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Effects of scorpion venom on some physiological processes in cockroach
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EFFECTS OF SCORPION VENOM ON SOME PHYSIOLOGICAL PROCESSES IN COCKROACH $ASTRA BABU, K. P. Mu1tAL1 K1ttsl~rA DASS and S. A. T. VENKATACIiARI Department of Zoology, Sri Venkateswara University, Tirupati, India (Accepted jor publication 161uiy 1970) AbetnactThe venom of the scorpion Heteronxtrus juivipes has six protein fnictions all of which exhibit cathodic mobility. Soon after injection of 0005 ml of the venom into the cockroach, the heart beat and spiracuhtr movements stopped and the legs show sagging and erratic movements without coordination . Muscle action potentials fail within 10 sec, while those of the nerve cord disappear in 30 to 40 sec . The activities of SDH, LDH and AChE are inhibited to a greater extent in muscle homogenates than in nerve cord homogenates. A gradual drop in oxygen consumption along with a drop in heat production of the cockroach was noted after venom injection . INTRODUCTION
AN ATTEMPT was made to study the effects of venom obtained frovo, the south Indian scorpion Heterometrwsfulvipes onsomephysiological activities ofthecockroachPeriplaneta americana. MATERIAL AND METHODS
Scorpions were obtained around the University campus and kept in glass containers. After applying electric shocks to the post-abdomen with a Grass S~l stimulator, the venom was drawn into an Agla-micrometer syringe and transferred to microbeakers that were kept on ice. Throughout the investigation the venom was used without dilution. The electrophoretic migration of protein was conducted using Whatman No. 1 Filter paper stripes of 1 in. width fitted to a 12 in. cell at a pH of 8~6 (Veronal buffer ; 005 M). A gradient of 200 V was applied for 16 to 18 hr. The stripes were then dried in an oven and the colour was developed using the amido black staining technique. Electrical activity in muscle and nerve was studied using conventional silver chloride electrodes both for stimulation and recording. Square wave pulses of 0~1 and 1 ~0 mast duration were delivered from a Grass S-4 stimulator. The action potentials picked up by recording electrodes were fed to a Grass P-9 preamplifier and displayed on a Tektronix type 502-A dual beam oscilloscope. All the recordings were made in air at 28°. Oxygen consumption was measured using an aerial respirometer (WELSH and SMITH, 1961) employing Brady's fluid in the manometer. Heat production was measured using thermocouples made with constantan wire fitted to a Cambridge spot galvanometer after standardization. The activity of succinate (succinate : FAD oxidoreductase, E.C.1.3.99.1) and lactate (lactate : NAD oxidoreductase, E.C.1 .1.1 .2']') dehydrogenases in muscle and nerve homogenates were estimated following the triphenyltetrazolium chloride reduction method (SRIKAIVTHAN and KRISFINA MOORTHY, 19$5), aS SuggCSted by GOVINDAPPA and SWAMI (19b5). The incubation mixture contained 1 ml of tissue homogenate (2 per cent w/v for 119
120
SASIRA BABU, K. P. MURALI KRISHNA DASS and S. A. T. VENKATACHARI
muscle and 1 per cent w/v for nerve), in 0'25 M sucrose; 1 ml of phosphate buffer of pH 7~2 ; 0~5 ml of sodium succinate or sodium lactate. Venom, 0005 ml, was added to the experimental flasks, whereas 0005 ml of 025 M sucrose was added to the controls . Incubation at 37° was stopped after 2 hr with 6 ml of toluene. The O.D . was read at 495 m~ employing silica cuvettes of 10 mm light path. The acetylcholinesterase activity was estimated after the method of Mgrenr.F (1951), as modified by Murali Krishna Dass (unpublished work). The incubation mixture contained 1 ml of the homogenate 1 ml of buffer-substrate (0~ 1 M phosphate buffer of pH 7~2 and 0~4 M acetylcholine chloride dissolved in a HCl solution of pH 4~2 mixed in 4 :1 proportion to give a final ACh concentration of 008 M and pH 7~2). Venom was added to the experimental flasks, while controls contained0 005 ml of025 Msucrose solution . Afterincubation for 4 hr at 37°, the reaction was stopped by adding 1 ml of6 N HC1. The unhydrolyzed ACh reacted with 2 ml of hydroxylamine hydrochloride and on addition of 1 ml of 10 per cent Fe C1 s solution gives a brown colour. The intensity ofthe colour was read against AChblank on the spectrophotometer at 540 mp. using silica cuvettes of 10 mm light path. Electrophoretic pattern of the venom
RESULTS
Electrophoretic separation of the venom at pH 8~6 revealed six protein fractions (Fig. 1), all of which exhibited cathodic mobility . Effects of the venom on the cockroach
The immediate effect was stoppage of the heart beat following injection of 0005 ml of venom into the abdomen. The normal 120 beats per min dropped to zero after a few seconds of sporadic and weak bursts . The spiracular movements also became erratic and stopped about 30 min after the injection. There was a loss of body tonus and normal leg movements. The legs showed a gradual sagging and erratic movements followed by complete immobilization . The effect was more pronounced when the venom was injected directly into the leg musculature. Effects ofvenom on muscle action potentials
Following exposure of the muscle to the venom, contractions disappeared . Washing the muscle in Ringers solution for 10 min and allowing it to recover did not restore normal activity. Stimulating an exposed muscle gave rise to action potentials (Fig. 2a) even at high repetitive frequencies. The application of 0005 ml of venom to coxal muscle resulted in a stepwise drop of response (Fig. 2 b-d), which was totally abolished in 10 sec. Effects ofvenom on nerve action potentials
On applying venom directly to the nerve cord a gradual inhibition of spontaneous activity was noted. Complete disappearance occurred within 2 min. On applying a drop of venom on the sixth ganglion, the entire spike complex gradually disappeared. Complete block of synaptic transmission was achieved with 35 to 40 sec. Conduction of the giant fibres in the ventral nerve cord was also blocked by the venom. The compound action potentials obtained on stimulation of the cord (Fig. 3a) decreased gradually in amplitude (Fig. 3 b-d) on addition of the venom. Complete abolition of the spikes was obtained in about 30 to 40 sec. As in the muscle, the effect of the venom on nerve activity was irreversible .
FIG. I . AN ELECTROPHORETOGRAM OF SCORPION VENOM.
The vertical streak near + shows the point of application of venom. Note the cathodic mobility of all the protein fractions at this pH .
b
c
FIG. 2. ACTION POTENTIALS FROM EXPOSED COXAL LEG MUSCLFS OF COCKROACH BEFORE AFTER (b-d~ ADDITION OF VENOM.
(a~
AND
Note the drop in amplitude of the spike potential. b~ were taken successively with an interval approximately 3 sec.
Tox. f.p . 120
FIG . 3 . ACTION POTENTIALS RECORDED FROM GIANT FIBRE3 IN THE VENTRAL NERVE CORD OF COCKROACH BEFORE (a~ AND AFTER (ßdÎ THE ADDITION OF VENOM .
Note the drop in spike amplitude. b-d were taken successively at 10 sec intervals.
121
Effects of Scorpion Venom in Cockroach
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SASIRA BABU, K . P . MURALI KRISHNA DASS and S . A. T . VENKATACHARI
Enzymic changes in cockroach tissues In the presence of0005 ml of venom, the activity of succinate dehydrogenase in muscle homogenate decreased by 94 per cent (p < 001), while in nerve cord homogenate, a decrease in activity of 40 per cent was not statistically significant . In the presence of the venom, lactate dehydrogenase (LDH) activity in muscle homogenate significantly decreased (36 per cent) while in nerve cord a decrease of 21 per cent was not significant . Acetylcholinesterase activity appeared to be inhibited both in muscle (24 per cent, p < 005) and nerve cord (15 per cent), although the latter effect was not significant . E ~ect ofvenom on oxygen consumption A cockroach (1 g body weight) consumed on the average 005 ml of oxygen per min. When 0005 ml of venom was injected into the cockroach there was an immediate increase in O$ consumption to about 0~ 1 ml per min which returned to normal about 5 min after injection. Afterwards, Os consumption gradually decreased to a subnormal level, reaching 001 ml per min in about 40 min (Fig. 4).
TIME W MINUTES FIG . 4 . GRAPH SHOWING THE RATE OF OXYGEN CONSUMPTION IN VENOM IIYIECTED COCKROACH . Note the fall in O, consumption to subnormal levels .
Effect of venom on body temperature Two ends of a thermocouple were introduced into two cockroaches through their anal openings. One was used as the control and the other one was used as the experimental animal. On injecting venom into the experimental animal, the cockroach temperature fell gradually by 055° in about 40 min (Fig. 5). The temperature drop remained at such a level for some time, and then returned to 28 ° at which time the animal died. DISCUSSION
In the present investigation it has been shown that venom of H. fulvipes contains six protein fractions similar to that found in H. scaber (OOMMEN and KuRUP, 1964), Tityus batiensis (FISCHER and Bol~r, 1957) and Buthusjudaicus (WEISSMANN et al., 1958) . However, in H. fulvipes all six fractions have ca.thodic mobility at pH 8~6. A preliminary study ofthe protein fractions on mice (Oo~rr and KuitUr, 1964) suggested that most of the toxicity resided in the cathodic fractions. Recently, the electrophoretic patterns of venoms from six species of Israeli scorpions (Nrrznx and SHUIAV, 1966) indicated that the relative toxicity of the venom depends on the proportion of the cathodic protein fraction . In the cockroach the effect of scorpion venom is most marked on muscle tissue. This is reflected in the spontaneous heart stoppage . We also observed arrest of spiracular movements in animals viewed under the microscope .
123
Effects of Scorpion Venom in Cockroach b z
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0.2 10
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T11~E AFTER N,ECTION OF POISON N MINUTES FIa.
S. GRAPH SHOWING 1iIE DECREASE WBODY TEI~PRAiURE SIJHSEQUENi' TO Tf~ INIECI'ION OF VENOM IIVhO TFIE BODY OF COCKROACH.
Note the maximum decrease in body temperature about 40 min after injection.
The nerve action potentials were abolished slower than those of the muscle . This may partly be due to thethick sheath around the nerve cord, which could retard toxin permeation. In this respect, the present findings are comparable to those of PiEK (1966x) who studied the effect of digger wasp venom on the neuromuscular system of the locust . The effect of scorpion venom on the muscular and nervous tissues of cockroach seems to be rapid and irreversible, even after repeated washings with Ringers solution . In this respect it differs from the venom of braconid wasps (PIEK, 1966b) in which case it has been shown that the effect is brought about 1S min after venom application, and restoration of original activity is achieved after washing the tissue in saline for about 10 min . Metabolic studies also show that muscle is more readily affected than nerve cord. The activity levels of both SDH and LDH are inhibited to a much greater extent in the muscle homogenates than in the nerve cord homogenates (Table 1). In nerve cord homogenates the extent of inhibition was not statistically significant. It appears that the venom attacks the energy producing systems in the muscle more readily than in the nerve. This is also true in regard to AChE activity (Table 1). The decreased activity of succinate dehydrogenase could lead to the decreased oxygen consumption of the animal. The initial increased oxygen consumption after injection of venom may be due to a shock effect since the 08 consumption then decreases, gradually reaching subnormal levels (Fig. 5). Stoppage of the heart beat and spiracular movements may also result in lowered oxygen consumption. Reduced oxygen consumption leads to lowering of the energy produced by tissues. Observed fall in body temperature by 055° (Fig. 6) within 40 min after injection ofvenom also points in the same direction. Acknowledgemeets-We thank Drs. K. S. SwAru and F. F. FARAOALLA for their helpful suggestions in preparing the manuscript. We also thank Prof. T. H. Bvtiocx and Prof. K. PAMPAPATFII RAO for permitting us to use the electropl>~ysiological equipment obtained by the latter throughajoint research programme. REFERENCES F. G. and Holov, H. (1937) Die Gifte der brasilianischen Skorpione Tityus serrulatus und Tityus bakierrsis. Z. Physlol. Chem. 306, 269. GOVII~APPA, S. and SwAtra, K. S. (1965) Electropboretic charactenistics of subcellular components and their relation to enzyme activities in the amphibian muscle fibers. Indian J. exp. Biol. 3, 209. Flsct~R,
124
SASIRA BABU, K. P. MURALI KRISHNA DASS and S. A. T. VENKATACHARI
ME~rcwt.a, R. L . (1951) The colorimetric microestimation of the human blood ; cholinesterases in its application to poisoning by organic phosphate insecticides. J. econ. Ent. 44, 883 . Nrrzntv, M, and SxvLOV, A. (1966) Electrophoretic patterns of the venoms of siz species of Israeli scorpions. Toxicon 4, 17 . Oo~~rr, P. K. and Kuxur, P. A. (1964) Proteins and amino acid contents and physiological properties of the venom of the South Indian scorpion, Heterometrus scaber. Irul. J. exp . Biol. 2, 78. Pte, T. (1966a) Site of action of thevenom of thedigger wasp Phitanthus triarrgulum Fon the fast neuromuscular systems of the locust . Toxicon 4, 191 . Prnac, T . (1966b) Site of action of the venom of Microbracon hebetor (Say) (Braconidae, Hymenoptera). J. Insect Physiol. 12, 561 .
SmKnxTxnx, T. N. and KxBxN~ Mooxlxv, C. R. (1955) Tetrawlium tests for dehydrogenases . J. scient . Ind. Res .14, 206.
W nrrtv, A., Sxvcov, A. and Sx~rxnz, E. (1958) Elxtrophoresis of the venom and haemolymph of the scorpion, Buthus judaicu.~ E. Sim. Experlentta. 14, 173. WErsx, J. H. and SMrtx, R. I. (1961) Laboratory ExercLses in Invertebrate Physiology. Minneapolis : Burgess.
, t9~t, VoL 9, vv. tts-iu. rarcemoo rrm. r~taa m aro.t adc.io
EFFECTS OF SCORPION VENOM ON SOME PHYSIOLOGICAL PROCESSES IN COCKROACH $ASTRA BABU, K. P. Mu1tAL1 K1ttsl~rA DASS and S. A. T. VENKATACIiARI Department of Zoology, Sri Venkateswara University, Tirupati, India (Accepted jor publication 161uiy 1970) AbetnactThe venom of the scorpion Heteronxtrus juivipes has six protein fnictions all of which exhibit cathodic mobility. Soon after injection of 0005 ml of the venom into the cockroach, the heart beat and spiracuhtr movements stopped and the legs show sagging and erratic movements without coordination . Muscle action potentials fail within 10 sec, while those of the nerve cord disappear in 30 to 40 sec . The activities of SDH, LDH and AChE are inhibited to a greater extent in muscle homogenates than in nerve cord homogenates. A gradual drop in oxygen consumption along with a drop in heat production of the cockroach was noted after venom injection . INTRODUCTION
AN ATTEMPT was made to study the effects of venom obtained frovo, the south Indian scorpion Heterometrwsfulvipes onsomephysiological activities ofthecockroachPeriplaneta americana. MATERIAL AND METHODS
Scorpions were obtained around the University campus and kept in glass containers. After applying electric shocks to the post-abdomen with a Grass S~l stimulator, the venom was drawn into an Agla-micrometer syringe and transferred to microbeakers that were kept on ice. Throughout the investigation the venom was used without dilution. The electrophoretic migration of protein was conducted using Whatman No. 1 Filter paper stripes of 1 in. width fitted to a 12 in. cell at a pH of 8~6 (Veronal buffer ; 005 M). A gradient of 200 V was applied for 16 to 18 hr. The stripes were then dried in an oven and the colour was developed using the amido black staining technique. Electrical activity in muscle and nerve was studied using conventional silver chloride electrodes both for stimulation and recording. Square wave pulses of 0~1 and 1 ~0 mast duration were delivered from a Grass S-4 stimulator. The action potentials picked up by recording electrodes were fed to a Grass P-9 preamplifier and displayed on a Tektronix type 502-A dual beam oscilloscope. All the recordings were made in air at 28°. Oxygen consumption was measured using an aerial respirometer (WELSH and SMITH, 1961) employing Brady's fluid in the manometer. Heat production was measured using thermocouples made with constantan wire fitted to a Cambridge spot galvanometer after standardization. The activity of succinate (succinate : FAD oxidoreductase, E.C.1.3.99.1) and lactate (lactate : NAD oxidoreductase, E.C.1 .1.1 .2']') dehydrogenases in muscle and nerve homogenates were estimated following the triphenyltetrazolium chloride reduction method (SRIKAIVTHAN and KRISFINA MOORTHY, 19$5), aS SuggCSted by GOVINDAPPA and SWAMI (19b5). The incubation mixture contained 1 ml of tissue homogenate (2 per cent w/v for 119
120
SASIRA BABU, K. P. MURALI KRISHNA DASS and S. A. T. VENKATACHARI
muscle and 1 per cent w/v for nerve), in 0'25 M sucrose; 1 ml of phosphate buffer of pH 7~2 ; 0~5 ml of sodium succinate or sodium lactate. Venom, 0005 ml, was added to the experimental flasks, whereas 0005 ml of 025 M sucrose was added to the controls . Incubation at 37° was stopped after 2 hr with 6 ml of toluene. The O.D . was read at 495 m~ employing silica cuvettes of 10 mm light path. The acetylcholinesterase activity was estimated after the method of Mgrenr.F (1951), as modified by Murali Krishna Dass (unpublished work). The incubation mixture contained 1 ml of the homogenate 1 ml of buffer-substrate (0~ 1 M phosphate buffer of pH 7~2 and 0~4 M acetylcholine chloride dissolved in a HCl solution of pH 4~2 mixed in 4 :1 proportion to give a final ACh concentration of 008 M and pH 7~2). Venom was added to the experimental flasks, while controls contained0 005 ml of025 Msucrose solution . Afterincubation for 4 hr at 37°, the reaction was stopped by adding 1 ml of6 N HC1. The unhydrolyzed ACh reacted with 2 ml of hydroxylamine hydrochloride and on addition of 1 ml of 10 per cent Fe C1 s solution gives a brown colour. The intensity ofthe colour was read against AChblank on the spectrophotometer at 540 mp. using silica cuvettes of 10 mm light path. Electrophoretic pattern of the venom
RESULTS
Electrophoretic separation of the venom at pH 8~6 revealed six protein fractions (Fig. 1), all of which exhibited cathodic mobility . Effects of the venom on the cockroach
The immediate effect was stoppage of the heart beat following injection of 0005 ml of venom into the abdomen. The normal 120 beats per min dropped to zero after a few seconds of sporadic and weak bursts . The spiracular movements also became erratic and stopped about 30 min after the injection. There was a loss of body tonus and normal leg movements. The legs showed a gradual sagging and erratic movements followed by complete immobilization . The effect was more pronounced when the venom was injected directly into the leg musculature. Effects ofvenom on muscle action potentials
Following exposure of the muscle to the venom, contractions disappeared . Washing the muscle in Ringers solution for 10 min and allowing it to recover did not restore normal activity. Stimulating an exposed muscle gave rise to action potentials (Fig. 2a) even at high repetitive frequencies. The application of 0005 ml of venom to coxal muscle resulted in a stepwise drop of response (Fig. 2 b-d), which was totally abolished in 10 sec. Effects ofvenom on nerve action potentials
On applying venom directly to the nerve cord a gradual inhibition of spontaneous activity was noted. Complete disappearance occurred within 2 min. On applying a drop of venom on the sixth ganglion, the entire spike complex gradually disappeared. Complete block of synaptic transmission was achieved with 35 to 40 sec. Conduction of the giant fibres in the ventral nerve cord was also blocked by the venom. The compound action potentials obtained on stimulation of the cord (Fig. 3a) decreased gradually in amplitude (Fig. 3 b-d) on addition of the venom. Complete abolition of the spikes was obtained in about 30 to 40 sec. As in the muscle, the effect of the venom on nerve activity was irreversible .
FIG. I . AN ELECTROPHORETOGRAM OF SCORPION VENOM.
The vertical streak near + shows the point of application of venom. Note the cathodic mobility of all the protein fractions at this pH .
b
c
FIG. 2. ACTION POTENTIALS FROM EXPOSED COXAL LEG MUSCLFS OF COCKROACH BEFORE AFTER (b-d~ ADDITION OF VENOM.
(a~
AND
Note the drop in amplitude of the spike potential. b~ were taken successively with an interval approximately 3 sec.
Tox. f.p . 120
FIG . 3 . ACTION POTENTIALS RECORDED FROM GIANT FIBRE3 IN THE VENTRAL NERVE CORD OF COCKROACH BEFORE (a~ AND AFTER (ßdÎ THE ADDITION OF VENOM .
Note the drop in spike amplitude. b-d were taken successively at 10 sec intervals.
121
Effects of Scorpion Venom in Cockroach
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122
SASIRA BABU, K . P . MURALI KRISHNA DASS and S . A. T . VENKATACHARI
Enzymic changes in cockroach tissues In the presence of0005 ml of venom, the activity of succinate dehydrogenase in muscle homogenate decreased by 94 per cent (p < 001), while in nerve cord homogenate, a decrease in activity of 40 per cent was not statistically significant . In the presence of the venom, lactate dehydrogenase (LDH) activity in muscle homogenate significantly decreased (36 per cent) while in nerve cord a decrease of 21 per cent was not significant . Acetylcholinesterase activity appeared to be inhibited both in muscle (24 per cent, p < 005) and nerve cord (15 per cent), although the latter effect was not significant . E ~ect ofvenom on oxygen consumption A cockroach (1 g body weight) consumed on the average 005 ml of oxygen per min. When 0005 ml of venom was injected into the cockroach there was an immediate increase in O$ consumption to about 0~ 1 ml per min which returned to normal about 5 min after injection. Afterwards, Os consumption gradually decreased to a subnormal level, reaching 001 ml per min in about 40 min (Fig. 4).
TIME W MINUTES FIG . 4 . GRAPH SHOWING THE RATE OF OXYGEN CONSUMPTION IN VENOM IIYIECTED COCKROACH . Note the fall in O, consumption to subnormal levels .
Effect of venom on body temperature Two ends of a thermocouple were introduced into two cockroaches through their anal openings. One was used as the control and the other one was used as the experimental animal. On injecting venom into the experimental animal, the cockroach temperature fell gradually by 055° in about 40 min (Fig. 5). The temperature drop remained at such a level for some time, and then returned to 28 ° at which time the animal died. DISCUSSION
In the present investigation it has been shown that venom of H. fulvipes contains six protein fractions similar to that found in H. scaber (OOMMEN and KuRUP, 1964), Tityus batiensis (FISCHER and Bol~r, 1957) and Buthusjudaicus (WEISSMANN et al., 1958) . However, in H. fulvipes all six fractions have ca.thodic mobility at pH 8~6. A preliminary study ofthe protein fractions on mice (Oo~rr and KuitUr, 1964) suggested that most of the toxicity resided in the cathodic fractions. Recently, the electrophoretic patterns of venoms from six species of Israeli scorpions (Nrrznx and SHUIAV, 1966) indicated that the relative toxicity of the venom depends on the proportion of the cathodic protein fraction . In the cockroach the effect of scorpion venom is most marked on muscle tissue. This is reflected in the spontaneous heart stoppage . We also observed arrest of spiracular movements in animals viewed under the microscope .
123
Effects of Scorpion Venom in Cockroach b z
0.8 0 .8
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0.2 10
20
30
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1
1
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1
80
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T11~E AFTER N,ECTION OF POISON N MINUTES FIa.
S. GRAPH SHOWING 1iIE DECREASE WBODY TEI~PRAiURE SIJHSEQUENi' TO Tf~ INIECI'ION OF VENOM IIVhO TFIE BODY OF COCKROACH.
Note the maximum decrease in body temperature about 40 min after injection.
The nerve action potentials were abolished slower than those of the muscle . This may partly be due to thethick sheath around the nerve cord, which could retard toxin permeation. In this respect, the present findings are comparable to those of PiEK (1966x) who studied the effect of digger wasp venom on the neuromuscular system of the locust . The effect of scorpion venom on the muscular and nervous tissues of cockroach seems to be rapid and irreversible, even after repeated washings with Ringers solution . In this respect it differs from the venom of braconid wasps (PIEK, 1966b) in which case it has been shown that the effect is brought about 1S min after venom application, and restoration of original activity is achieved after washing the tissue in saline for about 10 min . Metabolic studies also show that muscle is more readily affected than nerve cord. The activity levels of both SDH and LDH are inhibited to a much greater extent in the muscle homogenates than in the nerve cord homogenates (Table 1). In nerve cord homogenates the extent of inhibition was not statistically significant. It appears that the venom attacks the energy producing systems in the muscle more readily than in the nerve. This is also true in regard to AChE activity (Table 1). The decreased activity of succinate dehydrogenase could lead to the decreased oxygen consumption of the animal. The initial increased oxygen consumption after injection of venom may be due to a shock effect since the 08 consumption then decreases, gradually reaching subnormal levels (Fig. 5). Stoppage of the heart beat and spiracular movements may also result in lowered oxygen consumption. Reduced oxygen consumption leads to lowering of the energy produced by tissues. Observed fall in body temperature by 055° (Fig. 6) within 40 min after injection ofvenom also points in the same direction. Acknowledgemeets-We thank Drs. K. S. SwAru and F. F. FARAOALLA for their helpful suggestions in preparing the manuscript. We also thank Prof. T. H. Bvtiocx and Prof. K. PAMPAPATFII RAO for permitting us to use the electropl>~ysiological equipment obtained by the latter throughajoint research programme. REFERENCES F. G. and Holov, H. (1937) Die Gifte der brasilianischen Skorpione Tityus serrulatus und Tityus bakierrsis. Z. Physlol. Chem. 306, 269. GOVII~APPA, S. and SwAtra, K. S. (1965) Electropboretic charactenistics of subcellular components and their relation to enzyme activities in the amphibian muscle fibers. Indian J. exp. Biol. 3, 209. Flsct~R,
124
SASIRA BABU, K. P. MURALI KRISHNA DASS and S. A. T. VENKATACHARI
ME~rcwt.a, R. L . (1951) The colorimetric microestimation of the human blood ; cholinesterases in its application to poisoning by organic phosphate insecticides. J. econ. Ent. 44, 883 . Nrrzntv, M, and SxvLOV, A. (1966) Electrophoretic patterns of the venoms of siz species of Israeli scorpions. Toxicon 4, 17 . Oo~~rr, P. K. and Kuxur, P. A. (1964) Proteins and amino acid contents and physiological properties of the venom of the South Indian scorpion, Heterometrus scaber. Irul. J. exp . Biol. 2, 78. Pte, T. (1966a) Site of action of thevenom of thedigger wasp Phitanthus triarrgulum Fon the fast neuromuscular systems of the locust . Toxicon 4, 191 . Prnac, T . (1966b) Site of action of the venom of Microbracon hebetor (Say) (Braconidae, Hymenoptera). J. Insect Physiol. 12, 561 .
SmKnxTxnx, T. N. and KxBxN~ Mooxlxv, C. R. (1955) Tetrawlium tests for dehydrogenases . J. scient . Ind. Res .14, 206.
W nrrtv, A., Sxvcov, A. and Sx~rxnz, E. (1958) Elxtrophoresis of the venom and haemolymph of the scorpion, Buthus judaicu.~ E. Sim. Experlentta. 14, 173. WErsx, J. H. and SMrtx, R. I. (1961) Laboratory ExercLses in Invertebrate Physiology. Minneapolis : Burgess.