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Anaesthetics for the squid Sepioteuthis sepioidea (mollusca: cephalopoda)
Camp. Biochem. Physiol. Vol.
103C,No. I, pp. 121-123,1992
0306-4492/92 $5.00 + 0.00 0 1992 Pergamon Press Ltd
Printedin Great Britain
ANAESTHETICS FOR THE SQUID SZi’Z’ZOTEUTHIs XWZOZDEA (MOLLUSCA: CEPHALOPODA) MAURKIO GARCiA-FRANC0 Laboratory of Aquatic Biology, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108, Japan Fax: 81-3-34715794 (Received 3 February
1992; accepted 6 March
1992)
Abstract-l.
Specimens of Sepioreufhis sepioidea were exposed to different reagents commonly used as anaesthetics for cold blooded animals. 2. Although some reagents a&ted the specimens, the anesthesia state was only achieved when using ethanol, magnesium sulfate or magnesium chloride. 3. The optimal ranges of concentrations necessary to achieve the anesthesia were: 1 to 3% for ethanol, 1.5 to 2% for magnesium chloride and 3 to 4% for magnesium sulfate.
INTRODUCTION The search for potentially cultivable mollusk species is directly linked to the use of anesthetics capable of improving various handling techniques such as tagging and marking, weighing and sexing, artificial induced reproduction and particularly the transportation of live organisms. The anesthetics agents can be physical or chemical (McFarland, 1960; Norris et al., 1960; Ross and Ross, 1984). One of the physical methods is the hypothermia which is used alone or in combination with chemical anesthetics for transport of living organisms. Another is electroshock (Djourno, 1963), whose use is probably limited to manipulations involving rapid handling. The use of chemical reagents is more popular among aquatic organisms. However, in mollusks and cephalopods in particular, there are comparatively few researches done related to anesthetics. Consequently, this study was aimed at identifying the reagents, their concentrations and exposure periods that would induce anesthesia, allowing the easy manipulation of Sepioteuthis sepioidea. MATERIALSAND METHODS Samples of both sexes of S. sepioidea were obtained from Mochima Bay, Sucre State in Venezuela. Specimens of 42.2 to 290.9 g in total weight (7.6 to 17.1 cm in mantle length) were used in this study. The reagents tested were selected on the basis of their low price, availability and previous studies reported on cold blooded animals (Huf, 1934; Lever er al., 1963; Brady and Carbone, 1973). These included: ethanol, magnesium chloride, magnesium sulfate, chloroform (trichloroethane), chloral hydrate and carbon dioxide as soda (Schweppes carbonated water). The narcotic solutions were made by diluting the chemicals in filtrated sea water. To avoid the concentration reducing by evaporation of volatile substances, a cover was used on the aquaria and was only withdrawn to introduce or remove the squid. All the solutions in these exposure aquaria, except soda, were aerated continuously through an air-stone in order to keep the oxygen saturation higher in the water. All tests were conducted at room temperature.
For each test, the specimens were submerged in the solution. The initial exposure time was determined by considering that the squid was in a state of “narcosis” when losing its movement control and tentacles adherence capacities. After exposure, each squid was submitted to activity tests by holding them out of water in my hands or/and in a scoop net for no longer than 90 set and transferring them to a recuperation circular tank with running sea water. Then, recuperation time was determined as they completely recovered their lost capacities. In addition, behavioral data were taken on hands contact reaction, swimming capacity, corporal motility, ink secretion, coloration changes and tendency to sink to check the squid response to stress. Based on the recovery, exposure time was increased or decreased, searching for the range between the narcosis starting and lethal times. The initial concentration for each chemical was based on earlier experiments (Van Eeden, 1958 and Bell, 1964) and concentrations were tested by trial and error based on the previous recoveries. Anesthesia is a physiological state of protoplasm characterized by a reversible insensitiveness of the cell, tissue or organism (McFarland, 1959). Agents, usually described as general anesthetics, reversibly depress the sensory centers of the brain to various degrees and finally eliminate reflex action. Excessive dosage or prolonged exposure leads to involvement of the vital respiratory and vasomotor centers usually causing death (Bell, 1964). Based on these statements in this study, squid in the state of anesthesia were considered as those under “narcosis” that also showed total muscle relaxation. Anaesthesia is the absence of body hardness, flexibility of tentacles and elimination of external coloration patterns (transparent mantle) by inhibiting the myotibrilla contraction of chromatophores.
RESULTS Chloroform
This chemical caused the squid’s death in short exposure times and low concentrations. Even with 1% or less, they react violently when placed in contact with the solution hitting themselves against the container walls, and secreting ink continuously. After less than a minute the specimens sank with a discolored and rigid body, fin borders corrugated and shrunken and rolled tentacles separated from 121
122
MAURICIO GARCiA-FRANC0
of the myofibrillas of the chromatophores. This mantle tetanus might cause death by asphyxia, inhibiting the specimens from water and gas exchanges in the pallial cavity due to the rigidity of the mantle, unless they were immediately transferred to a well oxygenated tank and given artificial breathing by gently compressing and relaxing the mantle manually.
each other. Even if immediately after narcosis the specimens were transferred to the recuperation tank their death was inevitable. In addition, working at the tropical temperatures suitable for this species, the evaporation of chloroform is so rapid that the narcotic mixture concentration can not be controlled. Chloral hydrate
Using 1% or higher concentrations the squids sank with discolored bodies in less than 5 sec. However, the achieved effect is so impetuous that mortality while in the recuperation tank is high. With 0.5% or lower concentrations, even if narcosis can be attained before lOsec, the reaction of specimens to the solution is violent with turbulent movements followed by fin borders corrugation, muscular spasms of mantle, excreting ink continuously which clotted when it came in contact with the sedative and thus impeding its reutilization. In addition, even in 0.25% solution the squids can die from 30 sec. There is only a small interval between the minimal exposure time to obtain narcosis and the lethal exposure time: this would give an insufficient safety margin. On the other hand, there are no anesthesia symptoms, and after short periods of narcosis a long recuperation time is necessary.
Ethanol
This substance has been shown to have very low power when used as anaesthetic in fishes (McFarland, 1960), however in Sepioteuthis sepioidea it had a reasonable anesthetizing capacity. In spite of being a volatile substance, anesthesia was well achieved without any pernicious side effects. The optimum results were obtained between 1% and 3% concentrations (Fig. 1). Magnesium chloride
At first, the animals reacted to the MgCl, by expelling ink and faster and continuously changing the mantle coloration patterns. However, after a short time, the squid gradually reached the anaesthetic state losing any external coloration. Due to the transparency of the body surface, it was easy to observe the pallial cavity organs. The optimal results were between 1.5% and 2% concentrations (Fig. 1).
Soda (carbon dioxide)
Effects were attained only from 1.25% causing narcosis for short periods. Moreover even increasing the duration of the exposure, the state of the squids never changed from narcosis to anesthesia or died. Solutions above 10% are lethal. CO,, together with chloral hydrate, have a spasmogenic side effect but more pronounced in CO,. In this case, besides the rigidity of the body, corrugation of fin borders and spasms of mantle and tentacles, the squid did not present discoloration as in the others cases of narcosis. On the contrary, their body became dark brown in color due to an uninterrupted contraction 20
ETHANOL
I
4
On coming in contact with MgCI, the specimens reacted in a similar way as with magnesium chloride. After a short violent reaction, they reached anaesthesia becoming completely transparent and having their muscles fully relaxed. Subsequently they progressively regained the coloration patterns as they recovered. One difference is that with magnesium sulfate, in most cases, they defecated during exposure and in some cases regurgitated the stomach content or expelled eggs.
1
MC,
I
2
Magnesium sulfate
6
I.
8
10
I--II’
4
Exposure
6
6
10
*
12
w4SO4
4.6
a
10
(min)
1. Regression curves at the different exposure and recuperation times, Ethanol 1% (Y = 1.23+0.18X), 2% (Y =0.39e’,‘9x) and 3% (Y =0.89e3,‘MX);MgCI, 1.5% (Y = -5.9+ 1.74X) and 2% (Y = -51.66 + 32.59LnX) and MgSO, 3% (Y = 0.65e”.“X) and 4% (Y = - 18.17 + 3.97X). Fig.
..‘.. Point from where anesthesia was achieved. .-.-.-.
Point from where mortality was achieved.
Anaesthetics for the squid Sepioteuthis sepioidea
The optimal results were between 3% and 4% concentrations, having a narrower range of exposure time comparing with those of ethanol and magnesium chloride (Fig. 1). DISCUSSIONS
Chloral hydrate and chloroform are effective as a tranquilizer on other mollusks as freshwater snails (Huf, 1934 and Van Eeden, 1958). However, on Sepioteuthis sepioidea the anesthesia was never achieved, having acute fatal effects. CO2 too, can not be considered as anesthetics for this squid, causing harm to them in low concentrations and short exposures. CO, can induce violent reactions or cause irreversible side effects that resulted in death. CO* provokes symptoms similar to the tetanus-like clonospasms that had been demonstrated in the chromatophore masculature of other cephalopods as LoIigo and Sepia (Hill and Solandt, 1934; Nicol, 1964). Tetanus is a sustained muscular contraction caused by a series of stimuli repeated so rapidly that the individual muscular responses are fused (SchmidtNielsen, 1976). This can be caused by alkalosis resulting in a reduction of ionized calcium in body fluids, e.g., excessive amounts of sodium bicarbonate (Stedman, 1982). In the present study, this effect might be caused by alkalosis as the Schweppes soda is a basic carbonated water containing sodium bicarbonate. Furthermore, it had been demonstrated that cephalopod hemocyanin shows a large normal Bohr effect (Ghiretti, 1966). An increment in pH during alkalosis might make the liberation of oxygen in the tissues difficult (Wolvekamp et al., 1942). In addition, this condition was aggravated by the lack of O,, because the soda was the only solution kept without aeration in order to achieve the anaesthetic effects, as diluted CO, does in fishes, by inducing asphyxia from reduction of oxygen binding capacity of hemoglobin (Bell, 1964). Ethanol can be considered as a good drug for Sepioteuthis sepioidea inducing anaesthesia without side effects. Probably ethanol reversibly blocks nerve impulse transmission in squids (Brady and Carbone, 1973) annulling the reflex arc, a fact inherent to the anesthesia state. With both magnesium chloride and magnesium sulfate, anesthesia can be reached with optimum results. The relaxation was deep, reaching the chromatophores myofibrillas resulting in the transparency of the mantle. The pallial cavity was visible, that besides handling a squid for weighing and measuring, it was possible to determine its sex and the sexual maturation through the morphometric study of their gonads. The magnesium ion (Mg2+) in elevated concentrations at cellular level acts as an anaesthetic (Schmidt-Nielsen, 1976). In molluscan muscle Mg*+ presents an antagonistic action to Ca2+ inhibiting the neuromuscular transmission and excitation
123
(Kobayashi, 1974). As with ethanol, using magnesium salts, Sepioteuthis sepioidea reached anaesthesia in a relatively short period of exposure. This method is less complicated using only one step, as opposed with other techniques for mollusks (Huf, 1934; McGraw, 1958 and Lever et al., 1963). REFERENCES
Bell G. R. (1964) A guide to the properties, characteristics and uses of some general anaesthetics for fish. Bull. Fish. Res. Bd Can. 148, l-4. Plus the table as a guide is attached. Brady R. 0. and Carbone E. (1973) Comparison of the effects of A9-tetrahydrocannabinol, 1I-hydroxy-A’-tetrahydrocannabinol and ethanol on the electrophysiological activity of the giant axon of the squid. Neuropharmacology 12, 601-605. Djourno M. A. (1963) Nouvelles mtthodses d’anesthesie gtntrale par le courant tlectrique. Bulletin de I’academie nationale de medicine 147, 1666168. Ghiretti F. (1966) Molluscan hemocyanin. In Physiology of Mollusca (edited by K. M. Wilbur and C. M. Yonge), pp. 2333248, Vol. 2. Academic Press, London. Hill A. V. and Solandt D. Y. (1934) Mvoarams from the chromatophores of Sepia. J. Physiol. (L&d.) 83, 13-14. Huf E. (1934) Uber den einfluB der narkose auf den wasserund mineralhausalt bei siiI3wassertieren. Pfliigere Arch 235, 129-140. Kobayashi M. (1974) Antagonistic action of Ca and Mg on the neuromuscular transmission and excitation in a molluscan muscle (radula protactor). J. camp. Physiol. 94, 17-24.
Lever J., Jager J. C. and Westerveld A. (1963) A new anesthetization technique for fresh water snails, tested on Lymnaea stagnalis. Malacologia 1, 33 l-337. McFarland W. N. (1959) A study of the effects of anesthetics on the behavior and physiology of fishes. Publications of the Institute of Marine Science 6, 23-55.
McFarland W. N. (1960) The use of anesthetics for the handling and transport of fishes. California Fish Game 46, 407-43 1. McGraw B. M. (1958) Relaxation of snails before fixation. Nature 181, 575.
Nicol J. A. C. (1964) Special effecters: luminous organs, chromatophores, pigments and poison glands. In Physiology of Mollusca (Edited by K. M. Wilbur and C. M. Yonge), pp. 353-381, Vol. 1. Academic Press, London. Norris K. S., Brocato F. and Calandrino F. (1960) A survey of fish transportation methods and equipment. California Fish Game 46, 5-33.
ROSS L. G. and Ross B. (1984) Anaesthetic and Sedative Techniques for Fish, pp. l-34. Institute of Aquaculture, University of Stirling, Scotland. Schmidt-Nielsen K. (1976) Fisiologia Animal (Spanish), pp. l-499. Ediciones Omega, Barcelona. Stedman L. T. (1982) Illustrated Stedman’s Medical Dictionary, pp. I-1678, 24th edition. Williams and Wilkins, Baltimore. Van Eeden J. A. (1958) Two useful techniques in freshwater malacology. Proceedings of the Malacological Society 33, 6467.
Wolvekamp H. P., Baerends G. P., Kok B. and Mommaerts W. F. H. M. (1942) 0, and CO, binding properties of the blood of the cuttlefish (Sepia ojkinalis) and the common squid (Loligo oulgaris). Arch. neerl. physiol. 26, 203-2 18.
103C,No. I, pp. 121-123,1992
0306-4492/92 $5.00 + 0.00 0 1992 Pergamon Press Ltd
Printedin Great Britain
ANAESTHETICS FOR THE SQUID SZi’Z’ZOTEUTHIs XWZOZDEA (MOLLUSCA: CEPHALOPODA) MAURKIO GARCiA-FRANC0 Laboratory of Aquatic Biology, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108, Japan Fax: 81-3-34715794 (Received 3 February
1992; accepted 6 March
1992)
Abstract-l.
Specimens of Sepioreufhis sepioidea were exposed to different reagents commonly used as anaesthetics for cold blooded animals. 2. Although some reagents a&ted the specimens, the anesthesia state was only achieved when using ethanol, magnesium sulfate or magnesium chloride. 3. The optimal ranges of concentrations necessary to achieve the anesthesia were: 1 to 3% for ethanol, 1.5 to 2% for magnesium chloride and 3 to 4% for magnesium sulfate.
INTRODUCTION The search for potentially cultivable mollusk species is directly linked to the use of anesthetics capable of improving various handling techniques such as tagging and marking, weighing and sexing, artificial induced reproduction and particularly the transportation of live organisms. The anesthetics agents can be physical or chemical (McFarland, 1960; Norris et al., 1960; Ross and Ross, 1984). One of the physical methods is the hypothermia which is used alone or in combination with chemical anesthetics for transport of living organisms. Another is electroshock (Djourno, 1963), whose use is probably limited to manipulations involving rapid handling. The use of chemical reagents is more popular among aquatic organisms. However, in mollusks and cephalopods in particular, there are comparatively few researches done related to anesthetics. Consequently, this study was aimed at identifying the reagents, their concentrations and exposure periods that would induce anesthesia, allowing the easy manipulation of Sepioteuthis sepioidea. MATERIALSAND METHODS Samples of both sexes of S. sepioidea were obtained from Mochima Bay, Sucre State in Venezuela. Specimens of 42.2 to 290.9 g in total weight (7.6 to 17.1 cm in mantle length) were used in this study. The reagents tested were selected on the basis of their low price, availability and previous studies reported on cold blooded animals (Huf, 1934; Lever er al., 1963; Brady and Carbone, 1973). These included: ethanol, magnesium chloride, magnesium sulfate, chloroform (trichloroethane), chloral hydrate and carbon dioxide as soda (Schweppes carbonated water). The narcotic solutions were made by diluting the chemicals in filtrated sea water. To avoid the concentration reducing by evaporation of volatile substances, a cover was used on the aquaria and was only withdrawn to introduce or remove the squid. All the solutions in these exposure aquaria, except soda, were aerated continuously through an air-stone in order to keep the oxygen saturation higher in the water. All tests were conducted at room temperature.
For each test, the specimens were submerged in the solution. The initial exposure time was determined by considering that the squid was in a state of “narcosis” when losing its movement control and tentacles adherence capacities. After exposure, each squid was submitted to activity tests by holding them out of water in my hands or/and in a scoop net for no longer than 90 set and transferring them to a recuperation circular tank with running sea water. Then, recuperation time was determined as they completely recovered their lost capacities. In addition, behavioral data were taken on hands contact reaction, swimming capacity, corporal motility, ink secretion, coloration changes and tendency to sink to check the squid response to stress. Based on the recovery, exposure time was increased or decreased, searching for the range between the narcosis starting and lethal times. The initial concentration for each chemical was based on earlier experiments (Van Eeden, 1958 and Bell, 1964) and concentrations were tested by trial and error based on the previous recoveries. Anesthesia is a physiological state of protoplasm characterized by a reversible insensitiveness of the cell, tissue or organism (McFarland, 1959). Agents, usually described as general anesthetics, reversibly depress the sensory centers of the brain to various degrees and finally eliminate reflex action. Excessive dosage or prolonged exposure leads to involvement of the vital respiratory and vasomotor centers usually causing death (Bell, 1964). Based on these statements in this study, squid in the state of anesthesia were considered as those under “narcosis” that also showed total muscle relaxation. Anaesthesia is the absence of body hardness, flexibility of tentacles and elimination of external coloration patterns (transparent mantle) by inhibiting the myotibrilla contraction of chromatophores.
RESULTS Chloroform
This chemical caused the squid’s death in short exposure times and low concentrations. Even with 1% or less, they react violently when placed in contact with the solution hitting themselves against the container walls, and secreting ink continuously. After less than a minute the specimens sank with a discolored and rigid body, fin borders corrugated and shrunken and rolled tentacles separated from 121
122
MAURICIO GARCiA-FRANC0
of the myofibrillas of the chromatophores. This mantle tetanus might cause death by asphyxia, inhibiting the specimens from water and gas exchanges in the pallial cavity due to the rigidity of the mantle, unless they were immediately transferred to a well oxygenated tank and given artificial breathing by gently compressing and relaxing the mantle manually.
each other. Even if immediately after narcosis the specimens were transferred to the recuperation tank their death was inevitable. In addition, working at the tropical temperatures suitable for this species, the evaporation of chloroform is so rapid that the narcotic mixture concentration can not be controlled. Chloral hydrate
Using 1% or higher concentrations the squids sank with discolored bodies in less than 5 sec. However, the achieved effect is so impetuous that mortality while in the recuperation tank is high. With 0.5% or lower concentrations, even if narcosis can be attained before lOsec, the reaction of specimens to the solution is violent with turbulent movements followed by fin borders corrugation, muscular spasms of mantle, excreting ink continuously which clotted when it came in contact with the sedative and thus impeding its reutilization. In addition, even in 0.25% solution the squids can die from 30 sec. There is only a small interval between the minimal exposure time to obtain narcosis and the lethal exposure time: this would give an insufficient safety margin. On the other hand, there are no anesthesia symptoms, and after short periods of narcosis a long recuperation time is necessary.
Ethanol
This substance has been shown to have very low power when used as anaesthetic in fishes (McFarland, 1960), however in Sepioteuthis sepioidea it had a reasonable anesthetizing capacity. In spite of being a volatile substance, anesthesia was well achieved without any pernicious side effects. The optimum results were obtained between 1% and 3% concentrations (Fig. 1). Magnesium chloride
At first, the animals reacted to the MgCl, by expelling ink and faster and continuously changing the mantle coloration patterns. However, after a short time, the squid gradually reached the anaesthetic state losing any external coloration. Due to the transparency of the body surface, it was easy to observe the pallial cavity organs. The optimal results were between 1.5% and 2% concentrations (Fig. 1).
Soda (carbon dioxide)
Effects were attained only from 1.25% causing narcosis for short periods. Moreover even increasing the duration of the exposure, the state of the squids never changed from narcosis to anesthesia or died. Solutions above 10% are lethal. CO,, together with chloral hydrate, have a spasmogenic side effect but more pronounced in CO,. In this case, besides the rigidity of the body, corrugation of fin borders and spasms of mantle and tentacles, the squid did not present discoloration as in the others cases of narcosis. On the contrary, their body became dark brown in color due to an uninterrupted contraction 20
ETHANOL
I
4
On coming in contact with MgCI, the specimens reacted in a similar way as with magnesium chloride. After a short violent reaction, they reached anaesthesia becoming completely transparent and having their muscles fully relaxed. Subsequently they progressively regained the coloration patterns as they recovered. One difference is that with magnesium sulfate, in most cases, they defecated during exposure and in some cases regurgitated the stomach content or expelled eggs.
1
MC,
I
2
Magnesium sulfate
6
I.
8
10
I--II’
4
Exposure
6
6
10
*
12
w4SO4
4.6
a
10
(min)
1. Regression curves at the different exposure and recuperation times, Ethanol 1% (Y = 1.23+0.18X), 2% (Y =0.39e’,‘9x) and 3% (Y =0.89e3,‘MX);MgCI, 1.5% (Y = -5.9+ 1.74X) and 2% (Y = -51.66 + 32.59LnX) and MgSO, 3% (Y = 0.65e”.“X) and 4% (Y = - 18.17 + 3.97X). Fig.
..‘.. Point from where anesthesia was achieved. .-.-.-.
Point from where mortality was achieved.
Anaesthetics for the squid Sepioteuthis sepioidea
The optimal results were between 3% and 4% concentrations, having a narrower range of exposure time comparing with those of ethanol and magnesium chloride (Fig. 1). DISCUSSIONS
Chloral hydrate and chloroform are effective as a tranquilizer on other mollusks as freshwater snails (Huf, 1934 and Van Eeden, 1958). However, on Sepioteuthis sepioidea the anesthesia was never achieved, having acute fatal effects. CO2 too, can not be considered as anesthetics for this squid, causing harm to them in low concentrations and short exposures. CO, can induce violent reactions or cause irreversible side effects that resulted in death. CO* provokes symptoms similar to the tetanus-like clonospasms that had been demonstrated in the chromatophore masculature of other cephalopods as LoIigo and Sepia (Hill and Solandt, 1934; Nicol, 1964). Tetanus is a sustained muscular contraction caused by a series of stimuli repeated so rapidly that the individual muscular responses are fused (SchmidtNielsen, 1976). This can be caused by alkalosis resulting in a reduction of ionized calcium in body fluids, e.g., excessive amounts of sodium bicarbonate (Stedman, 1982). In the present study, this effect might be caused by alkalosis as the Schweppes soda is a basic carbonated water containing sodium bicarbonate. Furthermore, it had been demonstrated that cephalopod hemocyanin shows a large normal Bohr effect (Ghiretti, 1966). An increment in pH during alkalosis might make the liberation of oxygen in the tissues difficult (Wolvekamp et al., 1942). In addition, this condition was aggravated by the lack of O,, because the soda was the only solution kept without aeration in order to achieve the anaesthetic effects, as diluted CO, does in fishes, by inducing asphyxia from reduction of oxygen binding capacity of hemoglobin (Bell, 1964). Ethanol can be considered as a good drug for Sepioteuthis sepioidea inducing anaesthesia without side effects. Probably ethanol reversibly blocks nerve impulse transmission in squids (Brady and Carbone, 1973) annulling the reflex arc, a fact inherent to the anesthesia state. With both magnesium chloride and magnesium sulfate, anesthesia can be reached with optimum results. The relaxation was deep, reaching the chromatophores myofibrillas resulting in the transparency of the mantle. The pallial cavity was visible, that besides handling a squid for weighing and measuring, it was possible to determine its sex and the sexual maturation through the morphometric study of their gonads. The magnesium ion (Mg2+) in elevated concentrations at cellular level acts as an anaesthetic (Schmidt-Nielsen, 1976). In molluscan muscle Mg*+ presents an antagonistic action to Ca2+ inhibiting the neuromuscular transmission and excitation
123
(Kobayashi, 1974). As with ethanol, using magnesium salts, Sepioteuthis sepioidea reached anaesthesia in a relatively short period of exposure. This method is less complicated using only one step, as opposed with other techniques for mollusks (Huf, 1934; McGraw, 1958 and Lever et al., 1963). REFERENCES
Bell G. R. (1964) A guide to the properties, characteristics and uses of some general anaesthetics for fish. Bull. Fish. Res. Bd Can. 148, l-4. Plus the table as a guide is attached. Brady R. 0. and Carbone E. (1973) Comparison of the effects of A9-tetrahydrocannabinol, 1I-hydroxy-A’-tetrahydrocannabinol and ethanol on the electrophysiological activity of the giant axon of the squid. Neuropharmacology 12, 601-605. Djourno M. A. (1963) Nouvelles mtthodses d’anesthesie gtntrale par le courant tlectrique. Bulletin de I’academie nationale de medicine 147, 1666168. Ghiretti F. (1966) Molluscan hemocyanin. In Physiology of Mollusca (edited by K. M. Wilbur and C. M. Yonge), pp. 2333248, Vol. 2. Academic Press, London. Hill A. V. and Solandt D. Y. (1934) Mvoarams from the chromatophores of Sepia. J. Physiol. (L&d.) 83, 13-14. Huf E. (1934) Uber den einfluB der narkose auf den wasserund mineralhausalt bei siiI3wassertieren. Pfliigere Arch 235, 129-140. Kobayashi M. (1974) Antagonistic action of Ca and Mg on the neuromuscular transmission and excitation in a molluscan muscle (radula protactor). J. camp. Physiol. 94, 17-24.
Lever J., Jager J. C. and Westerveld A. (1963) A new anesthetization technique for fresh water snails, tested on Lymnaea stagnalis. Malacologia 1, 33 l-337. McFarland W. N. (1959) A study of the effects of anesthetics on the behavior and physiology of fishes. Publications of the Institute of Marine Science 6, 23-55.
McFarland W. N. (1960) The use of anesthetics for the handling and transport of fishes. California Fish Game 46, 407-43 1. McGraw B. M. (1958) Relaxation of snails before fixation. Nature 181, 575.
Nicol J. A. C. (1964) Special effecters: luminous organs, chromatophores, pigments and poison glands. In Physiology of Mollusca (Edited by K. M. Wilbur and C. M. Yonge), pp. 353-381, Vol. 1. Academic Press, London. Norris K. S., Brocato F. and Calandrino F. (1960) A survey of fish transportation methods and equipment. California Fish Game 46, 5-33.
ROSS L. G. and Ross B. (1984) Anaesthetic and Sedative Techniques for Fish, pp. l-34. Institute of Aquaculture, University of Stirling, Scotland. Schmidt-Nielsen K. (1976) Fisiologia Animal (Spanish), pp. l-499. Ediciones Omega, Barcelona. Stedman L. T. (1982) Illustrated Stedman’s Medical Dictionary, pp. I-1678, 24th edition. Williams and Wilkins, Baltimore. Van Eeden J. A. (1958) Two useful techniques in freshwater malacology. Proceedings of the Malacological Society 33, 6467.
Wolvekamp H. P., Baerends G. P., Kok B. and Mommaerts W. F. H. M. (1942) 0, and CO, binding properties of the blood of the cuttlefish (Sepia ojkinalis) and the common squid (Loligo oulgaris). Arch. neerl. physiol. 26, 203-2 18.