despite these behavioural mechanisms
which serve to re-
duce the degree of thermal stress to which they are exposed, both species may still experience a range of temperature which will have important physiological implications. The use of evaporative cooling to lower the body temperature may be crucial in enabling them to survive potentially lethal high temperatures (7). In both species, however, exposure to varying temperatures will affect other aspects of their physiology. The two species differ in the method used for aerial respi-
A. A. Eshky et al.
298
ration. LJ. inversa has expanded epibranchial spaces whose moist surfaces function as lungs whereas M. messor lacks
of differing sizes (fresh weight range = 1.15-5.9
lungs but, when emersed, circulates branchial water across antero-ventral body surfaces where it is reoxygenated
ried out over the temperature
(31,1,7). In the present paper, we consider the effects of temperature on aspects of the respiratory physiology of both U. inversa and M. messor to provide further information
on the
adaptations of these species to the often extreme conditions that characterize their habitat.
AND
Uca inwersa and Metopograpsus messor were collected
from
Ras Hatiba near Dah’ban approximately 50 km north of Jeddah, Saudi Arabia. The crabs were transported to the Marine Biological
Station
at Obhur, part of the Faculty of Ma-
were car-
using an im-
amount of sea water. The tanks were covered with black plastic sheets and placed in water baths at 15*C and the crabs left undisturbed for at least 2 hr before recordings commenced. Heart rate recordings were carried out continuously for a period of approximately 30 min and then the was slowly increased
next experimental
METHODS
range 15-40°C
pedance technique (11,27). Crabs were placed individually in small tanks containing a mud substratum and a small
temperature MATERIALS
g and 0.38-
17.5 g for U. inversa and M. messor, respectively)
temperature
(1°C
in 30 min) to the
and further recordings car-
ried out. In most cases, heart rate recordings were made at intervals of 5°C but, in other experiments, recordings were made at 2°C intervals. Since these experiments nary experiments
lasted for several hours, prelimi-
were carried out to establish whether the
rine Science, King Abdulaziz University. Both species were kept separately in large outdoor tanks (2 m diam.) con-
crabs showed any die1 rhythms of heart rate. In these experiments, heart rate recordings were carried out continuously
taining an appropriate mud substratum (from the local habi-
for periods over 24 hr on crabs held at 25°C under constant
tat) and were subjected to normal daylight and temperature cycles (the daytime temperature was approximately 40°C
illumination or in constant darkness. Although some variation was recorded in heart rate during these periods, no clear
falling to approximately 20°C at night). The mud in each tank was formed into a slope enabling the crabs to gain
rhythms could be established. In all further experiments, therefore, heart rate recordings were carried out in constant
access to sea water (salinity = 38%0) at the bottom of the tanks. Many U. inversa were observed to construct their
darkness to try to eliminate heart rate.
characteristic
burrows in the sediment and both species re-
mained in good condition
for many days.
Oxygen Transporting Properties and Ionic Composition of the Haemolymph
Oxygen Consumption Measurements
the effects of die1 changes in
of the rates of oxygen consumption
ried out using constant
pressure respirometers
were car(4). Crabs
(fresh weight range = 0.95-6.10 g and 2.75- 12.51 g for U. inversa and M. messor, respectively) were placed individually into each of the respirometers
and a few drops of sea
Values for the in viuo pH of the pre-branchial haemolymph of U. inversa and M. messor were obtained for crabs maintained at 25°C. The crabs (n = 10) were maintained
undis-
turbed at this temperature for several hours prior to haemolymph sampling to ensure that the pH values obtained were representative
of quiescent animals. The haemolymph
sam-
water (salinity = 38%0) were added to provide a source of water for the crabs and to maintain a high relative humidity in the respirometers. The respirometers were then placed
ples were taken by inserting the hypodermic needle of a chilled 1 ml syringe through the arthrodial membrane at the base of one of the pereiopods and the pH determined immedi-
in water baths at the appropriate temperature. The crabs were left undisturbed in the respirometers for at least 2 hr
ately by drawing it from the syringe into a capillary pH electrode (G299, Radiometer, Denmark) maintained at the same
before recordings commenced. Preliminary experiments demonstrated that this was sufficient to ensure that the
temperature and connected to a pH meter (Corning). Unfortunately, it was not possible to obtain values for the Paz of the pre- and post-branchial blood of these crabs since the
crabs had become quiescent
before recordings were carried
out. During this period the respirometers were not sealed to enable oxygen to diffuse into the chambers. At the start of each recording, the respirometers were sealed and changes in the manometer volume recorded at 5 min intervals over a period of 1 hr. At the end of the experiment the fresh weights of the crabs were obtained and weight specific rates of oxygen consumption (ko2) calculated. Rates of oxygen consumption were determined for both U. inversa and for M. messor over the temperature range 1540°C. The effect of temperature on the heart rate of these two species was also determined. Heart rate recordings of crabs
appropriate equipment was not available in Jeddah. Further haemolymph samples from both species were centrifuged (12,000 X g for 5 min) to remove haemocytes and any clotted proteins and then frozen at - 20°C. The samples were transferred still frozen to the University of Glasgow where the analyses were carried out. The oxygen carrying capacity of the haemocyanin (CHcOZ) was determined on individual haemolymph samples at 25°C using the method of Tucker (28) as modified by Bridges et al. (3). Following these determinations, the remaining haemolymph was pooled to provide a larger sample for ion analysis and for the construction of oxygen dissociation curves. The concentrations of the
Temperature and Respiration of Land Crabs
Na+, K+, Ca’+ and Mg”
299
30 -
ions in the serum were determined
by atomic absorption spectrophotometry (Philips 9200) following dilution of each sample with deionized water. The concentration
ofCl-
20 -
ions was determined by electrochemical
titration (Jenway Chloride meter). The concentration of Llactate in the haemolymph of both species was determined using the method of Gutman and Wahlefeld modifications of Graham et al. (8). In vitro oxygen dissociation
curves for the haemolymph
of U. inversa and M. messor were constructed spectrophotometrically using a diffusion chamber (34). Dissociation curves were constructed
at different
pHs to examine
the
magnitude of the Bohr factor in these species. The pH of the haemolymph was altered by varying the proportion of carbon dioxide in the gas mixtures supplied by gas mixing pumps (Wiisthoff, Bochum, Germany). The pH of the haemolymph
was
measured
by
tonometering
The pH of the haemolymph
was determined
haemolymph
that had been frozen (-20°C).
using
This was un-
avoidable since the samples had to be transferred frozen from Saudi Arabia to Glasgow for the analyses to be carried out. Pooled haemolymph samples were used in this study due to the limited amount of haemolymph that was available but this procedure had the advantage that it overcame any variations in the concentrations of haemolymph ions or L-lactate between samples taken on different occasions. Morris (15) and Lallier and Truchot (12) have shown that long-term storage of haemolymph samples at very low temperature (-SO’C) may affect the cooperativity (n50) of the haemocyanin. In other studies, however, it was found that storage at -20°C did not affect the cooperativity of the haemocyanin of other species (34,25). The effect of temperarure on the oxygen affinity of the haemocyanin
was also investigated
by constructing
oxygen
dissociation curves over a range of temperatures between 15 and 35°C. The effect of temperature on the oxygen affinity of the haemocyanin can be quantified by calculating the change in enthalpy (AH) accompanying oxygenation of the haemocyanin at a constant pH (7.9) using the following equation: BH = -2.303
x R x d log P50 (kJ_mol-,) Al/T1 -
T2
RESULTS Effect of Temperature
on &lo, and Heart
s-
E t cy .P
l-
at
the PsOusing the capillary pH electrode of the BMS2 which was connected to a pH meter (Corning 255 ion analyser). The PsOwas estimated from saturation values between 25% and 75% according to the Hill equation. Oxygen dissociation curves were constructed
h 7 G 7 F
another
haemolymph sample (100 ,~l) in a Radiometer BMS2 against the same gas mixtures supplied to the diffusion chamber.
10 _
(10) with the
Rate
The effect of temperature on the relationships between X402 and fresh body weight for U. inversa and for M. messor is
0.5 1
0.5
I
I
I
I11111~
1
10
5
Fresh weight (g) FIG. 1. The effect of temperature on the relationship between the rate of oxygen consumption (MO*) and fresh body weight of Uca inversa. The temperatures used were 15 (W), 20 (0), 25 (O), 30 (A), 35 (A) and40”C (0). The regression equations fitted to these data are given in Table 1.
shown in Figs. 1 and 2, respectively. As in other decapods, there is an inverse relationship between h;io, and body weight in both species. The regression equations describing the lines fitted to these data are presented in Table
1. Co-
variance analysis of the data for both U. inversa and M. messor showed that, although there were no significant differences (p > 0.05) between the slopes of the regression lines, there were highly significant differences
(p < 0.001)
between their elevations. Values for MO* were calculated for 5°C increases in temperature over the range of temperatures
used (15-40°C).
Values for Uoz for ‘standard’ crabs (fresh weight 3.0 g and 7.0 g in U. inwersa and M. messor, respectively) were obtained by interpolation from the regression equations in Table 1. Q10 values calculated from these data for U. inversa were between 2.4-3.2 over the temperature range 15-35°C but at higher temperatures (between 35 and 40°C) the QIO increased to 4.82 (Table 2). The QIo values for the hoz of M. messor were slightly lower (1.8-2.1 in the temperature range 15-35°C) but at high temperatures (between 35 and 40°C) QIo declined slightly (Table 2). Both species were clearly stressed at these high temperatures since a number of animals died when exposed to 40°C. As a result, fewer data were obtained and their reliability must also be questioned.
A. A. Eshky et al.
300
20 -
10 A ‘; r 7 B = E S N
5-
.l/10
20
FIG. 3. The effect of temperature on the rate of oxygen consumption (MO,) (as loglo) of Uca inversa (0) and Metopograpsus messor (0). The data have been calculated for ‘Stan. dard’ crabs of 3.0 and 7.0 g fresh weight, respectively, using the regression equations in Table 1.
2-
I’,
I
,
I
10
5
2
20
Calculation
Fresh weight (g) FIG. 2. The effect of temperature on t&e relationship between the rate of oxygen consumption (MOM)and fresh body weight of Metopograpsus messor. The temperatures used were 15 (U), 20 (0), 25 (O), 30 (A), 35 (A) and 40°C (0). The regression equations fitted to these data are given in Tae ble 1.
TABLE 1. .Regression equations describing the relationships between Mo2 and fresh weight for U. inversa and M. messor u. inversa
15°C 20°C 25°C 30°C 35°C 40°C
40
Temperature (“C)
.P
Temp.
30
a 0.093 0.32 0.544 0.821 1.016 1.308
b -0.377 -0.432 -0.377 -0.488 -0.494 - 0.390
M. me.war r*
n
a
b
r2
n
0.724 0.794 0.691 0.781 0.744 0.704
17 18 25 20 15 18
0.535 0.672 0.840 0.999 1.125 1.258
-0.437 -0.442 -0.466 -0.463 -0.428 -0.453
0.929 0.908 0.943 0.906 0.901 0.918
16 27 34 30 33 15
TABLE 2. Q10 values for oxygen consumption heart rate (fh) of U. inversa and M. messor
(&IO,) and for
U. inversa GO,
1%20°C 20-25°C 25-30°C 30-35°C 35-40°C
2.52 3.17 2.81 2.42 4.82
M. messor fll
3.01 2.99 1.83 1.78 1.32
the temperature range 1%
ture on Go? is greater in the former species. This is demonstrated more clearly in Fig. 3 in which log Go, is plotted against temperature. The effect of temperature
on the relationships
between
heart rate and fresh body weight for LJ. inversa and M. mesSOTis shown in Fig. 4. The regression equations of the lines fitted to these data are given in Table 3. Covariance analysis of these data showed that there were no significant differences (p > 0.05) between the slopes of the regression lines but there were highly significant differences (p < 0.001) between their elevations. Thus, as was the case for k40z, temperature does not affect the relationship between heart rate and fresh body weight. It is interesting to note, however, that the slopes of the regression lines obtained for M. messor were significantly lower than those for U. inversa. The QE values for heart rate were similar in both U. ineiersa and M. messor (Table 2). In addition, both species showed a gradual reduction in the value of QIo with increasing temperature.
Equations take the form log Y = a + h log X.
Temperature
ofQlo values over
40°C gave values of 3.04 and 1.92 for U. inversa and M. messor, respectively, indicating that the effect of tempera-
hi0,
1.84 1.97 2.10 2.05 1.67
fll
3.34 2.16 1.60 2.18 1.43
Oxygen Transporting
Properties
of the Haemocyanin
The mean in viva pH of the prebranchial haemolymph of U. &versa and M. messor measured at 25°C was 7.856 2 0.11 and 7.921 + 0.09, respectively. The concentrations of the major inorganic ions and of L-lactate in the haemolymph of U. inversa and M. messor are shown in Table 4. The most notable difference between the two species was that the concentrations of Na+ and Cl- ions were significantly higher in the haemolymph of M. messor. The mean oxygen carrying capacity of the haemocyanin (i.e., bound
Temperature and Respiration of Land Crabs
301
TABLE 4. Concentrations of the major inorganic ions and of L.-lactate in the haemolymph of U. &versa and M. messor U. iiwersa (mmol.l-‘)
Na+ K+
M. messor (mmol.l-’ )
558.3 10.9 8.5 28.7 572.0 0.42
Ca*+
MgI+ ClIact-
622.1 13.2 9.3 28.6 615.0 0.51
Values ate the means of two replicates from the same pooled haemolymph sample.
oxygen) was 0.76 + 0.32 and 0.89 -+ 0.26 mmol.lk’ inversa and M. messor, respectively.
in U.
The relationships between PSOand pH and the cooperativity (nso) and pH for the haemocyanin of U. inversa and M. messor are presented in Figs. 5 and 6, respectively.
B
oxygen
600-
affinity of the haemocyanin
The
of both species was
quite high (Pso = 6.4 and 12.4 Torr for U. inversa and M. 400.
A 200 -
5040-
nr
loo -
30-
20 -
Fresh weight(g)
hi0
FIG. 4. The effect of temperature on the relationships bee tween heart rate and fresh body weight of (A) Uca invem and (b) Metopogmpsus messor. The temperatures used were 15 (O), 20 (O), 25 (A), 30 (0), 35 (M) and 40°C (A). The regression equations fitted to these data are given in Table 2.
10 864 4-
2
7.4
7.6
7.6
7.7
7.0
7.9
9.0
8.1
7.4
7.5
7.6
7.7
7.0
7.9
6.0
8.1
TABLE 3. Regression equations describing the relationships between heart rate and fresh weight for U. inversa and M. messor hf. messor
U. invefsa Temp.
a
15°C 20°C 25°C 30°C 35°C 40°C
2.057 2.309 2.473 2.629 2.708 2.770
b -0.382 -0.408 -0.253 -0.304 -0.207 -0.211
r2
D
a
b
rz
R
0.601 0.623 0.676 0.666 0.651 0.674
27 26 27 23 21 7
2.047 2.253 2.490 2.551 2.724 2.791
-0.161 -0.095 -0.177 -0.128 -0.133 -0.123
0.746 0.575 0.748 0.677 0.712 0.637
16 22 16 16 13 6
Equations take the form log Y = a + b log X.
FIG. 5. The effect of temperature on the relationship between (A) the oxygen affinity (P,,) and pH and (B) the cooperativity (nso) and pH for the haemocyanin of Uca thinversa. The temperatures used were 15 (0), 20 (A), 25 (O), 30 (a) and 35% (A).
A. A. Eshky et al.
302
A
TABLE 5. Values for the Bohr coefficient of the haemocya. nin of U. inversa and M. messor at different temperatures U. inversa
Temperature
-1.14 -1.16 -1.07 - 1.05 -1.07
20°C 25°C 30°C 35°C 40°C
M. messor -0.92 -0.94 -0.96 -0.82 -0.89
lo0p50
6-
M. messor and in U. inuersa, respectively. The cooperativity
4-
of the haemocyanin
differed between
ntu values for the haemocyanin
lower than those of M. messor. In neither species was there any clear relationship between nSOand pH nor was n50 af-
2-
fected by temperature 7.6 717
the two species; the
of LJ. inversa were generally
710
719
S!O
811
012
0:s
014
0:s
B
(Figs. 5 and 6).
DISCUSSION Both U. inversa and M. messor showed the expected
4-
nounced
increase
pro-
in MO? and heart rate during exposure
to increasing temperature. Temperature did not, however, affect the relationship between Mel and body weight or be-
"50
tween heart rate and body weight in either species. This is lI 7.6 7.7
7.0
7.9
0.0
0.1
0.2
0.3
0.4
1 0.5
PB FIG. 6. The effect of temperature on the relationship bed tween (A) the oxygen affinity (l’s,) and pH and (B) the cooperativity (nso) and pH for the haemocyanin of Meroe pograpsus messor. The temperatures used were 15 (0), 20 (A), 25 (O), 30 (0) and 35°C (A).
in agreement with the results of a number of previous studies on decapod Crustacea (32,33,6). Several studies of the effects of temperature on the metabolic rate of intertidal invertebrates, including decapods, have shown that, for some species, values of Qlo are lower in the temperature range normally experienced but increase at temperatures outside this range (2,22,30,32,33). This reduction in the sensitivity of metabolic rate to changing temperature has been interpreted as a mechanism by which energy could be conserved despite the increase in environmental temperature (23). Such an effect was not seen during the present study for,
messor, respectively,
pH = 7.9, temperature
= 30°C).
The
in both U. inversa and M. messor, values for Qrd remained
differences in P5ubetween the two species cannot be attributed to differences in the L-lactate concentration of the
approximately constant over a wide range of temperature. The Q,o values for heart rate also showed little change over the temperature range used in these studies except at the lowest temperatures (15-20°C) when significantly higher
haemolymph since there was no significant difference (p > 0.05) in the concentrations of L-lactate between the two species (0.42 2 0.12 and 0.51 2 0.16 mmol.l-’ in U. inversa and M. messor, respectively). The magnitude of the Bohr effect was greater in LJ. inuersa than in M. messor (Table 5). Covariance analysis showed that in neither species was there any significant (p > 0.05) effect of temperature on the Bohr value. Temperature did have a significant effect, however, on the oxygen affinity of the haemocyanin of these two crabs. As in other decapods, the oxygen affinity of the haemocyanin of both species decreased significantly with increasing temperature. Values for AH were quite similar between the two species, namely, -51.7 kJ.mol-’ and -57.4 kJ.mol-’ (for a temperature increase from 25-30°C; pH = 7.9) in
Qlo values were recorded. The difference in Qlo values for Moz between the two species may reflect differences in the range of temperatures that they normally experience in the field. Although both species occurred at the same location, differences in their life style and behaviour may result in the two species experiencing somewhat different temperature regimes (7). The adoption of a burrowing mode of life by U. inversa enables it to avoid exposure to temperature extremes. In our observations (7), crabs emerged from their burrows early in the morning but did not emerge during the hottest times of the day or at night. The temperature within the burrow showed much less die1 variation than that of the sediment surface
Temperature
and Respiration
of Land Crabs
or of the air just above the sediment
303
surface (7). In contrast,
It is equally difficult to discern any clear trends in the
M. messor appears to be subject to a greater range of temper-
magnitude of the Bohr effect or of the oxygen carrying ca-
ature despite showing behavioural
pacity of the haemolymph
patterns, such as seeking
shade among the mangrove bushes, that may limit the temperature range experienced (7). In previous studies of these species, the upper lethal tem-
with increasing terrestrialization.
There is some evidence in the literature that the Bohr effect is smaller in air-breathing crabs than aquatic species but there are several exceptions (27,19). The Bohr value
peratures for U. inversa appeared to be just above 40°C
(- 1.16 at 25°C) for the haemocyanin
(5,7). A similar value was suggested by the present study: a number of crabs, particularly the smaller ones, did not
than in many terrestrial species, for example, -0.13 in Holthusiana rrans~ersa (21), -0.67 in Ocrpode saratan (l?), -0.38 and -0.55 in Gecurcoidea natalis and in G. Iulandii,
survive exposure to this temperature
within the respirome-
ter. The importance of evaporative cooling in enabling the body temperature of both species to be kept below lethal limits has been demonstrated
previously (7), but this mech-
anism could not be used to lower body temperature
during
respectively
(16),
and -0.43
of U. inversa is higher
in Coenobita
cIy@eatw (18)
but an even higher value ( - 1.67) has been reported for U. @gilator (19). Changes
in temperature
can have important
effects on
exposure to high temperatures in the respirometers since the addition of a small amount of sea water to the respirometers
the oxygen transporting These may be manifested
ensured that the air inside the chamber was completely saturated. This may have been a contributing factor to the high
is, changes in the heat of oxygenation (AH) which reduces oxygen affinity, and indirect effects such as the changes in haemolymph pH which in turn decrease oxygen affinity via
mortalities
recorded at the highest temperatures
used.
properties of the haemolymph. through both direct effects, that
the Bohr effect. The values of dH for U. inversa and M.
There is now a reasonably extensive literature on the oxygen transporting properties of the haemocyanin of semi-
messor (-57.4
terrestrial
that the oxygen affinity of the haemocyanin
crabs. Attempts
of air and bimodal
to compare
breathers
the oxygen affinity
have proved difficult since
much of the earlier information was obtained before the role of organic compounds (e.g., L-lactate and urate) in altering haemocyanin oxygen affinity was understood (2620). Mangum (14) failed to show a significant trend in the oxygen affinity of the haemocyanin
of a range of air and bimodal
and -51.7
kJ.moll’,
respectively)
indicate
of the two spe-
cies shows a high temperature sensitivity. The dH value for the haemocyanin of M. messor is similar to that for Leptograpsus vuriegutus (-67.4 kJ. mol-*) (9) and for Holthusiana transversa (-54.4 kJ.moll’) (21) both of which, like M. messor, have haemocyanins
an amphibious life style. In contrast, the of more terrestrial species show a lower tem-
breathers but a recent study by Morris and Bridge (19) in which comparisons of the haemocyanin oxygen affinity
perature sensitivity, for example, Coenobim clypeatw -42.8 kJ.moll’ (18), Ocrpode saratan -25.8 kJ.moll’ (17) and the
were made at constant pH and L-lactate concentration has indicated that, although there is considerable variation, the
anomuran
oxygen affinity of the haemocyanin of bimodal breathers is generally higher than that of aquatic species. The trend of
other more terrestrial species that have been examined. There is evidence, however, that species which inhabit en-
increasing
vironments
oxygen affinity with increasing
was demonstrated grapsid crabs (19). Both U. inversa haemocyanins
terrestrialization
more clearly in an ecological
series of
and M. messor were found to possess
having quite high oxygen affinities
(Pso =
6.4 and 12.4 Torr at pH 7.9, temperature = 30°C). The differences between the species could not be attributed to differences in the L-lactate concentration of the haemolymph which did not differ significantly in the pooled samples used in the analyses. (Urate concentrations were not measured.) These data support the trend noted by Morris and Bridges (19) since U. inversa is a more terrestrial species than M. messor which, at the study site, regularly spends time in the shallow water (but not totally submerged)
at
the base of Avicennia bushes (7). There are few compara, tive data for other grapsid crabs but the PsO value for M. messor falls approximately in the middle of the range of values obtained for a number of grapsid crabs exhibiting different degrees of terrestriality (19). Similarly, the PsO for U. inversa is almost identical to that of U. pugilator (8.9 Torr) (19).
Birgus [atro -38.5
kJ.mol-i
(18). The tempera-
ture sensitivity of U. inversa is therefore higher than in some
in which they are regularly exposed to changes
in temperature show a reduced temperature sensitivity of the haemocyanin whereas others that experience smaller temperature
fluctuations,
or that show behavioural
adapta-
tions to avoid extreme temperatures, have haemocyanins which show higher temperature sensitivities. The data for U. inversa provide further support for this relationship since the adoption of a burrowing lifestyle by this species has been demonstrated to provide protection from environmental extremes; the body temperature of crabs in natural burrows showed much smaller die1 variation than the temperature of the air outside the burrow (7). Similarly, M. messor may also overcome means any potential
by behavioural
problems associated with the posses-
sion of a respiratory pigment having a high temperature sensitivity. It appears to avoid prolonged immersion particularly during the day when exposure to increasing temperatures in water may compromise its ability to take up oxygen from this often hypoxic medium. M. messor was frequently observed to shelter in the Avicennin bushes where temperatures in the shade may be several degrees
A. A. Eshky et al.
304
lower than in water or on the mud surface (7). Even at night when some crabs were seen to move across the immersed mud flats, most left the water by climbing into the mangrove bushes. By leaving the water and emerging into shaded air, the crabs experience a less extreme thermal environment and oxygen uptake is facilitated. Even if the air temperature was not significantly
lower, the reduction in oxygen affinity
at higher temperatures would aid oxygen release to the tissues without adversely affecting the pigment’s ability to take up oxygen from the air. This work was funded by a grant from King Abdu&i~
References 1. Alexander, S.J.; Ewer, D.W. A comparative
2.
3.
4. 5. 6.
7.
8.
9.
10.
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14.
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study of some aspects of the biology and ecology of Sesarma catenata Ort. and Cyclogra~sw @nctatu.s M. Edw., with additional observations on Sesarmn meinerti De Man. Zool. Afr. 4:1-35;1969. Boyden, C.R. Aerial respiration of the cockle Cerastoderma eduk in relation to temperature. Comp. Biochem. Physiol. 43A:697-712;1972. Bridges, C.R.; Bicudo, J.E.P.W.; Lykkeboe, G. Oxygen content measurement in blood containing haemocyanin. Comp. Biochem. Physiol. 62A:457-462;1979. Davies, P.S. A constant pressure respirometer for mediumsized animals. Oikos 17:108-l 12;1966. Edney, E.B. The water and heat relationships of fiddler crabs (Uca spp.). Trans. R. Sot. S. Africa. 36:71-91;1961. Emmerson, W.D. Oxygen consumption in Pulaemon pacijicur (Stimpson) (Decapoda: Palaemonidae) in relation to temperature, size and season. Comp. Biochem. Physiol. 81A:71-78; 1985. Eshky, A.A.; Atkinson, R.J.A.; Taylor, A.C. The physiological ecology of brachyuran crabs from Saudi Arabian Aoicenniu mangrove. Mar. Ecol. Prog. Ser. 126:83-95;1995. Graham, R.A.; Mangum, C.P.; Terwilliger, R.C.; Terwilliger, N.B. The effect of organic acids on oxygen binding of hemocyanin from the crab Cancer magister. Comp. Biochem. Physiol. 74A:45-50;1983. Greenaway, P.; Morris, S.; Sanders, N.; Adamczewska, A. Blood gas transport and oxygen consumption in a supralittoral crab, Leptograpsus variegatus (Crustacea: Brachyura) Aust. J. Mar. Freshwat. Res. 43:1573-1584;1992. Gutmann, I.; Wahlefeld, A.W. L-(+)-lactate. Determination with lactate dehydrogenase and NAD. In: Bergmeyer, H.U. (ed). Methods of enzymatic analysis, 2nd ed. New York: Academic Press; 1974:1464-1468. Hoggarth, K.R.; Trueman, E.R. Techniques for recording the activity of aquatic invertebrates. Nature, London. 213:10501051;1967. Lallier, F.; Truchot, J.P. Modulation of haemocyanin oxygenaffinity by I-lactate and urate in the prawn Penaeus japonicus. J. Exp. Biol. 147:133-146;1989. Macnae, W. A general account of the fauna and flora of mangrove swamps and forests in the Indo-west-Pacific region. Adv. Mar. Biol. 6:73-270;1968. Mangum, C.P. On the relationship between Pso and the mode of gas exchange in tropical crustaceans. Pac. Sci. 36:403-410; 1982.
15. Morris, S. Effects of freezing on the function and association state of crustacean haemocyanin. J. Exp. Biol. 138:535-539; 1988. 16. Morris, S. Respiratory gas exchange and transport in crustaceans: ecological determinants. Mem. Qd Mus. 31:241-261; 1991. 17. Morris, S.; Bridges, C.R. An investigation of haemocyanin oxygen affinity in the semi-terrestrial crab Ocypode saratan Forsk. J. Exp. Biol. 117:119-132;1985. 18. Morris, S.; Bridges, C.R. Oxygen binding by the hemocyanin of the terrestrial hermit crab Coenobita clypeatus (Herbst)the effect of physiological parameters in vitro. Physiol. Zoiil. 59:606-615;1986. 19. Morris, S.; Bridges, CR. Properties of respiratory pigments in bimodal breathing animals: air and water breathing by fish and crustaceans. Am. Zool. 34:216-228;1994. 20 Morris, S.; Bridges, C.R.; Grieshaber, M.K. A new role for uric acid: modulator of haemocyanin oxygen affinity in crustaceans. J. Exp. Zool. 235:135-139;1985. 21. Morris, S.; Greenaway, I’.; McMahon, B.R. Oxygen and carbon dioxide transport by the haemocyanin of an amphibious crab, Holthuisanu transversa. J. Comp. Physiol. 157:873-882;1988. 22. Newell, R.C. Effect of fluctuations in temperature on the metabolism of intertidal invertebrates. Amer. Zool. 9:293-307; 1969. 23. Newell, R.C. Factors affecting the respiration of intertidal invertebrates. Amer. Zool. 13:513-528;1973. maenas to 24. Taylor, A.C. The respiratory responses of Car&w declining oxygen tension. J. Exp. Biol. 65:309-322;1976. 25. Taylor, A.C.; Funge-Smith, S.J. Temperature and salinity effects on oxygen transport by the hemolymph of the freshwater prawn Macrobrachium rosenbergii (de Man). Physiol. Zo61. 67: 26. 27.
28.
29.
30. 31. 32.
33.
34.
639-658;1994. Truchot, J.P. Lactate increases the oxygen aft;nity of crab hemocyanin. J. Exp. Zoo]. 214:205-208;1980. Truchot, J.P. Respiratory function of arthropod hemocyanins. In: Mangum, C.P. (ed). Blood and tissue oxygen carriers: advances in comparative and environmental physiology. Berlin: Springer-Verlag; 1992:377-410. Tucker, V.A. Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23:410414;1967. Vannini, M.; Valmori, P. Researches on the coast of Somalia. The shore and dune of Sar Uanle. 3 1. Ocypodidae and Gecarcinidae (Decapoda Brachyura). Monitore Zool. Ital. (N.S.) 14:199-226;1981. Vernberg, W.B.; Vemberg, F.J. Environmental physiology of marine animals. New York: Springer Verlag; 1972. Verwey, J. Einiges uber die Biologie Ost-lndischer Mangrovekrabben. Treubia 12:167-261 + PI VI-XIV. Wallace, J.C. Activity and metabolic rate in the shore crab, Carcinus maenas (L.). Comp. Biochem. Physiol. 41A:523533;1972. Wernick, A.M. The oxygen consumption of the hermit crab Clibanarius piittatus (Bose) (Decapoda, Diogenidae) in relation to temperature and size. Revta Bras. Biol. 42:267-273; 1982. Zainal, K.A.Y., Taylor, A.C.; Atkinson R.J.A. The effect of temperature and hypoxia on the respiratory physiology of the squat lobsters, Munida rugosa and Munida sarsi (Anomura, Galatheidae). Comp. Biochem. Physiol. lOlA:557-567; 1992.
1996
ISSN 0300-9629/96/$15.00 PI1 SO300.9629(96)00007-2
ELSEVIER
The Effects of Temperature on Aspects of Respiratory Physiology of the Semidterrestrial Crabs, Uca inversa (Hoffmann) and Metopogrupsus messor (Forsksl) from the Red Sea A. A. Eshky,’ A. C. Taylor,2 and R. _I. A. Atkinson3 ‘FACULTYOF MARINE SCIENCE,KING ABDULAZIZUNIVERSITY,JEDDAH,SAUDI ARABIA, *INSTITUTEOF BIOMEDICAL AND LIFESCIENCES,GRAHAM KERRBUILDING,UNIVERSITYOF GLASGOW,GLASGOW, G12 8QQ, UK, AND ‘UNIVERSITY MARINE BIOLOGICAL STATION, MILLPORT,ISLEOF CUMBRAE,KA28 OEG, UK
ABSTRACT. The effects of temperature on aspects of the respiratory physiology of two semi-terrestrial crabs, Uca inoersa and Metopoppsus messor have been studied. When exposed to increasing temperatures in the laboratory both species show a pronounced increase in both the rate of oxygen consumption (tiol) and in heart rate; Q10 values were approximately 2-3 over the temperature range 15-40°C for both Mo2 and heart rate. Temperature did not, however, affect the relationships between h;lo2 and fresh body weight and between heart rate and fresh body weight. The haemocyanin of both species was found to have a high oxygen affinity (PsO = 6.4 and 12.4 Torr for U. inversa and M. messor, respectively, pH = 7.9, temperature = 30°C) and moderately high Bohr values (- 1.07 and -0.96 for U. inversa and M. messor, respectively). An increase in temperature resulted in a significant decrease in the oxygen affinity of the haemocyanin of both species (AH = -51.7 and -57.4 kJ.mol-’ for U. inversa and M. messor, respectively). Changes in temperature did not have a significant effect on either the Bohr value or the cooperativity of the pigment. COMP BIOCHEMPHYSIOL114A;4:297-304, 1996. KEY WORDS. Crustacea, Decapoda, Uca, Metopogmpsus, temperature, cyanin
INTRODUCTION Crabs are important
members of mangrove
ecosystems
in
tropical regions (3 1,13). One of the major problems that they face in this environment is that they may be exposed to temperatures that are potentially lethal. In a previous study (7) we discussed aspects of the ecology and behaviour in Red Sea mangroves of the two crabs used in the present study, the ocypodid Uca (Amphiucu) inversa inversa (Hoffmann) and the grapsid, Metopograpsus messor (Forsksl). The ecology of these two species is quite distinct; LJ. inversa occupies burrows in the upper eulittoral zone whereas M. mesSOTis found amongst mangrove trees (Auicennia marina) although it may also occur on rocky shores (29). Eshky et al. (7) showed that U. inversa was more tolerant of thermal stress than M. messor and that both species used behavioural and physiological means to survive exposure to high temperatures. In the case of U. inuersa, the burrow provided an essential refuge from environmental extremes since measurements
of the microclimatic
conditions
within
Address reprint requests to: A.C. Taylor, Institute of Biomedical and Life Sciences, Graham Kerr Building, University of Glasgow, Glasgow, G12 8QQ UK. Received 14 September 1995; revised 24 November 1995; accepted 5 December 1995.
oxygen consumption,
heart rate, haemo-
the burrow showed that the temperature within the burrow was consistently less variable than that of the sediment surface or of the air just above the sediment. Similarly, the relative humidity inside the burrow was much greater than that of the air outside and the burrow characteristically gave access to standing water. In contrast, M. messor constructs burrows only infrequently.
Instead, this species makes stra-
tegic use of the mangrove vegetation
to minimize the effects
of thermal stress; M. messor was observed to utilize the shade provided by the Avicennia plants during the the hottest times of the day but then foraged more widely on the mud plain during cooler periods. Although this species was observed to spend brief periods in shallow water at the base of the mangrove bushes, our observations indicate that it tends to avoid periods of prolonged immersion. Despite these behavioural mechanisms
which serve to re-
duce the degree of thermal stress to which they are exposed, both species may still experience a range of temperature which will have important physiological implications. The use of evaporative cooling to lower the body temperature may be crucial in enabling them to survive potentially lethal high temperatures (7). In both species, however, exposure to varying temperatures will affect other aspects of their physiology. The two species differ in the method used for aerial respi-
A. A. Eshky et al.
298
ration. LJ. inversa has expanded epibranchial spaces whose moist surfaces function as lungs whereas M. messor lacks
of differing sizes (fresh weight range = 1.15-5.9
lungs but, when emersed, circulates branchial water across antero-ventral body surfaces where it is reoxygenated
ried out over the temperature
(31,1,7). In the present paper, we consider the effects of temperature on aspects of the respiratory physiology of both U. inversa and M. messor to provide further information
on the
adaptations of these species to the often extreme conditions that characterize their habitat.
AND
Uca inwersa and Metopograpsus messor were collected
from
Ras Hatiba near Dah’ban approximately 50 km north of Jeddah, Saudi Arabia. The crabs were transported to the Marine Biological
Station
at Obhur, part of the Faculty of Ma-
were car-
using an im-
amount of sea water. The tanks were covered with black plastic sheets and placed in water baths at 15*C and the crabs left undisturbed for at least 2 hr before recordings commenced. Heart rate recordings were carried out continuously for a period of approximately 30 min and then the was slowly increased
next experimental
METHODS
range 15-40°C
pedance technique (11,27). Crabs were placed individually in small tanks containing a mud substratum and a small
temperature MATERIALS
g and 0.38-
17.5 g for U. inversa and M. messor, respectively)
temperature
(1°C
in 30 min) to the
and further recordings car-
ried out. In most cases, heart rate recordings were made at intervals of 5°C but, in other experiments, recordings were made at 2°C intervals. Since these experiments nary experiments
lasted for several hours, prelimi-
were carried out to establish whether the
rine Science, King Abdulaziz University. Both species were kept separately in large outdoor tanks (2 m diam.) con-
crabs showed any die1 rhythms of heart rate. In these experiments, heart rate recordings were carried out continuously
taining an appropriate mud substratum (from the local habi-
for periods over 24 hr on crabs held at 25°C under constant
tat) and were subjected to normal daylight and temperature cycles (the daytime temperature was approximately 40°C
illumination or in constant darkness. Although some variation was recorded in heart rate during these periods, no clear
falling to approximately 20°C at night). The mud in each tank was formed into a slope enabling the crabs to gain
rhythms could be established. In all further experiments, therefore, heart rate recordings were carried out in constant
access to sea water (salinity = 38%0) at the bottom of the tanks. Many U. inversa were observed to construct their
darkness to try to eliminate heart rate.
characteristic
burrows in the sediment and both species re-
mained in good condition
for many days.
Oxygen Transporting Properties and Ionic Composition of the Haemolymph
Oxygen Consumption Measurements
the effects of die1 changes in
of the rates of oxygen consumption
ried out using constant
pressure respirometers
were car(4). Crabs
(fresh weight range = 0.95-6.10 g and 2.75- 12.51 g for U. inversa and M. messor, respectively) were placed individually into each of the respirometers
and a few drops of sea
Values for the in viuo pH of the pre-branchial haemolymph of U. inversa and M. messor were obtained for crabs maintained at 25°C. The crabs (n = 10) were maintained
undis-
turbed at this temperature for several hours prior to haemolymph sampling to ensure that the pH values obtained were representative
of quiescent animals. The haemolymph
sam-
water (salinity = 38%0) were added to provide a source of water for the crabs and to maintain a high relative humidity in the respirometers. The respirometers were then placed
ples were taken by inserting the hypodermic needle of a chilled 1 ml syringe through the arthrodial membrane at the base of one of the pereiopods and the pH determined immedi-
in water baths at the appropriate temperature. The crabs were left undisturbed in the respirometers for at least 2 hr
ately by drawing it from the syringe into a capillary pH electrode (G299, Radiometer, Denmark) maintained at the same
before recordings commenced. Preliminary experiments demonstrated that this was sufficient to ensure that the
temperature and connected to a pH meter (Corning). Unfortunately, it was not possible to obtain values for the Paz of the pre- and post-branchial blood of these crabs since the
crabs had become quiescent
before recordings were carried
out. During this period the respirometers were not sealed to enable oxygen to diffuse into the chambers. At the start of each recording, the respirometers were sealed and changes in the manometer volume recorded at 5 min intervals over a period of 1 hr. At the end of the experiment the fresh weights of the crabs were obtained and weight specific rates of oxygen consumption (ko2) calculated. Rates of oxygen consumption were determined for both U. inversa and for M. messor over the temperature range 1540°C. The effect of temperature on the heart rate of these two species was also determined. Heart rate recordings of crabs
appropriate equipment was not available in Jeddah. Further haemolymph samples from both species were centrifuged (12,000 X g for 5 min) to remove haemocytes and any clotted proteins and then frozen at - 20°C. The samples were transferred still frozen to the University of Glasgow where the analyses were carried out. The oxygen carrying capacity of the haemocyanin (CHcOZ) was determined on individual haemolymph samples at 25°C using the method of Tucker (28) as modified by Bridges et al. (3). Following these determinations, the remaining haemolymph was pooled to provide a larger sample for ion analysis and for the construction of oxygen dissociation curves. The concentrations of the
Temperature and Respiration of Land Crabs
Na+, K+, Ca’+ and Mg”
299
30 -
ions in the serum were determined
by atomic absorption spectrophotometry (Philips 9200) following dilution of each sample with deionized water. The concentration
ofCl-
20 -
ions was determined by electrochemical
titration (Jenway Chloride meter). The concentration of Llactate in the haemolymph of both species was determined using the method of Gutman and Wahlefeld modifications of Graham et al. (8). In vitro oxygen dissociation
curves for the haemolymph
of U. inversa and M. messor were constructed spectrophotometrically using a diffusion chamber (34). Dissociation curves were constructed
at different
pHs to examine
the
magnitude of the Bohr factor in these species. The pH of the haemolymph was altered by varying the proportion of carbon dioxide in the gas mixtures supplied by gas mixing pumps (Wiisthoff, Bochum, Germany). The pH of the haemolymph
was
measured
by
tonometering
The pH of the haemolymph
was determined
haemolymph
that had been frozen (-20°C).
using
This was un-
avoidable since the samples had to be transferred frozen from Saudi Arabia to Glasgow for the analyses to be carried out. Pooled haemolymph samples were used in this study due to the limited amount of haemolymph that was available but this procedure had the advantage that it overcame any variations in the concentrations of haemolymph ions or L-lactate between samples taken on different occasions. Morris (15) and Lallier and Truchot (12) have shown that long-term storage of haemolymph samples at very low temperature (-SO’C) may affect the cooperativity (n50) of the haemocyanin. In other studies, however, it was found that storage at -20°C did not affect the cooperativity of the haemocyanin of other species (34,25). The effect of temperarure on the oxygen affinity of the haemocyanin
was also investigated
by constructing
oxygen
dissociation curves over a range of temperatures between 15 and 35°C. The effect of temperature on the oxygen affinity of the haemocyanin can be quantified by calculating the change in enthalpy (AH) accompanying oxygenation of the haemocyanin at a constant pH (7.9) using the following equation: BH = -2.303
x R x d log P50 (kJ_mol-,) Al/T1 -
T2
RESULTS Effect of Temperature
on &lo, and Heart
s-
E t cy .P
l-
at
the PsOusing the capillary pH electrode of the BMS2 which was connected to a pH meter (Corning 255 ion analyser). The PsOwas estimated from saturation values between 25% and 75% according to the Hill equation. Oxygen dissociation curves were constructed
h 7 G 7 F
another
haemolymph sample (100 ,~l) in a Radiometer BMS2 against the same gas mixtures supplied to the diffusion chamber.
10 _
(10) with the
Rate
The effect of temperature on the relationships between X402 and fresh body weight for U. inversa and for M. messor is
0.5 1
0.5
I
I
I
I11111~
1
10
5
Fresh weight (g) FIG. 1. The effect of temperature on the relationship between the rate of oxygen consumption (MO*) and fresh body weight of Uca inversa. The temperatures used were 15 (W), 20 (0), 25 (O), 30 (A), 35 (A) and40”C (0). The regression equations fitted to these data are given in Table 1.
shown in Figs. 1 and 2, respectively. As in other decapods, there is an inverse relationship between h;io, and body weight in both species. The regression equations describing the lines fitted to these data are presented in Table
1. Co-
variance analysis of the data for both U. inversa and M. messor showed that, although there were no significant differences (p > 0.05) between the slopes of the regression lines, there were highly significant differences
(p < 0.001)
between their elevations. Values for MO* were calculated for 5°C increases in temperature over the range of temperatures
used (15-40°C).
Values for Uoz for ‘standard’ crabs (fresh weight 3.0 g and 7.0 g in U. inwersa and M. messor, respectively) were obtained by interpolation from the regression equations in Table 1. Q10 values calculated from these data for U. inversa were between 2.4-3.2 over the temperature range 15-35°C but at higher temperatures (between 35 and 40°C) the QIO increased to 4.82 (Table 2). The QIo values for the hoz of M. messor were slightly lower (1.8-2.1 in the temperature range 15-35°C) but at high temperatures (between 35 and 40°C) QIo declined slightly (Table 2). Both species were clearly stressed at these high temperatures since a number of animals died when exposed to 40°C. As a result, fewer data were obtained and their reliability must also be questioned.
A. A. Eshky et al.
300
20 -
10 A ‘; r 7 B = E S N
5-
.l/10
20
FIG. 3. The effect of temperature on the rate of oxygen consumption (MO,) (as loglo) of Uca inversa (0) and Metopograpsus messor (0). The data have been calculated for ‘Stan. dard’ crabs of 3.0 and 7.0 g fresh weight, respectively, using the regression equations in Table 1.
2-
I’,
I
,
I
10
5
2
20
Calculation
Fresh weight (g) FIG. 2. The effect of temperature on t&e relationship between the rate of oxygen consumption (MOM)and fresh body weight of Metopograpsus messor. The temperatures used were 15 (U), 20 (0), 25 (O), 30 (A), 35 (A) and 40°C (0). The regression equations fitted to these data are given in Tae ble 1.
TABLE 1. .Regression equations describing the relationships between Mo2 and fresh weight for U. inversa and M. messor u. inversa
15°C 20°C 25°C 30°C 35°C 40°C
40
Temperature (“C)
.P
Temp.
30
a 0.093 0.32 0.544 0.821 1.016 1.308
b -0.377 -0.432 -0.377 -0.488 -0.494 - 0.390
M. me.war r*
n
a
b
r2
n
0.724 0.794 0.691 0.781 0.744 0.704
17 18 25 20 15 18
0.535 0.672 0.840 0.999 1.125 1.258
-0.437 -0.442 -0.466 -0.463 -0.428 -0.453
0.929 0.908 0.943 0.906 0.901 0.918
16 27 34 30 33 15
TABLE 2. Q10 values for oxygen consumption heart rate (fh) of U. inversa and M. messor
(&IO,) and for
U. inversa GO,
1%20°C 20-25°C 25-30°C 30-35°C 35-40°C
2.52 3.17 2.81 2.42 4.82
M. messor fll
3.01 2.99 1.83 1.78 1.32
the temperature range 1%
ture on Go? is greater in the former species. This is demonstrated more clearly in Fig. 3 in which log Go, is plotted against temperature. The effect of temperature
on the relationships
between
heart rate and fresh body weight for LJ. inversa and M. mesSOTis shown in Fig. 4. The regression equations of the lines fitted to these data are given in Table 3. Covariance analysis of these data showed that there were no significant differences (p > 0.05) between the slopes of the regression lines but there were highly significant differences (p < 0.001) between their elevations. Thus, as was the case for k40z, temperature does not affect the relationship between heart rate and fresh body weight. It is interesting to note, however, that the slopes of the regression lines obtained for M. messor were significantly lower than those for U. inversa. The QE values for heart rate were similar in both U. ineiersa and M. messor (Table 2). In addition, both species showed a gradual reduction in the value of QIo with increasing temperature.
Equations take the form log Y = a + h log X.
Temperature
ofQlo values over
40°C gave values of 3.04 and 1.92 for U. inversa and M. messor, respectively, indicating that the effect of tempera-
hi0,
1.84 1.97 2.10 2.05 1.67
fll
3.34 2.16 1.60 2.18 1.43
Oxygen Transporting
Properties
of the Haemocyanin
The mean in viva pH of the prebranchial haemolymph of U. &versa and M. messor measured at 25°C was 7.856 2 0.11 and 7.921 + 0.09, respectively. The concentrations of the major inorganic ions and of L-lactate in the haemolymph of U. inversa and M. messor are shown in Table 4. The most notable difference between the two species was that the concentrations of Na+ and Cl- ions were significantly higher in the haemolymph of M. messor. The mean oxygen carrying capacity of the haemocyanin (i.e., bound
Temperature and Respiration of Land Crabs
301
TABLE 4. Concentrations of the major inorganic ions and of L.-lactate in the haemolymph of U. &versa and M. messor U. iiwersa (mmol.l-‘)
Na+ K+
M. messor (mmol.l-’ )
558.3 10.9 8.5 28.7 572.0 0.42
Ca*+
MgI+ ClIact-
622.1 13.2 9.3 28.6 615.0 0.51
Values ate the means of two replicates from the same pooled haemolymph sample.
oxygen) was 0.76 + 0.32 and 0.89 -+ 0.26 mmol.lk’ inversa and M. messor, respectively.
in U.
The relationships between PSOand pH and the cooperativity (nso) and pH for the haemocyanin of U. inversa and M. messor are presented in Figs. 5 and 6, respectively.
B
oxygen
600-
affinity of the haemocyanin
The
of both species was
quite high (Pso = 6.4 and 12.4 Torr for U. inversa and M. 400.
A 200 -
5040-
nr
loo -
30-
20 -
Fresh weight(g)
hi0
FIG. 4. The effect of temperature on the relationships bee tween heart rate and fresh body weight of (A) Uca invem and (b) Metopogmpsus messor. The temperatures used were 15 (O), 20 (O), 25 (A), 30 (0), 35 (M) and 40°C (A). The regression equations fitted to these data are given in Table 2.
10 864 4-
2
7.4
7.6
7.6
7.7
7.0
7.9
9.0
8.1
7.4
7.5
7.6
7.7
7.0
7.9
6.0
8.1
TABLE 3. Regression equations describing the relationships between heart rate and fresh weight for U. inversa and M. messor hf. messor
U. invefsa Temp.
a
15°C 20°C 25°C 30°C 35°C 40°C
2.057 2.309 2.473 2.629 2.708 2.770
b -0.382 -0.408 -0.253 -0.304 -0.207 -0.211
r2
D
a
b
rz
R
0.601 0.623 0.676 0.666 0.651 0.674
27 26 27 23 21 7
2.047 2.253 2.490 2.551 2.724 2.791
-0.161 -0.095 -0.177 -0.128 -0.133 -0.123
0.746 0.575 0.748 0.677 0.712 0.637
16 22 16 16 13 6
Equations take the form log Y = a + b log X.
FIG. 5. The effect of temperature on the relationship between (A) the oxygen affinity (P,,) and pH and (B) the cooperativity (nso) and pH for the haemocyanin of Uca thinversa. The temperatures used were 15 (0), 20 (A), 25 (O), 30 (a) and 35% (A).
A. A. Eshky et al.
302
A
TABLE 5. Values for the Bohr coefficient of the haemocya. nin of U. inversa and M. messor at different temperatures U. inversa
Temperature
-1.14 -1.16 -1.07 - 1.05 -1.07
20°C 25°C 30°C 35°C 40°C
M. messor -0.92 -0.94 -0.96 -0.82 -0.89
lo0p50
6-
M. messor and in U. inuersa, respectively. The cooperativity
4-
of the haemocyanin
differed between
ntu values for the haemocyanin
lower than those of M. messor. In neither species was there any clear relationship between nSOand pH nor was n50 af-
2-
fected by temperature 7.6 717
the two species; the
of LJ. inversa were generally
710
719
S!O
811
012
0:s
014
0:s
B
(Figs. 5 and 6).
DISCUSSION Both U. inversa and M. messor showed the expected
4-
nounced
increase
pro-
in MO? and heart rate during exposure
to increasing temperature. Temperature did not, however, affect the relationship between Mel and body weight or be-
"50
tween heart rate and body weight in either species. This is lI 7.6 7.7
7.0
7.9
0.0
0.1
0.2
0.3
0.4
1 0.5
PB FIG. 6. The effect of temperature on the relationship bed tween (A) the oxygen affinity (l’s,) and pH and (B) the cooperativity (nso) and pH for the haemocyanin of Meroe pograpsus messor. The temperatures used were 15 (0), 20 (A), 25 (O), 30 (0) and 35°C (A).
in agreement with the results of a number of previous studies on decapod Crustacea (32,33,6). Several studies of the effects of temperature on the metabolic rate of intertidal invertebrates, including decapods, have shown that, for some species, values of Qlo are lower in the temperature range normally experienced but increase at temperatures outside this range (2,22,30,32,33). This reduction in the sensitivity of metabolic rate to changing temperature has been interpreted as a mechanism by which energy could be conserved despite the increase in environmental temperature (23). Such an effect was not seen during the present study for,
messor, respectively,
pH = 7.9, temperature
= 30°C).
The
in both U. inversa and M. messor, values for Qrd remained
differences in P5ubetween the two species cannot be attributed to differences in the L-lactate concentration of the
approximately constant over a wide range of temperature. The Q,o values for heart rate also showed little change over the temperature range used in these studies except at the lowest temperatures (15-20°C) when significantly higher
haemolymph since there was no significant difference (p > 0.05) in the concentrations of L-lactate between the two species (0.42 2 0.12 and 0.51 2 0.16 mmol.l-’ in U. inversa and M. messor, respectively). The magnitude of the Bohr effect was greater in LJ. inuersa than in M. messor (Table 5). Covariance analysis showed that in neither species was there any significant (p > 0.05) effect of temperature on the Bohr value. Temperature did have a significant effect, however, on the oxygen affinity of the haemocyanin of these two crabs. As in other decapods, the oxygen affinity of the haemocyanin of both species decreased significantly with increasing temperature. Values for AH were quite similar between the two species, namely, -51.7 kJ.mol-’ and -57.4 kJ.mol-’ (for a temperature increase from 25-30°C; pH = 7.9) in
Qlo values were recorded. The difference in Qlo values for Moz between the two species may reflect differences in the range of temperatures that they normally experience in the field. Although both species occurred at the same location, differences in their life style and behaviour may result in the two species experiencing somewhat different temperature regimes (7). The adoption of a burrowing mode of life by U. inversa enables it to avoid exposure to temperature extremes. In our observations (7), crabs emerged from their burrows early in the morning but did not emerge during the hottest times of the day or at night. The temperature within the burrow showed much less die1 variation than that of the sediment surface
Temperature
and Respiration
of Land Crabs
or of the air just above the sediment
303
surface (7). In contrast,
It is equally difficult to discern any clear trends in the
M. messor appears to be subject to a greater range of temper-
magnitude of the Bohr effect or of the oxygen carrying ca-
ature despite showing behavioural
pacity of the haemolymph
patterns, such as seeking
shade among the mangrove bushes, that may limit the temperature range experienced (7). In previous studies of these species, the upper lethal tem-
with increasing terrestrialization.
There is some evidence in the literature that the Bohr effect is smaller in air-breathing crabs than aquatic species but there are several exceptions (27,19). The Bohr value
peratures for U. inversa appeared to be just above 40°C
(- 1.16 at 25°C) for the haemocyanin
(5,7). A similar value was suggested by the present study: a number of crabs, particularly the smaller ones, did not
than in many terrestrial species, for example, -0.13 in Holthusiana rrans~ersa (21), -0.67 in Ocrpode saratan (l?), -0.38 and -0.55 in Gecurcoidea natalis and in G. Iulandii,
survive exposure to this temperature
within the respirome-
ter. The importance of evaporative cooling in enabling the body temperature of both species to be kept below lethal limits has been demonstrated
previously (7), but this mech-
anism could not be used to lower body temperature
during
respectively
(16),
and -0.43
of U. inversa is higher
in Coenobita
cIy@eatw (18)
but an even higher value ( - 1.67) has been reported for U. @gilator (19). Changes
in temperature
can have important
effects on
exposure to high temperatures in the respirometers since the addition of a small amount of sea water to the respirometers
the oxygen transporting These may be manifested
ensured that the air inside the chamber was completely saturated. This may have been a contributing factor to the high
is, changes in the heat of oxygenation (AH) which reduces oxygen affinity, and indirect effects such as the changes in haemolymph pH which in turn decrease oxygen affinity via
mortalities
recorded at the highest temperatures
used.
properties of the haemolymph. through both direct effects, that
the Bohr effect. The values of dH for U. inversa and M.
There is now a reasonably extensive literature on the oxygen transporting properties of the haemocyanin of semi-
messor (-57.4
terrestrial
that the oxygen affinity of the haemocyanin
crabs. Attempts
of air and bimodal
to compare
breathers
the oxygen affinity
have proved difficult since
much of the earlier information was obtained before the role of organic compounds (e.g., L-lactate and urate) in altering haemocyanin oxygen affinity was understood (2620). Mangum (14) failed to show a significant trend in the oxygen affinity of the haemocyanin
of a range of air and bimodal
and -51.7
kJ.moll’,
respectively)
indicate
of the two spe-
cies shows a high temperature sensitivity. The dH value for the haemocyanin of M. messor is similar to that for Leptograpsus vuriegutus (-67.4 kJ. mol-*) (9) and for Holthusiana transversa (-54.4 kJ.moll’) (21) both of which, like M. messor, have haemocyanins
an amphibious life style. In contrast, the of more terrestrial species show a lower tem-
breathers but a recent study by Morris and Bridge (19) in which comparisons of the haemocyanin oxygen affinity
perature sensitivity, for example, Coenobim clypeatw -42.8 kJ.moll’ (18), Ocrpode saratan -25.8 kJ.moll’ (17) and the
were made at constant pH and L-lactate concentration has indicated that, although there is considerable variation, the
anomuran
oxygen affinity of the haemocyanin of bimodal breathers is generally higher than that of aquatic species. The trend of
other more terrestrial species that have been examined. There is evidence, however, that species which inhabit en-
increasing
vironments
oxygen affinity with increasing
was demonstrated grapsid crabs (19). Both U. inversa haemocyanins
terrestrialization
more clearly in an ecological
series of
and M. messor were found to possess
having quite high oxygen affinities
(Pso =
6.4 and 12.4 Torr at pH 7.9, temperature = 30°C). The differences between the species could not be attributed to differences in the L-lactate concentration of the haemolymph which did not differ significantly in the pooled samples used in the analyses. (Urate concentrations were not measured.) These data support the trend noted by Morris and Bridges (19) since U. inversa is a more terrestrial species than M. messor which, at the study site, regularly spends time in the shallow water (but not totally submerged)
at
the base of Avicennia bushes (7). There are few compara, tive data for other grapsid crabs but the PsO value for M. messor falls approximately in the middle of the range of values obtained for a number of grapsid crabs exhibiting different degrees of terrestriality (19). Similarly, the PsO for U. inversa is almost identical to that of U. pugilator (8.9 Torr) (19).
Birgus [atro -38.5
kJ.mol-i
(18). The tempera-
ture sensitivity of U. inversa is therefore higher than in some
in which they are regularly exposed to changes
in temperature show a reduced temperature sensitivity of the haemocyanin whereas others that experience smaller temperature
fluctuations,
or that show behavioural
adapta-
tions to avoid extreme temperatures, have haemocyanins which show higher temperature sensitivities. The data for U. inversa provide further support for this relationship since the adoption of a burrowing lifestyle by this species has been demonstrated to provide protection from environmental extremes; the body temperature of crabs in natural burrows showed much smaller die1 variation than the temperature of the air outside the burrow (7). Similarly, M. messor may also overcome means any potential
by behavioural
problems associated with the posses-
sion of a respiratory pigment having a high temperature sensitivity. It appears to avoid prolonged immersion particularly during the day when exposure to increasing temperatures in water may compromise its ability to take up oxygen from this often hypoxic medium. M. messor was frequently observed to shelter in the Avicennin bushes where temperatures in the shade may be several degrees
A. A. Eshky et al.
304
lower than in water or on the mud surface (7). Even at night when some crabs were seen to move across the immersed mud flats, most left the water by climbing into the mangrove bushes. By leaving the water and emerging into shaded air, the crabs experience a less extreme thermal environment and oxygen uptake is facilitated. Even if the air temperature was not significantly
lower, the reduction in oxygen affinity
at higher temperatures would aid oxygen release to the tissues without adversely affecting the pigment’s ability to take up oxygen from the air. This work was funded by a grant from King Abdu&i~
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