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Succinic dehydrogease activity in a fresh-water crab in relation to salinity stress
Comp. B;ochem. Physiol., 1966, Vol. 19, pp. 645 to 648. Pergamon Press Ltd. Pnnted zn Great Britain
SUCCINIC DEHYDROGENASE ACTIVITY IN A FRESH-WATER CRAB IN RELATION TO SALINITY STRESS* R. R A M A M U R T H I Department of Biology, University of Oregon, Eugene, Oregon (Received 9 April 1966)
A b s t r a c t - - A study of the succinic dehydrogenase activity in the giU and the
hepatopancreas of a fresh-water crab in relation to sex, size and salinity revealed the foUowing trends: 1. The enzyme activity is about 3"5 times greater in the gill than in the hepatopancreas. 2. In both the tissues studied the enzyme activity is greater in males. 3. The enzyme activity increases with increasing body size (body weight) irrespective of the sex of the indtviduals. 4. The enzyme activity increases with the increase in the salinity of the medium. INTRODUCTION IT IS well known that the variations in the salinity of the medium exert considerable influence on the activity and metabolism of poikilotherms (Florkin, 1960; Kinne, 1964; Ramamurthi, 1965). It has been suggested that the changes in the metabolic response to salinity are at least in part the reflections of the load on the organism for osmotic or ionic regulation (Ramamurthi, 1965). But the mechanisms underlying the shift in the pattern of metabolic response to varied osmotic stress are not clear. T h e purpose of the present study was to investigate the changes in the activity at the enzymatic level after the imposition of salinity stress. This report deals with the activity of the respiratory enzyme succinic dehydrogenase in the gill and hepatopancreas of the fresh-water field crab Paratelphusa hydrodromous as a function of salinity. Sex and size were also taken into consideration as these factors are known to influence a majority of rate functions. MATERIALS AND METHODS For the study of the influence of salinity, only male crabs falling into the most c o m m o n weight range of 19-21 g were used to avoid the discrepancies due to sex and size. Twelve crabs were placed in 50% sea water and after 45 min 6 crabs were taken out for the enzyme assay. T h e other 6 individuals were transferred to * This work was carried out at the Department of Zoology, Sn Venkateswara University, Tirupati, India. The work was supported in part by Grant GB3181 from the N.S.F., U.S.A. 645
646
R, RAMAMURTI-II
100% sea water and were left in that medium for 45 min before they were taken out. Ten per cent homogenates of the gill and hepatopancreas were prepared with ice-cold 0.1 M phosphate buffer, pH 7.0. 0.1 M sodium succinate was used as the substrate. Triphenyl tetrazolium chloride reduction method has been followed for the estimation of succinic dehydrogenase activity, using toluene for the extraction of formazan formed (Sri Kantan & Krishnamurti, 1955). Measurements of the optical density of the toluene layer were carried out in the U.V. Spectrophotometer (Hilger & Watts, England) at 495 m/x. The enzyme activity is expressed in terms of mg of formazan formed per g of the wet weight of the tissue per hr. RESULTS AND DISCUSSION It is common knowledge that succinic dehydrogenase is a very important enzyme in the T.C.A. cycle of Krebs. The presence of this enzyme in the gill and hepatopancreas of P. hydrodromous indicates the operation of a T.C.A. cycle in this crab. This enzyme has been reported previously amongst Crustacea in lobster heart muscle (Ball & Mayerhof, 1940), and in the neurosecretory cells of the thoracic ganglion of the fresh-water crab (Nayar & Parameswaran, 1955). The existence of the Krebs cycle in crustaceans has been demonstrated previously (Munday & Thompson, 1961; Scheer & Meenakshi, 1961). An examination of Fig. 1 reveals the following trends: 1. In both the tissues examined, the enzyme activity increases with the increasing body size (body weight) of the crabs, irrespective of sex. A similar trend was reported in the case of the hepatopancreas of the amphibious snail Pila globosa (Raghupathiramireddy, 1965, personal communication). 2. The enzyme activity is greater in males in both the tissues. It is more pronounced in the smaller individuals. This observation of higher enzyme activity in males closely parallels the trend shown for the oxygen consumption of the same crab (Ramamurthi, 1962); this was explained as due to the active growth process in males, more so in the case of younger individuals, on the basis of Orton's observation (Orton, 1936) that in some decapod Crustacea males grow at a greater rate than females. 3. The enzyme activity is approximately 3-5 times greater in the gill tissue than that in the hepatopancreas. This is understandable from the fact that the gill is the site of gaseous exchange and also that it is in direct contact with external medium playing a pivotal role in the salt and water fluxes of the animal. It is evident from Table 1 that transfer to saline media results in an increase in the enzyme activity in both the tissues studied. In full-strength sea water which presents a maximal chloride gradient to the crab, the steep rise in the succinic dehydrogenase activity may be related to the greater expenditure of energy for the chloride regulation. But the minimal enzyme activity in fresh water, in spite of the existence of a large chloride gradient, is puzzling, especially in view of the previous report (Ramamurthi, 1965) that the crab exhibits maximal O~ consumption in fresh water. If the metabolic homeostatic mechanism (Pampapathi Rao,
SUCCINICDEHYDROGENASEACTIVITYIN A FRESH-WATERCRAB
647
1958) is invoked to explain the low enzyme activity of the crab in its natural m e d i u m of fresh water, it is difficult to understand how the compensation of this particular enzyme activity would be helpful to the animal when its total O3 consumption is very high in that medium. As our knowledge of the various
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Weight,
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FIG. 1. Succinic dehydrogenase activity in the gill and hepatopancreas of P.
hydrodromous as a function of sex and size (body weight). A and & represent the gill tissue of males and females respectively. Q and © represent the hepatopancreas of males and females respectively. TABLE I--SuccINIC DEHYDROGENASEACTIVITYIN P. hydrodromous IN DIFFERENTSALINITIES Enzyme activity (mg of formazan formed/g/hr) Hepatopancreas S.D.
Chloride gradient (Ramamurthi, 1965) (mM/1: mediumblood)
0"228(6) _+0"021 0"362(6)_+0"035 0"674(6) _+0"032
- 255 +21"05 + 281
Medium Gill Tap water 50% seawater 100% sea water
S.D.
0-809 (6)* _+0"034 1"081 (6) +0"159 2"174 (6) _+0-186
* The figures in parentheses indicate the number of esttmations. Room temperature 28°+_ 1°C. Salinity of 100% sea water 33,°. metabolic pathways in crustaceans is fragmentary, it would be a matter of great interest to investigate whether the animal is obtaining energy through the operation of other pathways also, besides the one involving succinic dehydrogenase. I f this is the case, then the high O 2 consumption m a y fail to reflect in the succinic dehydrogenase activity when the crab is in fresh water.
648
R. RAMAMURTHI
Acknowledgements--Thls work was carried out during the tenure of a Postdoctoral Fellowship from the Government of India. I wish to express my thanks to Professor K. Pampapathi Rao of Sri Venkateswara University, Tirupat,, India, for his encouragement and to Professor Bradley T. Scheer for kindly going through the manuscript. M y thanks are due to Mrs. Elsie Mumbach and Mr. William Mumbach for their help in preparing this manuscript. REFERENCES BALL E. G. & MEYERHOF O. (1940) On the occurrence of iron-porphyrin compounds and succinlc dehydrogenase in marine organisms possessing the copper blood pigment hemocyanin. J . biol. Chem. 134, 483--493. FLORKIN M. (1960) Ecology and metabolism. In Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. 1, p. 395. Academic Press, New York and London. KINNE O. (1964) T h e effects of temperature and salinity on marine and brackish-water a n i m a l s - - I I . Salinity and temperature-salinity combinations. Oceanogr. mar. Biol. A. Rev. 2, 281-339. MUNDAY K. A. & THOMPSON B. O. (1961) T h e tricarboxylic cycle m Carcinus maenas. Proc. 5th int. Congr. Biochem., Moscow. Pergamon Press, Oxford. NAYAR K. K. & PARAMESWARANR. (1955) Succinic dehydrogenase in the neurosecretory cells of the thoracic ganglion of the crab. Curr. Sci. 24, 341. ORTO~ J. H. (1936) Experiments in the sea on rate of growth of some crustacean decapods. J. mar. Biol. Ass. U.K. 20, 673-689. PAMPAPATrtI RAO K. (1958) Oxygen consumption as a function of size and salinity m Metapenaeus monoceros Fab. from marine and brackish-water environments. J. exp. Biol. 35, 307-313. RAMAMURTHI R. (1962) Studies on the respiration of fresh-water poikllotherms in relation to osmotic stress. Doctoral dissertation submitted to Sri Venkateswara University, Tirupati, India. RAMAMURTHI R. (1965) Metabolic response to osmotic stress in some fresh-water polkilotherms. Curr. Sci. 34, 351. SCHEER B. T. &; MEENAKSHIV. R. (1961) T h e metabolism of carbohydrates in arthropods. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTIn A. W. ), pp. 65-88. University of Washington Press, Seattle. SRIKANTAN T. N. & KRISHNAMURTI C. R. (1955) Tetrazolium test for dehydrogenases. J. Sci. ind. Res. 14, 206-207.
SUCCINIC DEHYDROGENASE ACTIVITY IN A FRESH-WATER CRAB IN RELATION TO SALINITY STRESS* R. R A M A M U R T H I Department of Biology, University of Oregon, Eugene, Oregon (Received 9 April 1966)
A b s t r a c t - - A study of the succinic dehydrogenase activity in the giU and the
hepatopancreas of a fresh-water crab in relation to sex, size and salinity revealed the foUowing trends: 1. The enzyme activity is about 3"5 times greater in the gill than in the hepatopancreas. 2. In both the tissues studied the enzyme activity is greater in males. 3. The enzyme activity increases with increasing body size (body weight) irrespective of the sex of the indtviduals. 4. The enzyme activity increases with the increase in the salinity of the medium. INTRODUCTION IT IS well known that the variations in the salinity of the medium exert considerable influence on the activity and metabolism of poikilotherms (Florkin, 1960; Kinne, 1964; Ramamurthi, 1965). It has been suggested that the changes in the metabolic response to salinity are at least in part the reflections of the load on the organism for osmotic or ionic regulation (Ramamurthi, 1965). But the mechanisms underlying the shift in the pattern of metabolic response to varied osmotic stress are not clear. T h e purpose of the present study was to investigate the changes in the activity at the enzymatic level after the imposition of salinity stress. This report deals with the activity of the respiratory enzyme succinic dehydrogenase in the gill and hepatopancreas of the fresh-water field crab Paratelphusa hydrodromous as a function of salinity. Sex and size were also taken into consideration as these factors are known to influence a majority of rate functions. MATERIALS AND METHODS For the study of the influence of salinity, only male crabs falling into the most c o m m o n weight range of 19-21 g were used to avoid the discrepancies due to sex and size. Twelve crabs were placed in 50% sea water and after 45 min 6 crabs were taken out for the enzyme assay. T h e other 6 individuals were transferred to * This work was carried out at the Department of Zoology, Sn Venkateswara University, Tirupati, India. The work was supported in part by Grant GB3181 from the N.S.F., U.S.A. 645
646
R, RAMAMURTI-II
100% sea water and were left in that medium for 45 min before they were taken out. Ten per cent homogenates of the gill and hepatopancreas were prepared with ice-cold 0.1 M phosphate buffer, pH 7.0. 0.1 M sodium succinate was used as the substrate. Triphenyl tetrazolium chloride reduction method has been followed for the estimation of succinic dehydrogenase activity, using toluene for the extraction of formazan formed (Sri Kantan & Krishnamurti, 1955). Measurements of the optical density of the toluene layer were carried out in the U.V. Spectrophotometer (Hilger & Watts, England) at 495 m/x. The enzyme activity is expressed in terms of mg of formazan formed per g of the wet weight of the tissue per hr. RESULTS AND DISCUSSION It is common knowledge that succinic dehydrogenase is a very important enzyme in the T.C.A. cycle of Krebs. The presence of this enzyme in the gill and hepatopancreas of P. hydrodromous indicates the operation of a T.C.A. cycle in this crab. This enzyme has been reported previously amongst Crustacea in lobster heart muscle (Ball & Mayerhof, 1940), and in the neurosecretory cells of the thoracic ganglion of the fresh-water crab (Nayar & Parameswaran, 1955). The existence of the Krebs cycle in crustaceans has been demonstrated previously (Munday & Thompson, 1961; Scheer & Meenakshi, 1961). An examination of Fig. 1 reveals the following trends: 1. In both the tissues examined, the enzyme activity increases with the increasing body size (body weight) of the crabs, irrespective of sex. A similar trend was reported in the case of the hepatopancreas of the amphibious snail Pila globosa (Raghupathiramireddy, 1965, personal communication). 2. The enzyme activity is greater in males in both the tissues. It is more pronounced in the smaller individuals. This observation of higher enzyme activity in males closely parallels the trend shown for the oxygen consumption of the same crab (Ramamurthi, 1962); this was explained as due to the active growth process in males, more so in the case of younger individuals, on the basis of Orton's observation (Orton, 1936) that in some decapod Crustacea males grow at a greater rate than females. 3. The enzyme activity is approximately 3-5 times greater in the gill tissue than that in the hepatopancreas. This is understandable from the fact that the gill is the site of gaseous exchange and also that it is in direct contact with external medium playing a pivotal role in the salt and water fluxes of the animal. It is evident from Table 1 that transfer to saline media results in an increase in the enzyme activity in both the tissues studied. In full-strength sea water which presents a maximal chloride gradient to the crab, the steep rise in the succinic dehydrogenase activity may be related to the greater expenditure of energy for the chloride regulation. But the minimal enzyme activity in fresh water, in spite of the existence of a large chloride gradient, is puzzling, especially in view of the previous report (Ramamurthi, 1965) that the crab exhibits maximal O~ consumption in fresh water. If the metabolic homeostatic mechanism (Pampapathi Rao,
SUCCINICDEHYDROGENASEACTIVITYIN A FRESH-WATERCRAB
647
1958) is invoked to explain the low enzyme activity of the crab in its natural m e d i u m of fresh water, it is difficult to understand how the compensation of this particular enzyme activity would be helpful to the animal when its total O3 consumption is very high in that medium. As our knowledge of the various
09 OS 07
,,,
& A
A A
A
04
A
O-5 ~,
0,4 o" •
~E ~o
° •
o
o
o •
o
g o
E I
r I II
6
7
8
9 I0
Weight,
I
I
20
30
1
g
FIG. 1. Succinic dehydrogenase activity in the gill and hepatopancreas of P.
hydrodromous as a function of sex and size (body weight). A and & represent the gill tissue of males and females respectively. Q and © represent the hepatopancreas of males and females respectively. TABLE I--SuccINIC DEHYDROGENASEACTIVITYIN P. hydrodromous IN DIFFERENTSALINITIES Enzyme activity (mg of formazan formed/g/hr) Hepatopancreas S.D.
Chloride gradient (Ramamurthi, 1965) (mM/1: mediumblood)
0"228(6) _+0"021 0"362(6)_+0"035 0"674(6) _+0"032
- 255 +21"05 + 281
Medium Gill Tap water 50% seawater 100% sea water
S.D.
0-809 (6)* _+0"034 1"081 (6) +0"159 2"174 (6) _+0-186
* The figures in parentheses indicate the number of esttmations. Room temperature 28°+_ 1°C. Salinity of 100% sea water 33,°. metabolic pathways in crustaceans is fragmentary, it would be a matter of great interest to investigate whether the animal is obtaining energy through the operation of other pathways also, besides the one involving succinic dehydrogenase. I f this is the case, then the high O 2 consumption m a y fail to reflect in the succinic dehydrogenase activity when the crab is in fresh water.
648
R. RAMAMURTHI
Acknowledgements--Thls work was carried out during the tenure of a Postdoctoral Fellowship from the Government of India. I wish to express my thanks to Professor K. Pampapathi Rao of Sri Venkateswara University, Tirupat,, India, for his encouragement and to Professor Bradley T. Scheer for kindly going through the manuscript. M y thanks are due to Mrs. Elsie Mumbach and Mr. William Mumbach for their help in preparing this manuscript. REFERENCES BALL E. G. & MEYERHOF O. (1940) On the occurrence of iron-porphyrin compounds and succinlc dehydrogenase in marine organisms possessing the copper blood pigment hemocyanin. J . biol. Chem. 134, 483--493. FLORKIN M. (1960) Ecology and metabolism. In Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. 1, p. 395. Academic Press, New York and London. KINNE O. (1964) T h e effects of temperature and salinity on marine and brackish-water a n i m a l s - - I I . Salinity and temperature-salinity combinations. Oceanogr. mar. Biol. A. Rev. 2, 281-339. MUNDAY K. A. & THOMPSON B. O. (1961) T h e tricarboxylic cycle m Carcinus maenas. Proc. 5th int. Congr. Biochem., Moscow. Pergamon Press, Oxford. NAYAR K. K. & PARAMESWARANR. (1955) Succinic dehydrogenase in the neurosecretory cells of the thoracic ganglion of the crab. Curr. Sci. 24, 341. ORTO~ J. H. (1936) Experiments in the sea on rate of growth of some crustacean decapods. J. mar. Biol. Ass. U.K. 20, 673-689. PAMPAPATrtI RAO K. (1958) Oxygen consumption as a function of size and salinity m Metapenaeus monoceros Fab. from marine and brackish-water environments. J. exp. Biol. 35, 307-313. RAMAMURTHI R. (1962) Studies on the respiration of fresh-water poikllotherms in relation to osmotic stress. Doctoral dissertation submitted to Sri Venkateswara University, Tirupati, India. RAMAMURTHI R. (1965) Metabolic response to osmotic stress in some fresh-water polkilotherms. Curr. Sci. 34, 351. SCHEER B. T. &; MEENAKSHIV. R. (1961) T h e metabolism of carbohydrates in arthropods. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTIn A. W. ), pp. 65-88. University of Washington Press, Seattle. SRIKANTAN T. N. & KRISHNAMURTI C. R. (1955) Tetrazolium test for dehydrogenases. J. Sci. ind. Res. 14, 206-207.