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Hypoxia adaptation in the crayfish procambarus clarki
Camp. Biochem. Physiol. Vol. 79A, No. I, pp. 13-15, 1984
0300-9629/84 $3.00 + 0.00
Printedin Great Britain
0
HYPOXIA
ADAPTATION PROCAMBARUS
NICHOLAS A.
MAURO
and
1984 PergamonPress Ltd
IN THE CRAYFISH CLARKI CLAUDIA THOMPSON
Department of Biology, Hartwick College, Oneonta, NY 13820, U.S.A. Telephone: (607) 432-4200 (Received 10 November 1983)
Abstract-l. P. clurki is an oxyconformer, with an oxygen uptake rate of 144 + 4 Ml/g wet wt/hr at oxygen tensions above 90% saturation and an uptake rate of 18 + 3 ~1 g wet wt/hr at 15 torr. 2. Between 159 and 40 torr, blood pH decreases slightly from 7.77 k 0.03 to 7.65 k 0.04, and at 1.5torr, blood pH drops to 7.36 + 0.06. 3. At normoxia, blood lactate levels are low at 0.66 + 0.01 mM/l blood. After 2 and 5 hr exposure to 15 torr, blood lactate levels increase to 3.29 k 0.47 and 8.91 f. 0.14 mM/l blood, respectively. Upon return to normoxia, blood lactate levels decrease and are comparable to normoxic controls after 13 hr. 4. During mild hypoxia, P. clurki maintains adequate oxygen transport by utilizing a high 0, affinity hemocyanin in conjunction with a low metabolic demand by its tissues.
INTRODUCTION
ographic electrode (5420A), and the required oxygen tension obtained by bubbling either air or nitrogen into the water. Blood pH was determined with an Orion implantable electrode (916300). The electrode was inserted directly into the ventral muscle of the first abdominal segment and the pH values read with a Leeds and Northrup pH meter (7405). Blood lactate levels were determined in 0.5 ml of prebranchial blood obtained via hypodermic syringe through the sternum of the first abdominal segment. In some cases, blood samples were pooled from individuals. Blood samples were analysed with a Sigma lactate test kit, modified according to the procedures of Graham et al. (1983).
The pH modulation of hemocyanin oxygen affinity has an important function in the adaptation of freshwater crustaceans to hypoxia. In previously reported studies, short-term exposure (2-24 hr) to mild hypoxia (P,02 = 40 torr) induces a hyperventilatory response in several species, with a concomitant increase in blood pH (Dejours and Beeckenkamp, 1971; Sinha and Dejours, 1980; Wheatly and Taylor, 1981; Wilkes and McMahon, 1982a,b; McMahon and Wilkens, 1983). Since elevated pH increases hemocyanin oxygen affinity, the change in blood pH allows the blood to maintain its oxygen transport function despite a reduced oxygen tension gradient. This adaptive mechanism requires that a given species must possess a hemocyanin which is sensitive to pH modulation: however, not all species of freshwater crustaceans possess a hemocyanin with a Bohr shift. In contrast, the oxygen affinity of Procambarus clarki hemocyanin has little dependence on pH within the physiological range (Mangum, 1983). This observation suggests a different mechanism for adapting to hypoxia than is found in other freshwater crustaceans. This study examines and compares the hypoxia adaptation mechanisms of the crayfish P. clarki to previously described species.
RESULTS
Effects of hypoxia on oxygen uptake
In response to hypoxia, P. clarki is an oxyconformer (Fig. l), comparable to Procambarus simduns and Orthonectes immunis (Larimer and Gold,
160 1
I
z
140-
IZO-
IOO-
MATERIAL AND METHODS 80-
Animals were obtained from commercial sources and were maintained in the laboratory in well-aerated tanks for 2 weeks at 22°C prior to all experiments. Their weights ranged from 11 to 15 g. Oxygen uptake (riOJ of animals in the dark were determined by the depletion of oxygen in closed containers (Yellow Springs Instrument Co. 5420A). Animals were starved for 48 hr before being tested, and were allowed to adjust to aerated containers for 30 min before the beginning of measurements. For the purpose of blood pH and lactate determinations, animals were placed into 4-l tanks for varying time periods during which the oxygen tension of the water was closely monitored with a Yellow Springs Instrument Co. polar-
60-
40-
20-
0
I
I 20
I 40
I 60
I 80
I 100
I 120
I 140
Torr ( mm Hgl Fig. 1. Effects of hypoxia on oxygen uptake of P. clarki at 22°C. Mean + SE N = 6 for each exposure. 73
NICHOLAS A. MAURO
14
-
Hypoxfa -4
and
Normoxla-+
13IZ1, IO9a?65432Iz __
o-
CLAUDIA THOMPK~N
f
76 I, 75 f
74
72
7’II / 11 I II I II I I I1 0 12345,23456789101112
II
I I1
I
0
20
‘1
40
1’
60 100 60 Torr (mmlig)
1
120
11
140
160
Time (hr)
Fig. 2. Lactate production during 5 hr exposure to 15 torr followed by recovery at 159 torr. Mean f SE N = 6 for exposure.
1961; Wiens and Armitage, 1961). Above 90% saturation oxygen uptake (PO,) measures 144 f 4 pi/g wet wt/hr and decreases by 87.5% at 15 torr to 18 f 3 ~1 wet wt/hr. In obtaining these measurements, animals were not restrained and hypoxia was induced by organism depletion in closed system respirometers. Lactate production with hypoxia
At normoxia, the blood lactate level in P. clarki is low, measuring 0.66 f 0.01 mM/l blood. Following a 5-hr exposure to 40 torr, no significant increase (P > 0.5) in blood lactate occurs. After 2 and 5 hr exposures to 15 torr, blood lactate levels increase to 3.20 _t 0.47 and 8.91 f 0.14 mM/l blood, respectively (Fig. 2). Following reexposure to normoxia, there is a continuous decline in blood lactate until after 13 hr, when blood lactate levels become comparable to normoxic controls. The time needed to reestablish normoxic blood lactate levels in P. clarki is slower than that reported for M. rosenbergii and is comparable to the rates observed in the burrowing marine species Nephrops norvegicus and Atelecyclus rotundatus (Mauro and Malecha, 1984a; Bridges and Brand, 1980). Effects of hypoxia on bloodpH
and oxygen transport
After a 5-hr exposure to reduced oxygen tensions, blood pH remains fairly constant, between 159 and 40 torr ranging between 7.77 + 0.03 and 7.65 + 0.04. This response to hypoxia contrasts markedly with other crayfish species in which blood pH increases with exposure to mild hypoxia (Dejours and Beeckenkamp, 1971; Sinha and Dejours, 1980; Whea.tly and Taylor, 1981; Wilkes and McMahon, 1982a,b; McMahon and Wilkens, 1983). Down to 40 torr oxygen delivery to the tissues is adequate to fuel oxidative metabolism and impede lactate production. At 15 torr, oxygen transport is reduced beyond the point where oxidative metabolism can be maintained. Lactate accumulates and blood pH falls sharply to 7.36 f 0.06 (Figs 2, 3).
Fig. 3. Blood pH after 5 hr exposure to different oxygen tensions. Mean 5 SE N = 6 at each oxygen tension.
sustains a high oxygen affinity (P,, = 5; Mangum, 1983). This ability to maintain oxygen transport in response to a small internal oxygen tension gradient is not unique and has been described previously in other organisms (Mangum et al., 1975; Dejours, 1981). While this ability helps explain the adaptive strategy of P. clarki to hypoxia, the mechanism used by this species to sustain oxygen transport under normoxic conditions has yet to be investigated. Still, sustained high oxygen affinity of P. clarki hemocyanin does not by itself explain how this species adapts to hypoxia; a reduction in metabolic rate also plays a significant role. A comparison with the freshwater prawn Macrobrachium rosenbergii will better illustrate this point: at 40 torr, using specimens of comparable size, the oxygen uptake rate of M. rosenbergii is about four times that of P. clarki (Mauro and Malecha, 1984b). In addition, at 15 torr, the rate of lactate production is four times greater in M. rosenbergii than for P. clarki (Mauro and Malecha, 1984a). Clearly, the reduced metabolic rate of P. clarki, which contributes to the lack of change in blood lactate levels at 40 torr, also enhances its ability to withstand prolonged exposure to hypoxia. Both the high oxygen affinity of P. clarki hemocyanin and its low metabolic rate under hypoxia are adaptive to the species’ survival in hypoxic environments. Although most commonly found in shallow muddy water, P. clarki’s habitats range from swamps and marshes to lagoons and bayous (Penn, 1943). While not commonly found in burrows, P. clarki will burrow during the winter, or, in the case of females, will occupy burrows during the summer months. It is suggested that burrowing species of crustacea are more likely to encounter hypoxia in their natural environment and are better adapted physiologically for the rapid removal of lactate when aerobic conditions return (Bridges and Brand, 1980). In P. clarki, the rate of lactate removal may reflect this periodic burrowing behavior. REFERENCES
DISCUSSION
Under mild hypoxia, P. clarki maintains adequate oxygen transport by utilizing a hemocyanin that
Bridges C. R. and Brand A. R. (1980) The effects of hypoxia on oxygen consumption and blood lactate levels on some marine crustacea. Comp. Biochem. Physiol. 65A, 399409.
Hypoxia adaptation in crayfish Dejours P. (1981) Principles of Comparative Respiratory Physiology. Elsevier/North-Holland, New York. Dejours P. and Beeckenkamp H. (1977) Crayfish respiration as a function of water oxygenation. Resp. Physiol. 30, 241-251.
Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger N. B. (1983) The effects of organic acids on oxygen binding of hemocyanin from the crab Cancer magister. Comp. Biochem. Physiol. 14A, 45-50.
Larimer J. L. and Gold A. H. (1961) Responses of the crayfish Procambarus simulans to respiratory stress. Physiol. Zod. 134, 165-176. Mangum C. P. (1983) On the distribution of lactate sensitivity among the hemocyanins. Mar. Biol. Left. 4, 139-149.
Mangum C. P., Lykkeboe G. and Johansen K. (1975) Oxygen uptake and the role of hemoglobin in the east African swampworm Alma emini. Comp. Biochem. Physiol. 52, 477482.
Mauro N. A. and Malecha S. R. (1984a) The effects of hypoxia on blood pH and lactate levels in Macrobrachium rosenbergii (de Man) Comp. Biochem. Physiol. 77A, 627-630.
Mauro N. A. and Malecha S. R. (1984a) Intraspecific comparison of hemocyanin oxygen affinity and the effects of hypoxia on oxygen consumption in Macrobrachium
rosenbergii
15
(de Man) Comp. Biochem. Physiol. IIA, 631-633. McMahon B. R. and Wilkens J. L. (1983) Ventilation, perfusion, and oxygen uptake. In: Biology of Crustacea (Edited by Mantel L.). Academic Press, New York. Penn G. H.-(1943) Life’history of Louisiana red-crawfish. Ecolo&y 24, l-18. Sinha N. P. and Dejours P. (1980) Ventilation and blood acid-base balance of the crayfish as a function of water oxygenation (4&1500 torr). Comp. Biochem. Physiol. &A, 427-432. Wheatly M. G. and Taylor E. W. (1981) The effects of progressive hypoxia on heart rate, ventilation, respiratory gas exchange and acid-base status in the crayfish Austropotamobius pal&es. J. exp. Biol. 92, 125-141. Wiens A. W. and Armitage H. B. (1961) The oxygen consumption of the crayfish Orconectes immunis and Orconectes nais in response to temperature and oxygen saturation. Physiol. Zod. 34, 39-54. Wilkes P. R. H. and McMahon B. R. (1982a) Effects of maintained hypoxic exposure on the crayfish Orconectes rusticus I. Ventilatory, acid-base and cardiovascular adjustments. J. exp. Biol. 98, 119-137. Wilkes P. R. H. and McMahon B. R. (1982b) Effects of maintained hypoxic exposure on the crayfish Orconectes rusticus II. Modulation of hemocyanin oxygen affinity. J. exp. Biol. 98, 139-149.
0300-9629/84 $3.00 + 0.00
Printedin Great Britain
0
HYPOXIA
ADAPTATION PROCAMBARUS
NICHOLAS A.
MAURO
and
1984 PergamonPress Ltd
IN THE CRAYFISH CLARKI CLAUDIA THOMPSON
Department of Biology, Hartwick College, Oneonta, NY 13820, U.S.A. Telephone: (607) 432-4200 (Received 10 November 1983)
Abstract-l. P. clurki is an oxyconformer, with an oxygen uptake rate of 144 + 4 Ml/g wet wt/hr at oxygen tensions above 90% saturation and an uptake rate of 18 + 3 ~1 g wet wt/hr at 15 torr. 2. Between 159 and 40 torr, blood pH decreases slightly from 7.77 k 0.03 to 7.65 k 0.04, and at 1.5torr, blood pH drops to 7.36 + 0.06. 3. At normoxia, blood lactate levels are low at 0.66 + 0.01 mM/l blood. After 2 and 5 hr exposure to 15 torr, blood lactate levels increase to 3.29 k 0.47 and 8.91 f. 0.14 mM/l blood, respectively. Upon return to normoxia, blood lactate levels decrease and are comparable to normoxic controls after 13 hr. 4. During mild hypoxia, P. clurki maintains adequate oxygen transport by utilizing a high 0, affinity hemocyanin in conjunction with a low metabolic demand by its tissues.
INTRODUCTION
ographic electrode (5420A), and the required oxygen tension obtained by bubbling either air or nitrogen into the water. Blood pH was determined with an Orion implantable electrode (916300). The electrode was inserted directly into the ventral muscle of the first abdominal segment and the pH values read with a Leeds and Northrup pH meter (7405). Blood lactate levels were determined in 0.5 ml of prebranchial blood obtained via hypodermic syringe through the sternum of the first abdominal segment. In some cases, blood samples were pooled from individuals. Blood samples were analysed with a Sigma lactate test kit, modified according to the procedures of Graham et al. (1983).
The pH modulation of hemocyanin oxygen affinity has an important function in the adaptation of freshwater crustaceans to hypoxia. In previously reported studies, short-term exposure (2-24 hr) to mild hypoxia (P,02 = 40 torr) induces a hyperventilatory response in several species, with a concomitant increase in blood pH (Dejours and Beeckenkamp, 1971; Sinha and Dejours, 1980; Wheatly and Taylor, 1981; Wilkes and McMahon, 1982a,b; McMahon and Wilkens, 1983). Since elevated pH increases hemocyanin oxygen affinity, the change in blood pH allows the blood to maintain its oxygen transport function despite a reduced oxygen tension gradient. This adaptive mechanism requires that a given species must possess a hemocyanin which is sensitive to pH modulation: however, not all species of freshwater crustaceans possess a hemocyanin with a Bohr shift. In contrast, the oxygen affinity of Procambarus clarki hemocyanin has little dependence on pH within the physiological range (Mangum, 1983). This observation suggests a different mechanism for adapting to hypoxia than is found in other freshwater crustaceans. This study examines and compares the hypoxia adaptation mechanisms of the crayfish P. clarki to previously described species.
RESULTS
Effects of hypoxia on oxygen uptake
In response to hypoxia, P. clarki is an oxyconformer (Fig. l), comparable to Procambarus simduns and Orthonectes immunis (Larimer and Gold,
160 1
I
z
140-
IZO-
IOO-
MATERIAL AND METHODS 80-
Animals were obtained from commercial sources and were maintained in the laboratory in well-aerated tanks for 2 weeks at 22°C prior to all experiments. Their weights ranged from 11 to 15 g. Oxygen uptake (riOJ of animals in the dark were determined by the depletion of oxygen in closed containers (Yellow Springs Instrument Co. 5420A). Animals were starved for 48 hr before being tested, and were allowed to adjust to aerated containers for 30 min before the beginning of measurements. For the purpose of blood pH and lactate determinations, animals were placed into 4-l tanks for varying time periods during which the oxygen tension of the water was closely monitored with a Yellow Springs Instrument Co. polar-
60-
40-
20-
0
I
I 20
I 40
I 60
I 80
I 100
I 120
I 140
Torr ( mm Hgl Fig. 1. Effects of hypoxia on oxygen uptake of P. clarki at 22°C. Mean + SE N = 6 for each exposure. 73
NICHOLAS A. MAURO
14
-
Hypoxfa -4
and
Normoxla-+
13IZ1, IO9a?65432Iz __
o-
CLAUDIA THOMPK~N
f
76 I, 75 f
74
72
7’II / 11 I II I II I I I1 0 12345,23456789101112
II
I I1
I
0
20
‘1
40
1’
60 100 60 Torr (mmlig)
1
120
11
140
160
Time (hr)
Fig. 2. Lactate production during 5 hr exposure to 15 torr followed by recovery at 159 torr. Mean f SE N = 6 for exposure.
1961; Wiens and Armitage, 1961). Above 90% saturation oxygen uptake (PO,) measures 144 f 4 pi/g wet wt/hr and decreases by 87.5% at 15 torr to 18 f 3 ~1 wet wt/hr. In obtaining these measurements, animals were not restrained and hypoxia was induced by organism depletion in closed system respirometers. Lactate production with hypoxia
At normoxia, the blood lactate level in P. clarki is low, measuring 0.66 f 0.01 mM/l blood. Following a 5-hr exposure to 40 torr, no significant increase (P > 0.5) in blood lactate occurs. After 2 and 5 hr exposures to 15 torr, blood lactate levels increase to 3.20 _t 0.47 and 8.91 f 0.14 mM/l blood, respectively (Fig. 2). Following reexposure to normoxia, there is a continuous decline in blood lactate until after 13 hr, when blood lactate levels become comparable to normoxic controls. The time needed to reestablish normoxic blood lactate levels in P. clarki is slower than that reported for M. rosenbergii and is comparable to the rates observed in the burrowing marine species Nephrops norvegicus and Atelecyclus rotundatus (Mauro and Malecha, 1984a; Bridges and Brand, 1980). Effects of hypoxia on bloodpH
and oxygen transport
After a 5-hr exposure to reduced oxygen tensions, blood pH remains fairly constant, between 159 and 40 torr ranging between 7.77 + 0.03 and 7.65 + 0.04. This response to hypoxia contrasts markedly with other crayfish species in which blood pH increases with exposure to mild hypoxia (Dejours and Beeckenkamp, 1971; Sinha and Dejours, 1980; Whea.tly and Taylor, 1981; Wilkes and McMahon, 1982a,b; McMahon and Wilkens, 1983). Down to 40 torr oxygen delivery to the tissues is adequate to fuel oxidative metabolism and impede lactate production. At 15 torr, oxygen transport is reduced beyond the point where oxidative metabolism can be maintained. Lactate accumulates and blood pH falls sharply to 7.36 f 0.06 (Figs 2, 3).
Fig. 3. Blood pH after 5 hr exposure to different oxygen tensions. Mean 5 SE N = 6 at each oxygen tension.
sustains a high oxygen affinity (P,, = 5; Mangum, 1983). This ability to maintain oxygen transport in response to a small internal oxygen tension gradient is not unique and has been described previously in other organisms (Mangum et al., 1975; Dejours, 1981). While this ability helps explain the adaptive strategy of P. clarki to hypoxia, the mechanism used by this species to sustain oxygen transport under normoxic conditions has yet to be investigated. Still, sustained high oxygen affinity of P. clarki hemocyanin does not by itself explain how this species adapts to hypoxia; a reduction in metabolic rate also plays a significant role. A comparison with the freshwater prawn Macrobrachium rosenbergii will better illustrate this point: at 40 torr, using specimens of comparable size, the oxygen uptake rate of M. rosenbergii is about four times that of P. clarki (Mauro and Malecha, 1984b). In addition, at 15 torr, the rate of lactate production is four times greater in M. rosenbergii than for P. clarki (Mauro and Malecha, 1984a). Clearly, the reduced metabolic rate of P. clarki, which contributes to the lack of change in blood lactate levels at 40 torr, also enhances its ability to withstand prolonged exposure to hypoxia. Both the high oxygen affinity of P. clarki hemocyanin and its low metabolic rate under hypoxia are adaptive to the species’ survival in hypoxic environments. Although most commonly found in shallow muddy water, P. clarki’s habitats range from swamps and marshes to lagoons and bayous (Penn, 1943). While not commonly found in burrows, P. clarki will burrow during the winter, or, in the case of females, will occupy burrows during the summer months. It is suggested that burrowing species of crustacea are more likely to encounter hypoxia in their natural environment and are better adapted physiologically for the rapid removal of lactate when aerobic conditions return (Bridges and Brand, 1980). In P. clarki, the rate of lactate removal may reflect this periodic burrowing behavior. REFERENCES
DISCUSSION
Under mild hypoxia, P. clarki maintains adequate oxygen transport by utilizing a hemocyanin that
Bridges C. R. and Brand A. R. (1980) The effects of hypoxia on oxygen consumption and blood lactate levels on some marine crustacea. Comp. Biochem. Physiol. 65A, 399409.
Hypoxia adaptation in crayfish Dejours P. (1981) Principles of Comparative Respiratory Physiology. Elsevier/North-Holland, New York. Dejours P. and Beeckenkamp H. (1977) Crayfish respiration as a function of water oxygenation. Resp. Physiol. 30, 241-251.
Graham R. A., Mangum C. P., Terwilliger R. C. and Terwilliger N. B. (1983) The effects of organic acids on oxygen binding of hemocyanin from the crab Cancer magister. Comp. Biochem. Physiol. 14A, 45-50.
Larimer J. L. and Gold A. H. (1961) Responses of the crayfish Procambarus simulans to respiratory stress. Physiol. Zod. 134, 165-176. Mangum C. P. (1983) On the distribution of lactate sensitivity among the hemocyanins. Mar. Biol. Left. 4, 139-149.
Mangum C. P., Lykkeboe G. and Johansen K. (1975) Oxygen uptake and the role of hemoglobin in the east African swampworm Alma emini. Comp. Biochem. Physiol. 52, 477482.
Mauro N. A. and Malecha S. R. (1984a) The effects of hypoxia on blood pH and lactate levels in Macrobrachium rosenbergii (de Man) Comp. Biochem. Physiol. 77A, 627-630.
Mauro N. A. and Malecha S. R. (1984a) Intraspecific comparison of hemocyanin oxygen affinity and the effects of hypoxia on oxygen consumption in Macrobrachium
rosenbergii
15
(de Man) Comp. Biochem. Physiol. IIA, 631-633. McMahon B. R. and Wilkens J. L. (1983) Ventilation, perfusion, and oxygen uptake. In: Biology of Crustacea (Edited by Mantel L.). Academic Press, New York. Penn G. H.-(1943) Life’history of Louisiana red-crawfish. Ecolo&y 24, l-18. Sinha N. P. and Dejours P. (1980) Ventilation and blood acid-base balance of the crayfish as a function of water oxygenation (4&1500 torr). Comp. Biochem. Physiol. &A, 427-432. Wheatly M. G. and Taylor E. W. (1981) The effects of progressive hypoxia on heart rate, ventilation, respiratory gas exchange and acid-base status in the crayfish Austropotamobius pal&es. J. exp. Biol. 92, 125-141. Wiens A. W. and Armitage H. B. (1961) The oxygen consumption of the crayfish Orconectes immunis and Orconectes nais in response to temperature and oxygen saturation. Physiol. Zod. 34, 39-54. Wilkes P. R. H. and McMahon B. R. (1982a) Effects of maintained hypoxic exposure on the crayfish Orconectes rusticus I. Ventilatory, acid-base and cardiovascular adjustments. J. exp. Biol. 98, 119-137. Wilkes P. R. H. and McMahon B. R. (1982b) Effects of maintained hypoxic exposure on the crayfish Orconectes rusticus II. Modulation of hemocyanin oxygen affinity. J. exp. Biol. 98, 139-149.