Short Communications
143
These results strongly indicate that the toxic principle of the cabezon rce is similar to if not identical with dinogunellin. The toxic phospholipid is extractable from the rce or from a 10~ saline extract with a mixture of chloroform and methanol, but not with chloroform. This may be the reason why Fur-ntMAN et al. (1969, 1970) overlooked the phospholipid as a toxic moiety .
REFERENCES D~wsox, R. M. C. (1960) A hydrolytic procedtu+e for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75, 43 . Frn~x, F. A., Fu~lruv, G. J., Dur.t, D. L. and Mosllex, H. S. (1969) Toxins from eggs of fishes and amphibia . J. ogric. Fd Chem. 17, 417. Fur-mns.~t, F. A., FutntM~rr, G. J. and Rossex, J. S. (1970) Toxic effects produced by extracts of eggs of the cabezon Scorpaenichthys marmoratus . Toxicon 8, 53 . HAT.~xo, M. and HwsfnMOTO, Y. (1974) Properties of a tonic phospholipid in the northern blenny rce. Toxkon 12, 231. HAT.~NO, M., MARUMOTO, R. and Hasl~roTO, Y. (in press) Structure of a toxic phospholipid in the northern blenny roe. Proc . 4th Int. Symp. Animals, Plants and Microbial Toxins . HUHa9, C. L. and Wrcx, A. N. (1931) Toxicity of the roe of the cabezon Scorpaenichthys marmoratus. Calij. Fish. Game 37, 193. Pird3HURY, R. W. (1937) Avoidance of poisoning eggs of the marine fish Scorpaentchthys marmorat«s by predators. Copeia 3, 231.
Toxlron. 1976, Vol. 14, pD. 143-145 . Per~aawa Prees. Printed In Graat Britain.
VENOM EXTRACTION AND YIELDS FROM THE NORTH AMERICAN CORAL SNAKE, MICRURUS FULVIUS JAi~s D. Flx and S. A. MINTON, JR. Departments of Anatomy and Microbiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, U.S .A . (Acceptedjor publication 23 September 1975) TITS nRY venom yield ofthe North American coral snake,
Microrus fulvius, has been reported as 2-6 mg in an average adult snake of 58-62 cm (MINTON, 1957, 1974 ; RUSSELL, 1967) . McCoLLOUCH and GsNNARO (1963) reported yields of 1-13 mg from a series of 10 snakes 508-91 "4 cm in length . Individual snake length and yield were not recorded, but they stated there was little correlation between the size of the snake and its venom yield. In this communication we report venom yields from 14 coral snakes obtained from sources in Florida. They were measured using the `squeeze-box' method (QuINN, 1974), kept in plastic cages and force-fed about once every 2 weeks . Cage temperatures were maintained at ca. 24°, however, cages were warmed to ca. 37° about 30 min before venom extraction. Snakes to be `milked' were manipulated into a restraining block of polyurethane foam as shown in Fig . 1. The head and approximately 1 cm of neck were allowed to protrude. After immobilization, a small glass vial covered with a parafilm membrane was presented to the snake's head touching the nose. Snakes usually bit through the membrane pugnaciously and began to chew. Slight tugging on the vial caused increased chewing TOXICON 1976 Yol. If
14 4
Short Communications Polyurethane
Cut
Open slot
FIG . 1 . POLYURETHANE FOAM RESTRAINING BLOCK AND VENOM EXTRACITON.
a . A block of polyurethane foam, 45 cm long, 25 cm wide, and 12 an high is cut 9 an down the midfine with a razor blade (b) . c . The snake is positioned in the open slot. Bottom : venom extraction in restraining block. Moderate pressure with one hand safely immobilizes the snake without trauma .
activity and venom flow . The snake was induced to perform this activity three times and then returned to its cage . No attempt was made to press on or massage the venom glands. Bleeding from the oral mucous membrane and other signs of trauma to the snakes' head and neck were not observed. Individual venom samples were freeze-dried using a glass cryosorption pump apparatus (Fix, unpublished observations) which produces a stable, anhydrous powder within 8-12 hr. Dried samples were carefully scraped from the vials and weighed. An appreciable amount of dried venom could not be recovered from the vial or the inner surface of the parafilm, so our reported yields are slightly lower than the true amount of venom ejected. Yields from the 14 snakes are shown in Fig. 2. The two largest snakes were milked a second time 2 weeks after initial venom extraction . In the case of one of these snakes the second yield was identical with the first; in the other slightly less . We believe that the method described here produces a more physiological type of venom ejection than conventional milking, and the larger yields obtained are a truer measure of the snakes' capacity to inject venom. Statistically there exists a positive linear relationship between the length of the snake and the yield of dried venom, the coefficient of correlation being computed at 087 (Fig. 2). Approximately 76 ~ of the variation in venom yields can be accounted for by the differences in the lengths of the snakes . Whether the average length range of MicrurTts TOXICON 19:6 Vul, !4
Short Communications
t45
Length, cm FIO . 2 . DRY VENOM YIELDS OF 14 NORTH AM~ICwN coRwL sruxFS. y-least-square line, r-the ooelHcient of correlation. e-first and second yields from snake No. 13 . b and b'-first and second yields from snake No . 14.
fulvius is considered 58-83 cm (IvIuvTOx, 1974) or 51-76 cm (CoxnxT, 1975), 11 of our specimens fell within these limits . However, eight gave venom yields in excess of 6 mg and three were in excess of 12 mg, twice the upper limit of the often-cited average yield. Two large snakes in the 85-90 cm range gave yields of 20 mg or more on successive milkings 2 weeks apart. Coral snakes are not uncommon in parts of the southern United States, and occasionally inflict severe or fatal bites (NEILL, 1957; PnxRtsx and Knxx, 1967). While a majority of reported bites are accompanied by few or no symptoms and hence by little injection of venom, the potential ability of a large coral snake to inject 20 mg or more of venom has clinical implications. The available specific antivenin neutralizes 2 mg per 10 ml vial (Wyeth Inc., package insert), therefore more than 10 vials may be required to neutralize the maximum quantity of venom that could be injected by a large Microrusfulvius. Acknowledgement-We are indebted to William C3leason of Silver Springs, Florida for donating some of the coral snakes used . We thank Dr. Dwvm L. FELTEN of the Indiana University School of Medicine who generously permitted us to use his cryosorptive pump apparatus in this investigation. REFERENCES
CONANT, R. (1975) A Field Guide to Reptiles andAmphibians ofEastern and CentralNorth America. Boston : Houghton & Mi®in. McCoLLOUOII, N. D. and (3ENruRO, J. F. (1963) Coral snake bites in the United States. J. Fla med. Ass. 49, 968. Mn~rroN, S. A., JR. (1957) Snakebite. Scient. Amer .196,114 . MnaTON, S . A., JR. (1974) Yenom Diseases. Springfield : Charles C. Thomas. NP~i . W, T. (1957) Some misconceptions regarding the eastern coral snake, Micrurus fulvius. Herpetologica 13, 111. QunaN, H. (1974) Squeeze boz technique for measuring snakes . Herp. Rev. 5, 35 . PwRRISa, H. M. and I{wHN, M. S. (1967) Bites by coral snakes : reports of 11 representative cases. Am. J. med. Sci. ?S3, 561. Ri1~VFi .i ~ F. E. (1967) Pharmacology of animal venoms . Clin. Pharmac. Thor. 8, 849. TOXlCON 1976 Yof. I~
143
These results strongly indicate that the toxic principle of the cabezon rce is similar to if not identical with dinogunellin. The toxic phospholipid is extractable from the rce or from a 10~ saline extract with a mixture of chloroform and methanol, but not with chloroform. This may be the reason why Fur-ntMAN et al. (1969, 1970) overlooked the phospholipid as a toxic moiety .
REFERENCES D~wsox, R. M. C. (1960) A hydrolytic procedtu+e for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75, 43 . Frn~x, F. A., Fu~lruv, G. J., Dur.t, D. L. and Mosllex, H. S. (1969) Toxins from eggs of fishes and amphibia . J. ogric. Fd Chem. 17, 417. Fur-mns.~t, F. A., FutntM~rr, G. J. and Rossex, J. S. (1970) Toxic effects produced by extracts of eggs of the cabezon Scorpaenichthys marmoratus . Toxicon 8, 53 . HAT.~xo, M. and HwsfnMOTO, Y. (1974) Properties of a tonic phospholipid in the northern blenny rce. Toxkon 12, 231. HAT.~NO, M., MARUMOTO, R. and Hasl~roTO, Y. (in press) Structure of a toxic phospholipid in the northern blenny roe. Proc . 4th Int. Symp. Animals, Plants and Microbial Toxins . HUHa9, C. L. and Wrcx, A. N. (1931) Toxicity of the roe of the cabezon Scorpaenichthys marmoratus. Calij. Fish. Game 37, 193. Pird3HURY, R. W. (1937) Avoidance of poisoning eggs of the marine fish Scorpaentchthys marmorat«s by predators. Copeia 3, 231.
Toxlron. 1976, Vol. 14, pD. 143-145 . Per~aawa Prees. Printed In Graat Britain.
VENOM EXTRACTION AND YIELDS FROM THE NORTH AMERICAN CORAL SNAKE, MICRURUS FULVIUS JAi~s D. Flx and S. A. MINTON, JR. Departments of Anatomy and Microbiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, U.S .A . (Acceptedjor publication 23 September 1975) TITS nRY venom yield ofthe North American coral snake,
Microrus fulvius, has been reported as 2-6 mg in an average adult snake of 58-62 cm (MINTON, 1957, 1974 ; RUSSELL, 1967) . McCoLLOUCH and GsNNARO (1963) reported yields of 1-13 mg from a series of 10 snakes 508-91 "4 cm in length . Individual snake length and yield were not recorded, but they stated there was little correlation between the size of the snake and its venom yield. In this communication we report venom yields from 14 coral snakes obtained from sources in Florida. They were measured using the `squeeze-box' method (QuINN, 1974), kept in plastic cages and force-fed about once every 2 weeks . Cage temperatures were maintained at ca. 24°, however, cages were warmed to ca. 37° about 30 min before venom extraction. Snakes to be `milked' were manipulated into a restraining block of polyurethane foam as shown in Fig . 1. The head and approximately 1 cm of neck were allowed to protrude. After immobilization, a small glass vial covered with a parafilm membrane was presented to the snake's head touching the nose. Snakes usually bit through the membrane pugnaciously and began to chew. Slight tugging on the vial caused increased chewing TOXICON 1976 Yol. If
14 4
Short Communications Polyurethane
Cut
Open slot
FIG . 1 . POLYURETHANE FOAM RESTRAINING BLOCK AND VENOM EXTRACITON.
a . A block of polyurethane foam, 45 cm long, 25 cm wide, and 12 an high is cut 9 an down the midfine with a razor blade (b) . c . The snake is positioned in the open slot. Bottom : venom extraction in restraining block. Moderate pressure with one hand safely immobilizes the snake without trauma .
activity and venom flow . The snake was induced to perform this activity three times and then returned to its cage . No attempt was made to press on or massage the venom glands. Bleeding from the oral mucous membrane and other signs of trauma to the snakes' head and neck were not observed. Individual venom samples were freeze-dried using a glass cryosorption pump apparatus (Fix, unpublished observations) which produces a stable, anhydrous powder within 8-12 hr. Dried samples were carefully scraped from the vials and weighed. An appreciable amount of dried venom could not be recovered from the vial or the inner surface of the parafilm, so our reported yields are slightly lower than the true amount of venom ejected. Yields from the 14 snakes are shown in Fig. 2. The two largest snakes were milked a second time 2 weeks after initial venom extraction . In the case of one of these snakes the second yield was identical with the first; in the other slightly less . We believe that the method described here produces a more physiological type of venom ejection than conventional milking, and the larger yields obtained are a truer measure of the snakes' capacity to inject venom. Statistically there exists a positive linear relationship between the length of the snake and the yield of dried venom, the coefficient of correlation being computed at 087 (Fig. 2). Approximately 76 ~ of the variation in venom yields can be accounted for by the differences in the lengths of the snakes . Whether the average length range of MicrurTts TOXICON 19:6 Vul, !4
Short Communications
t45
Length, cm FIO . 2 . DRY VENOM YIELDS OF 14 NORTH AM~ICwN coRwL sruxFS. y-least-square line, r-the ooelHcient of correlation. e-first and second yields from snake No. 13 . b and b'-first and second yields from snake No . 14.
fulvius is considered 58-83 cm (IvIuvTOx, 1974) or 51-76 cm (CoxnxT, 1975), 11 of our specimens fell within these limits . However, eight gave venom yields in excess of 6 mg and three were in excess of 12 mg, twice the upper limit of the often-cited average yield. Two large snakes in the 85-90 cm range gave yields of 20 mg or more on successive milkings 2 weeks apart. Coral snakes are not uncommon in parts of the southern United States, and occasionally inflict severe or fatal bites (NEILL, 1957; PnxRtsx and Knxx, 1967). While a majority of reported bites are accompanied by few or no symptoms and hence by little injection of venom, the potential ability of a large coral snake to inject 20 mg or more of venom has clinical implications. The available specific antivenin neutralizes 2 mg per 10 ml vial (Wyeth Inc., package insert), therefore more than 10 vials may be required to neutralize the maximum quantity of venom that could be injected by a large Microrusfulvius. Acknowledgement-We are indebted to William C3leason of Silver Springs, Florida for donating some of the coral snakes used . We thank Dr. Dwvm L. FELTEN of the Indiana University School of Medicine who generously permitted us to use his cryosorptive pump apparatus in this investigation. REFERENCES
CONANT, R. (1975) A Field Guide to Reptiles andAmphibians ofEastern and CentralNorth America. Boston : Houghton & Mi®in. McCoLLOUOII, N. D. and (3ENruRO, J. F. (1963) Coral snake bites in the United States. J. Fla med. Ass. 49, 968. Mn~rroN, S. A., JR. (1957) Snakebite. Scient. Amer .196,114 . MnaTON, S . A., JR. (1974) Yenom Diseases. Springfield : Charles C. Thomas. NP~i . W, T. (1957) Some misconceptions regarding the eastern coral snake, Micrurus fulvius. Herpetologica 13, 111. QunaN, H. (1974) Squeeze boz technique for measuring snakes . Herp. Rev. 5, 35 . PwRRISa, H. M. and I{wHN, M. S. (1967) Bites by coral snakes : reports of 11 representative cases. Am. J. med. Sci. ?S3, 561. Ri1~VFi .i ~ F. E. (1967) Pharmacology of animal venoms . Clin. Pharmac. Thor. 8, 849. TOXlCON 1976 Yof. I~
