- Email: [email protected]
Ionic composition of the haemolymph of a chinese scorpion, buthus martensi
$3.00+ 0.00 0300-9629/88 Q 1988Pergamon Press plc
Camp. Biochem. Phvsiol. Vol. 91A, No. 2, pp. 323-325, 1988
Printed in Great B&in
IONIC COMPOSITION OF THE HAEMOLYMPH A CHINESE SCORPION, BUTHWS MARTENSI YOSHIAKI KI~A,*$
Susmu
TERAKAWA,* ICE Hsut
OF
and YONG-HUA Jlt
*Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, 444 Japan. Telephone 0564-54-l 111;and TDepartment of Neuropharmacology, Shanghai Institute of Physiology, Academia Sinica, 320 Yue-Yang, Rd., Shanghai, 200031, China
Abstract-1 . Ionic composition of the haemolymph of a Chinese scorpion, burgh ~arre~i Karsch, was determined. 2. Element analysis sbowed that the supematant of ultracentrifuged serum (435,OOOg,30 min) contained (in mM) 291.6 f 5.8 Na, 6.7 + 0.8 K, 3.6 + 0.4 Ca, 0.6 + 0.07 Mg, and 292.6 i 6.2 Cl. 3. Ion chromatography showed that fhe same supematant contained (in mM) 291 Na and 3.3 K. 4. The serum contained other minor components: 2.1 Cu, 0.04 Fe, 0.02 Mn, 0.57 Zn (mM), most of which were spun down by the ultracentrifugation. 5. No seasonal variation was observed in the composition. 6. Salines of various ionic compositions showed very little difference in ability to maintain nerve excitability and heart activity. 7. Based on these, we propose a physiological saline for the Chinese scorpion as: 292 NaCl, 3.3 KCl, 3.6 CaCI,, 0.6 MgCl,, 3 Tris-HCl (mM), pH 7.3.
compositions of scorpion haemol~ph or scorpion salines have been reported with a variety of values (see Table 1 and references list). Most of these reports were based on element analysis of crude scorpion haemolymph and a few of them were based on the ability of the saline to maintain physiological functions. The values range between 111 and 353 mM for Na and some of them show extraordinarily large statistical deviations. This is partly because of a wide latitude of the osmotic resistance of scorpion tissues. The elemental compositions of the crude haemolymph can poorly define the ionic environments for scorpion tissues unless the ratio of free ions to bound ones is known. Therefore, we measured both the elemental and ionic com~sition of the scorpion haemol~ph under several conditions to determine the proper physiological saline. Thus, we propose a salt mixture as the scorpion saline for physiological experiments. This saline maintained the neural and cardiac activities of the Chinese scorpion for more than 8 hr. Ionic
MATERIALS AND METHODS
The scorpion, Buthus martensi Karsch, found in Henan Province, China was used. We collected the haemolymph of some wild specimens in the fields. Tbe other specimens were purchased from local culture farms and were kept in our laboratory more than a year by feeding with crickets. The haemolymph was collected from these specimens throughout a year. The haemolymph was dripped into a polyethylene tube
(Eppendorf 3810, Hamburg, FRG) immediately after cutting the pedipalp. Syringes were not used because the location of the tip end was not clear. We drew the haemolymph with syringes initially and found that sometimes
SAuthor to whom all correspondence
should be addressed.
the haemol~ph looked turbid. By our method, we always obtained clear blue haemolymph owing to the open circulatory system. The haemolymph from each scorpion was spun immediately at low speed (1OOOa. 1 min. Tomv MC-ISA microcenirifugator, *Tokyo, Japan) to iemovk cells and cellular debris. In several samples, filtration with Millipore filter (0.22pm, Millipore, Bedford, MA, USA) was employed instead of the low-speed centrifugation. The pellet was very small and white. We obtained clear bluish supernatant or filtrate and analysed it as a scorpion serum. It was further ultracentrifuged (435,OOOg,30 min, Beckman TL-100, Palo Alto, CA, USA). We analysed the colorless supematant thus obtained as a scorpion saline. Element anafyses were done by double or triple combination of various methods including flame photometry, atomic absorption spectroscopy (atomic abso~tion/flame emission spectrophotometer AA670, Shimadzu, Kyoto, Japan, and sometimes atomic absorption spectrophotometer type 603, Perkin Elmer, Norwalk, CT, USA), ICP emission spectroscopy (Inductively Coupled Plasma emission spectrophotometer SPS-I 100, SEIKO Electronic, Tokyo, Japan) and chemical analysis. Ionic analyses were done by ion chromatography (Ion Chromatographic Analyzer IC500, Yokogawa Electric, Tokyo, Japan). Concentrations of Na and K were mostly measured by atomic absorption spectroscopy. Several samples were analysed with flame photometry by a clinical service company (SRL, Special Reference Laboratories, Tokyo, Japan). All data agreed very well. Concentrations of Ca and Mg were measured mostly by atomic absorption spectroscopy and ICP emission spectroscopy. Several samples were analysed by SRL (Ca by OCPC, Connerty and Briggs, 1966: and Mg by Xylidyl Blue, Mann and Yoe, 1956). The concentration of Cl was measured with a chloridimeter by SRL. Other metals were analyzed with the ICP emission spectrophotometer. The osmolarity was measured with an osmometer (Advanced Instrument INC. Model 3W, Needham Heights, MA, USA) and the pH was measured with a DH meter (Corning 130) using a microcombined pH -electrode (IWOSO, Iwaki, Tokyo, Japan). Neural activities were examined by measuring intracellular action potentials as described by Terakawa er al. (submitted for publication). 323
324
YOSHIAKI KIMURA et al.
All chemicals were purchased from Wako Pure Chemical Indust. (Osaka) or Katayama Chemical Indust. (Osaka) and were analytical grades. Milli Q water (Millipore) was used in all experiments. Standard solutions for elemental and ionic analyses were prepared both as separate solutions and as mixtures to check the possible cross interference among elements, which was not observed in all cases. Standard solutions for ICP emission spectroscopy were purchased from Merck (Merck Japan, Tokyo). RESULTS
The results of elemental and ionic analyses of the serum and the saline were listed in Tables 1 and 2. Sodium and potassium concentrations in the saline were 292 and 6.7 mM, respectively, according to the element analysis. A similar value for sodium was obtained by ionic analysis. However, ionic analysis of potassium showed only half the value obtained by the element analysis. Reliability of both analyses was checked using the various combination of standard solutions and the same samples. Therefore, we concluded that the ionic concentration of potassium was 3.3 mM and that the rest of potassium was in a non-ionic form, which was not removed by the ultracentrifugation. The actual form of the non-ion was left undetermined. The serum contained 6.3 mM calcium and 1.5 mM magnesium, whereas the saline contained 3.6 mM calcium and 0.6 mM magnesium. The difference of the values between the serum and the saline may be explained by calcium and magnesium trapped or bound to proteins, which were spun down by the ultracentrifugation. The copper concentration in the serum may reflect the amount of haemocyanin, which is present as the major oxygen-carrier in the haemolymph. The osmolarity was 558.7 f 4.9 mOsm/kg, which agreed well with the calculated osmolarity, 557.3 mOsm/kg. The pH of serum ranged between 7.2 and 7.3. All these data were obtained in winter and spring. The values in summer and autumn were 297.3 f 5.0 Na, 7.3 f 1.0 K, and 300.7 f 5.2 Cl (mM), which agree well statistically with the above values. Therefore, we concluded that there was no seasonal change in the composition of the scorpion haemolymph. The excitability of nerves and the rate of heart beat were observed in (1) our scorpion saline (in mM: 292 NaCl, 3.3 KCI, 3.6 CaCI, , 0.6 MgCl,, 3 Tris-HCl, pH 7.3, with or without 0.5 g/l glucose), (2) Padmanabhanaidu’s solution (147 NaCI, 1.7 KCI, 6.1 CaCl,, 10.4 MgCl,, 3 Tris-HC1, pH 7.3, 0.5 g/l glucose), (3) van Harreveld’s solution (205 NaCl, 5.7KC1, 13.9CaCl,, 2.6MgCI,, 3Tris-HCl, pH7.3) and (4) Ringer’s solution (109 NaCl, 2.3 KCl, 2.1 CaCI,, 1.2 MgCl,, 1.2 MgCI,, 1 Tris-HCl, pH 7.3).
Table 2. Minor inorganic components of the haemolymph of the scorpion, Bufhus martensi (mM) Element
Serum
Saline
CU Fe Mll Ztl
2.1 0.04 0.02 0.57
0.06 less than 0.0001 less than O.ooO1 0.02
Measured by inductively emission spectroscopy.
coupled
plasma
Ionic composition of a scorpion haemolymph In all cases, nerve excitability measured by the intracellular recording and heart activity were well maintained for more than 8 hr. DISCUSSION
The ionic composition of the haemol~ph of the scorpion, &thus ~artensi Karsch, was invariable throughout a year. Based on the ionic compositions measured, we propose a salt mixture as a physiological saline for the Chinese scorpion. This new scorpion saline will enable us to study physiology of scorpion more rigorously and elaborately. Our analyses elucidated the following: sodium and chloride are present in the scorpion haemolymph at concentrations much higher than those in most insects and mammals; the ionic concentration of potassium was about half the value measured by element analysis; the concentrations of calcium and magnesium in the saline were also about half those in the serum probably because of the bound ions; and the magnesium concentration was very low. Ionic compositions of scorpion haemolymph or physiological saline have been reported with a variety of values (Table 1). Kanugo (1955), Padmanabhanaidu (1967) and Zwicky (1968) determined their solutions so as to maintain the heart activity of the scorpions. Our results show that the scorpion’s nerve and heart are quite invulnerable and therefore the maintenance of these activities cannot be a good criterion to determine a physiological saline. Table 1 contains data with large variations for Opisthophthabnus (Robertson, 1982) and Heterometrus (Padmanabhanaidu, 1962, 1967). These scorpions both belong to the family Scorpionidae, one of six scorpion families. It remains a highly speculative problem whether the large variation reflects a particular characteristic of this family, a difficulty in measurement for this family, or other factors. The table contains data for Buthidae (Buthus, Purabuthus, Centrwoides, blurs and ~ndroctonus) and Vajovidae (Paruroctonus and Hadrurus). Data for the other three families are not available. The comparatively high osmolarity of the haemolymph of scorpions among terrestrial animals is now well established for Buthidae, and probably also for Vajovidae; this is easily explained by the high sodium and chloride concentrations. The physiological significance of these high salt concentrations is not well understood (Burton 1984). The extreme tolerance of scorpions for a lack of water (4 weeks without water and food in bottles while being carried in a desert, in our experience} might be related to the high osmolarity.
325
Acknowledgements-We thank Profs S. Ebashi and Z.-T. Mei for their assistance and also Mr K.-H. Tsai for his help in collecting scorpions. We are especially grateful to Mr. 0. Kumazaki and the General Technical Institute of Chubu Electric Power Supply Co. Ltd. for their generous help and for allowing us to use the ICP emission spectrophotometer and the ion chromatographjc analyser.
REFERENCES
Ahearn G. A. and Hadley N. F. (1976) Functional roles of luminal sodium and potassium in water transport across desert scorpion ileum. Nature 261, 66-68. _ Aheam G. A. and Hadlev N. F. (1977) Water Transoort in perfused scorpion ileum. Am. J.- Physial. 233, R1981R207. Bowerman R. F. (1972) A muscle receptor organ in the scorpion postabdomen. I. The sensory system. J. camp. Physiaf. 81, 133-146. Bowerman R. F. (1976) Ionic concentrations and pH of the h~ol~ph of the scorpions Hadrur~ arizonensis and Paruractonus mesaensis. Camp. Biochem. Physiol. 54A,
331-333. Bricteux-Grefoire S., Duchateau-Bosson Gh., Jeuniaux Ch., Schoffeniels E. and Florkin M. (1963) Constituants osmotiquement actifs du sang et des muscles de scorpion Androctonur australis L. Archs. Int. Physiol. Biochim. 71, 393-400.
Burton R. F. (1984) Haemolymph composition in spiders and scorpions. Comp. Biochem. Physiol. 78A, 613-616. Connerty H. V. and Briggs A. R. (1966) Determination of serum calcium by means of ~th~resolphthalein Complexone. Amer. J. c&t. Path. 45, 290. Farley R. D. (1987) Postsynaptic potentials and contraction pattern in the heart of the desert scorpion, Pururoctonus mesaensis. Comp. Biochem. Physiof. 86A, 12 l-l 3 1. Gilai A. and Pamas I. (1970) Neuromascular physiology of the closer muscles in the pedipalp of the scorpion Leiurus quinquestriatus. J. exp. Biol. 52, 325-344.
Kanungo M. S. (1955) Physiology of the heart of a scorpion. Nature 176, 980-981.
Mann C. K. and Yoe J. H. (1956) Spectrophotometric determination of Mg with Na-l-azo-2-hydroxy-3-(2,4-dimethyl carboxanilido)-naphthalene- 1-(t-hydroxy benzen5-sulfonate). Anal. Chem. 28, 202. Padmanabhanaidu B. (1962) Ionic composition of the blood of scorpion. Curr. Sci. 31, 21. Padmanabhanaidu B. (1966) Ionic composition of the blood and the blood volume of the scorpion, Heterometrus fiduipes. Coma. Biochem. Physiol. 17, 157-166.
Padmanabhanaidu B. (1967) Perfusion fluid for the scorpion, Heterometrus fulvipes. Nature 213, 410. Robertson H. G., Nicolson S. W. and Louw G. N. (I 982) Osmoregulation and temperature effects on water loss and oxygen consumption in two species of African scorpion. Camp. Biochek Physiol. ‘?l& 605609. Zwicky K. T. (1968) Innervation and nharmacolozv of the heart of Ur&&, a scorpion. Co&. ~~o~hern.-~h~sioZ. 24, 799-808.
Camp. Biochem. Phvsiol. Vol. 91A, No. 2, pp. 323-325, 1988
Printed in Great B&in
IONIC COMPOSITION OF THE HAEMOLYMPH A CHINESE SCORPION, BUTHWS MARTENSI YOSHIAKI KI~A,*$
Susmu
TERAKAWA,* ICE Hsut
OF
and YONG-HUA Jlt
*Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, 444 Japan. Telephone 0564-54-l 111;and TDepartment of Neuropharmacology, Shanghai Institute of Physiology, Academia Sinica, 320 Yue-Yang, Rd., Shanghai, 200031, China
Abstract-1 . Ionic composition of the haemolymph of a Chinese scorpion, burgh ~arre~i Karsch, was determined. 2. Element analysis sbowed that the supematant of ultracentrifuged serum (435,OOOg,30 min) contained (in mM) 291.6 f 5.8 Na, 6.7 + 0.8 K, 3.6 + 0.4 Ca, 0.6 + 0.07 Mg, and 292.6 i 6.2 Cl. 3. Ion chromatography showed that fhe same supematant contained (in mM) 291 Na and 3.3 K. 4. The serum contained other minor components: 2.1 Cu, 0.04 Fe, 0.02 Mn, 0.57 Zn (mM), most of which were spun down by the ultracentrifugation. 5. No seasonal variation was observed in the composition. 6. Salines of various ionic compositions showed very little difference in ability to maintain nerve excitability and heart activity. 7. Based on these, we propose a physiological saline for the Chinese scorpion as: 292 NaCl, 3.3 KCl, 3.6 CaCI,, 0.6 MgCl,, 3 Tris-HCl (mM), pH 7.3.
compositions of scorpion haemol~ph or scorpion salines have been reported with a variety of values (see Table 1 and references list). Most of these reports were based on element analysis of crude scorpion haemolymph and a few of them were based on the ability of the saline to maintain physiological functions. The values range between 111 and 353 mM for Na and some of them show extraordinarily large statistical deviations. This is partly because of a wide latitude of the osmotic resistance of scorpion tissues. The elemental compositions of the crude haemolymph can poorly define the ionic environments for scorpion tissues unless the ratio of free ions to bound ones is known. Therefore, we measured both the elemental and ionic com~sition of the scorpion haemol~ph under several conditions to determine the proper physiological saline. Thus, we propose a salt mixture as the scorpion saline for physiological experiments. This saline maintained the neural and cardiac activities of the Chinese scorpion for more than 8 hr. Ionic
MATERIALS AND METHODS
The scorpion, Buthus martensi Karsch, found in Henan Province, China was used. We collected the haemolymph of some wild specimens in the fields. Tbe other specimens were purchased from local culture farms and were kept in our laboratory more than a year by feeding with crickets. The haemolymph was collected from these specimens throughout a year. The haemolymph was dripped into a polyethylene tube
(Eppendorf 3810, Hamburg, FRG) immediately after cutting the pedipalp. Syringes were not used because the location of the tip end was not clear. We drew the haemolymph with syringes initially and found that sometimes
SAuthor to whom all correspondence
should be addressed.
the haemol~ph looked turbid. By our method, we always obtained clear blue haemolymph owing to the open circulatory system. The haemolymph from each scorpion was spun immediately at low speed (1OOOa. 1 min. Tomv MC-ISA microcenirifugator, *Tokyo, Japan) to iemovk cells and cellular debris. In several samples, filtration with Millipore filter (0.22pm, Millipore, Bedford, MA, USA) was employed instead of the low-speed centrifugation. The pellet was very small and white. We obtained clear bluish supernatant or filtrate and analysed it as a scorpion serum. It was further ultracentrifuged (435,OOOg,30 min, Beckman TL-100, Palo Alto, CA, USA). We analysed the colorless supematant thus obtained as a scorpion saline. Element anafyses were done by double or triple combination of various methods including flame photometry, atomic absorption spectroscopy (atomic abso~tion/flame emission spectrophotometer AA670, Shimadzu, Kyoto, Japan, and sometimes atomic absorption spectrophotometer type 603, Perkin Elmer, Norwalk, CT, USA), ICP emission spectroscopy (Inductively Coupled Plasma emission spectrophotometer SPS-I 100, SEIKO Electronic, Tokyo, Japan) and chemical analysis. Ionic analyses were done by ion chromatography (Ion Chromatographic Analyzer IC500, Yokogawa Electric, Tokyo, Japan). Concentrations of Na and K were mostly measured by atomic absorption spectroscopy. Several samples were analysed with flame photometry by a clinical service company (SRL, Special Reference Laboratories, Tokyo, Japan). All data agreed very well. Concentrations of Ca and Mg were measured mostly by atomic absorption spectroscopy and ICP emission spectroscopy. Several samples were analysed by SRL (Ca by OCPC, Connerty and Briggs, 1966: and Mg by Xylidyl Blue, Mann and Yoe, 1956). The concentration of Cl was measured with a chloridimeter by SRL. Other metals were analyzed with the ICP emission spectrophotometer. The osmolarity was measured with an osmometer (Advanced Instrument INC. Model 3W, Needham Heights, MA, USA) and the pH was measured with a DH meter (Corning 130) using a microcombined pH -electrode (IWOSO, Iwaki, Tokyo, Japan). Neural activities were examined by measuring intracellular action potentials as described by Terakawa er al. (submitted for publication). 323
324
YOSHIAKI KIMURA et al.
All chemicals were purchased from Wako Pure Chemical Indust. (Osaka) or Katayama Chemical Indust. (Osaka) and were analytical grades. Milli Q water (Millipore) was used in all experiments. Standard solutions for elemental and ionic analyses were prepared both as separate solutions and as mixtures to check the possible cross interference among elements, which was not observed in all cases. Standard solutions for ICP emission spectroscopy were purchased from Merck (Merck Japan, Tokyo). RESULTS
The results of elemental and ionic analyses of the serum and the saline were listed in Tables 1 and 2. Sodium and potassium concentrations in the saline were 292 and 6.7 mM, respectively, according to the element analysis. A similar value for sodium was obtained by ionic analysis. However, ionic analysis of potassium showed only half the value obtained by the element analysis. Reliability of both analyses was checked using the various combination of standard solutions and the same samples. Therefore, we concluded that the ionic concentration of potassium was 3.3 mM and that the rest of potassium was in a non-ionic form, which was not removed by the ultracentrifugation. The actual form of the non-ion was left undetermined. The serum contained 6.3 mM calcium and 1.5 mM magnesium, whereas the saline contained 3.6 mM calcium and 0.6 mM magnesium. The difference of the values between the serum and the saline may be explained by calcium and magnesium trapped or bound to proteins, which were spun down by the ultracentrifugation. The copper concentration in the serum may reflect the amount of haemocyanin, which is present as the major oxygen-carrier in the haemolymph. The osmolarity was 558.7 f 4.9 mOsm/kg, which agreed well with the calculated osmolarity, 557.3 mOsm/kg. The pH of serum ranged between 7.2 and 7.3. All these data were obtained in winter and spring. The values in summer and autumn were 297.3 f 5.0 Na, 7.3 f 1.0 K, and 300.7 f 5.2 Cl (mM), which agree well statistically with the above values. Therefore, we concluded that there was no seasonal change in the composition of the scorpion haemolymph. The excitability of nerves and the rate of heart beat were observed in (1) our scorpion saline (in mM: 292 NaCl, 3.3 KCI, 3.6 CaCI, , 0.6 MgCl,, 3 Tris-HCl, pH 7.3, with or without 0.5 g/l glucose), (2) Padmanabhanaidu’s solution (147 NaCI, 1.7 KCI, 6.1 CaCl,, 10.4 MgCl,, 3 Tris-HC1, pH 7.3, 0.5 g/l glucose), (3) van Harreveld’s solution (205 NaCl, 5.7KC1, 13.9CaCl,, 2.6MgCI,, 3Tris-HCl, pH7.3) and (4) Ringer’s solution (109 NaCl, 2.3 KCl, 2.1 CaCI,, 1.2 MgCl,, 1.2 MgCI,, 1 Tris-HCl, pH 7.3).
Table 2. Minor inorganic components of the haemolymph of the scorpion, Bufhus martensi (mM) Element
Serum
Saline
CU Fe Mll Ztl
2.1 0.04 0.02 0.57
0.06 less than 0.0001 less than O.ooO1 0.02
Measured by inductively emission spectroscopy.
coupled
plasma
Ionic composition of a scorpion haemolymph In all cases, nerve excitability measured by the intracellular recording and heart activity were well maintained for more than 8 hr. DISCUSSION
The ionic composition of the haemol~ph of the scorpion, &thus ~artensi Karsch, was invariable throughout a year. Based on the ionic compositions measured, we propose a salt mixture as a physiological saline for the Chinese scorpion. This new scorpion saline will enable us to study physiology of scorpion more rigorously and elaborately. Our analyses elucidated the following: sodium and chloride are present in the scorpion haemolymph at concentrations much higher than those in most insects and mammals; the ionic concentration of potassium was about half the value measured by element analysis; the concentrations of calcium and magnesium in the saline were also about half those in the serum probably because of the bound ions; and the magnesium concentration was very low. Ionic compositions of scorpion haemolymph or physiological saline have been reported with a variety of values (Table 1). Kanugo (1955), Padmanabhanaidu (1967) and Zwicky (1968) determined their solutions so as to maintain the heart activity of the scorpions. Our results show that the scorpion’s nerve and heart are quite invulnerable and therefore the maintenance of these activities cannot be a good criterion to determine a physiological saline. Table 1 contains data with large variations for Opisthophthabnus (Robertson, 1982) and Heterometrus (Padmanabhanaidu, 1962, 1967). These scorpions both belong to the family Scorpionidae, one of six scorpion families. It remains a highly speculative problem whether the large variation reflects a particular characteristic of this family, a difficulty in measurement for this family, or other factors. The table contains data for Buthidae (Buthus, Purabuthus, Centrwoides, blurs and ~ndroctonus) and Vajovidae (Paruroctonus and Hadrurus). Data for the other three families are not available. The comparatively high osmolarity of the haemolymph of scorpions among terrestrial animals is now well established for Buthidae, and probably also for Vajovidae; this is easily explained by the high sodium and chloride concentrations. The physiological significance of these high salt concentrations is not well understood (Burton 1984). The extreme tolerance of scorpions for a lack of water (4 weeks without water and food in bottles while being carried in a desert, in our experience} might be related to the high osmolarity.
325
Acknowledgements-We thank Profs S. Ebashi and Z.-T. Mei for their assistance and also Mr K.-H. Tsai for his help in collecting scorpions. We are especially grateful to Mr. 0. Kumazaki and the General Technical Institute of Chubu Electric Power Supply Co. Ltd. for their generous help and for allowing us to use the ICP emission spectrophotometer and the ion chromatographjc analyser.
REFERENCES
Ahearn G. A. and Hadley N. F. (1976) Functional roles of luminal sodium and potassium in water transport across desert scorpion ileum. Nature 261, 66-68. _ Aheam G. A. and Hadlev N. F. (1977) Water Transoort in perfused scorpion ileum. Am. J.- Physial. 233, R1981R207. Bowerman R. F. (1972) A muscle receptor organ in the scorpion postabdomen. I. The sensory system. J. camp. Physiaf. 81, 133-146. Bowerman R. F. (1976) Ionic concentrations and pH of the h~ol~ph of the scorpions Hadrur~ arizonensis and Paruractonus mesaensis. Camp. Biochem. Physiol. 54A,
331-333. Bricteux-Grefoire S., Duchateau-Bosson Gh., Jeuniaux Ch., Schoffeniels E. and Florkin M. (1963) Constituants osmotiquement actifs du sang et des muscles de scorpion Androctonur australis L. Archs. Int. Physiol. Biochim. 71, 393-400.
Burton R. F. (1984) Haemolymph composition in spiders and scorpions. Comp. Biochem. Physiol. 78A, 613-616. Connerty H. V. and Briggs A. R. (1966) Determination of serum calcium by means of ~th~resolphthalein Complexone. Amer. J. c&t. Path. 45, 290. Farley R. D. (1987) Postsynaptic potentials and contraction pattern in the heart of the desert scorpion, Pururoctonus mesaensis. Comp. Biochem. Physiof. 86A, 12 l-l 3 1. Gilai A. and Pamas I. (1970) Neuromascular physiology of the closer muscles in the pedipalp of the scorpion Leiurus quinquestriatus. J. exp. Biol. 52, 325-344.
Kanungo M. S. (1955) Physiology of the heart of a scorpion. Nature 176, 980-981.
Mann C. K. and Yoe J. H. (1956) Spectrophotometric determination of Mg with Na-l-azo-2-hydroxy-3-(2,4-dimethyl carboxanilido)-naphthalene- 1-(t-hydroxy benzen5-sulfonate). Anal. Chem. 28, 202. Padmanabhanaidu B. (1962) Ionic composition of the blood of scorpion. Curr. Sci. 31, 21. Padmanabhanaidu B. (1966) Ionic composition of the blood and the blood volume of the scorpion, Heterometrus fiduipes. Coma. Biochem. Physiol. 17, 157-166.
Padmanabhanaidu B. (1967) Perfusion fluid for the scorpion, Heterometrus fulvipes. Nature 213, 410. Robertson H. G., Nicolson S. W. and Louw G. N. (I 982) Osmoregulation and temperature effects on water loss and oxygen consumption in two species of African scorpion. Camp. Biochek Physiol. ‘?l& 605609. Zwicky K. T. (1968) Innervation and nharmacolozv of the heart of Ur&&, a scorpion. Co&. ~~o~hern.-~h~sioZ. 24, 799-808.