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Comparative lethality of several Latrodectus venoms
Tox!con, 1964, Vol . 2. pp. 201-203 .
Per~amon Preaa Ltd . Painted in Greet Hntam
COMPARATIVE LETHALITY OF SEVERAL LATRODECTUS VENOMS J. D. McCitorre Florida Presbyterian College, St. Petersburg, Florida, U.S .A. Abetrad-Four apociea of the blacdc-widow spider genus Latradectns are known to occur in North America. They are L . rn~actans ~etans, L. geometrieus, L. vnrtolus and L. blsleopf. The oomparafive lethality of the venons of these species and of the European sub-species L. rractana trcdeclmguttatus was evaluated. All have very potent venoma although there are species differentxa in venom lethality and the average amount of venom obtained. INTRODUCTION
Ix ><-ns revision of the black-widow genus Latrodectus, Lsvt [1] recognized three North American species L. mactans mactans (Fabricius, 1775), L. geometricus C. L. Koch, 1841, and L. curacaviensis (Muller, 177 . Subsequent field work, laboratory studies, and the acquisition of additional specimens have revealed that the North American specimens considered to be L. curacaviensis actually belong to two species, L. variohts Walckenaer, 1837 and L. bishopi Koston, 1938 [2]. This paper presents the results of a study of the comparative lethality of the venoms of each of the four North American species and of the European subspecies L. mactans tredecimguttatus . MATERIALS AND METHODS
The venoms were prepared by dissecting out the venom glands from groups of 20-30 mature female spiders and homogenizing them in distilled water with a Servall microhomogenizer. . The homogenate was then centrifuged at 7200 rev/min for 20 min. The glands and homogenate were kept cold during these operations. The clear, viscous supernatant obtained by centrifugation was then lyophilized in a serum bottle with a Virtis dry ice freeze-dryer. This lyophilized material, henceforth referred to as venom, was weighed and then was stored in a dessicator kept at -1 ~5° in a deep freeze . Kt~Ax et al. [3] have reported that there is seasonal variation in the lethality of L. mactans mactans venom. In order to eliminate this variable, all of the venoms used were collected during the Summer of 1963 . Also, all of the spiders used, with the exception of the L. macrons tredecimguttatus which were obtained from Israel, came from the same geographical area, the state of Florida. The method of Lrrcl~.n and WILCOXON [4] was used to determine the LDao of each venom in mice. Groups of six 20-30 g white mice of a single strain obtained from a commercial source were injected intraperitoneally with varying doses of venom dissolved in a constant volume (0~2 mI) of physiological saline . Death or survival at the end of 48 hr was considered the endpoint. In this study 48 hr was used for the following reason. The ütijected mice passed through two fairly distinct crises. The fast occurnd within 201
J. D. McCRONE
202
6-8 hr after injection, the other 188 hr after injection. Autopsy of the animals that died within 6-8 hr revealed no gross pathological changes, although the lungs were pale and inüated. Those animals which died later, however, showed marked gross pathological changes. The lungs had a reddish marbled appearance with numerous petechiae and there was often extensive hemorrhaging in the abdominal cavity . If an animal survived these two crises and lived for 48 hr it would eventually recover in most cases. RESULTS
The average amount of venom obtained per spider for each species and the LD6o for each venom are given in Table l . The average amount obtained per spider was calculated TAaLE
1.
AVaIeAa$ AlSOUNT
Species L. mactans t~tatus L. gonmetricus
OF
VBNOM
No . of spiders used 30 255
OH'rAINBD
AND LETHALITY
DATA FOR SEVERAL La1rOdLCINS
Avera®e Amount of Mouse LD,O in mg/kg venom obtained (95 ~ Confidence (mg/spider) interval)
SP8CIES .
Lethality factor (LD, o s/avera®e amount of venom obtained)
0238
0~59 (0~33-1 ~06)
0~404
00970
043 (031-0~59)
0226
L. tiarblus
74
0254
1 ~80 (120-2 ~70)
0l41
L. maetans mactans
49
0l90
1 ~30 (120-2 ~70)
0106
L. bfshopi
70
0l57
220 (129-3~74)
0~071
by dividing the total amount ofvenom extracted from each species by the number of spiders used of that species. Since the effectiveness of a venom is a function of both the amount of venom obtained and the venom lethality, a lethality factor similar to the toxicity factor used by WfnzTEMOxs and IRAN [5] was calculated for each species (Table 1). This value represents the number of LD 6o per average amount of venom obtained . DISCUSSION
The presence of gland material in the venom gland extract probably results in a very conservative estimate of the actual lethality of the venoms studied ; however, the LD 6o values reported here do give a fairly accurate estimation of the relative lethality of the venoms . It is difficult and probably meaningless to compare my lethality values with those previously published by other workers because of the marked differences in experimental conditions. An exception is the LD6o value given by BEITINI and Tosctll-1ÏtONTALI [6] for the L. mactans trea!ecimguttattts found in Italy. Using slightly different methods, they obtained an LD6o value for mice of 0 ~9 mg per kg while I obtained a value of 0"59 mg
Comparative Lethality of Severnl latrodectus Venons
203
per kg. The small difference may be due to one or more .of the following factors. First, the spiders canoe from different geographical areas. Secoàd, the difference could just be the result of experimental error since their value falls within my 95 per cent confidence interval. Third, they used a shorter assay period which would tend to raise their value. The most surprising result of this study is the finding that the brown widow L. geometricus has one of the most lethal of the Latrodectus venous. It has never been considered a particularly dangerous species although there have been reports of people being bitten with fairly severe results [7, 8j. Two factors probably account for the fact that more cases have not been reported . One is that the average amount ofvenom obtained per spider is very low, the other is that L. geometricus is much less aggmssive and irritable than any of the other species studied. SUMMARY
The average amount of venom obtained per spider, the LDso vahu itt mice of the venom, and a lethality factor was determined for Latrodectus mactats macrons, L. macrons tredecimguttatus, L. variolus, L. bishopi and L. geometricus using lyophilized venom gland extract. (2). All have very potent venons, but there are species differences in the three attributes that were measured . (1).
Ackrrowledgmuus-I wish to thank Mr. M. L. Nsrzt oFt+ for txhnical aid. Dr. H. W. Lsvl of the Museum of Comparative Zoology, Harvard University and Dr. A. Ssvtov of the Hebrew University, Jerusalem kindly helped me obtain ogg sacs of L mactau tredecirnquttatw. This study was supported by Public Health Service Cirant C3M 11206-01 from the National Institute of (leaeral Medical 3cd~oes. RBFBRENCBS [1] Lsvr, H. W., The spider genus Latrodectur (Amusas, Theridüdae) . 7Yars. Amer. nier. Soc., T1, 7,1959. [2] McCx~oxs, J. D. and LBVr, H. W., North American widow epideas of theLatrodatrv cmwcavlensis group (Araneae : Theridüdae). Psyche (in press). [3] IC~aex, H. L., W ei and H>m~v, R A., Seasonal variation in venom of black widow spiders. Amer.1. Tlrop. Med. Hyg., 10, 477, 1960. [4] Lrrc~~n, J. T. and Wu eoxox, F., A simplified method of evaluating dosaeffect experiments. 1. Pharnmcol., 96, 99, 1949. [S] Wi~rr>~oaB, F. W. and IC~a.~x, H. L., Medtaaify Emportant scorpions fie tira Pacffrc Aras in Yerromoar and Poisonous Animais and Noxious Piaule of the Pacf/rc Region, H. L. IC~~ sud W. V. MxcF~eur~, Eds. New York : Macneillan, p. 107, 1963 . [6] lisrzau, S. and Taecm-Fnoxrus, N., Sioch l and Toxicological aspects of Iatrodectw tredecinr guttatus venom. XI. Iru. Songr. F. Ertom. Wetn Werh. B., 3, 1960. [7] Bra, W. 7., The black widow and five other venomous spiders in the United States . Ark. Agr. exp. Sta. Bui., 608, 1959 . [8l Mt ePx~esox, J. W. E., A poisonous spider. 1. mad. Assoc. S. ~., 3, 691, 1929 .
Per~amon Preaa Ltd . Painted in Greet Hntam
COMPARATIVE LETHALITY OF SEVERAL LATRODECTUS VENOMS J. D. McCitorre Florida Presbyterian College, St. Petersburg, Florida, U.S .A. Abetrad-Four apociea of the blacdc-widow spider genus Latradectns are known to occur in North America. They are L . rn~actans ~etans, L. geometrieus, L. vnrtolus and L. blsleopf. The oomparafive lethality of the venons of these species and of the European sub-species L. rractana trcdeclmguttatus was evaluated. All have very potent venoma although there are species differentxa in venom lethality and the average amount of venom obtained. INTRODUCTION
Ix ><-ns revision of the black-widow genus Latrodectus, Lsvt [1] recognized three North American species L. mactans mactans (Fabricius, 1775), L. geometricus C. L. Koch, 1841, and L. curacaviensis (Muller, 177 . Subsequent field work, laboratory studies, and the acquisition of additional specimens have revealed that the North American specimens considered to be L. curacaviensis actually belong to two species, L. variohts Walckenaer, 1837 and L. bishopi Koston, 1938 [2]. This paper presents the results of a study of the comparative lethality of the venoms of each of the four North American species and of the European subspecies L. mactans tredecimguttatus . MATERIALS AND METHODS
The venoms were prepared by dissecting out the venom glands from groups of 20-30 mature female spiders and homogenizing them in distilled water with a Servall microhomogenizer. . The homogenate was then centrifuged at 7200 rev/min for 20 min. The glands and homogenate were kept cold during these operations. The clear, viscous supernatant obtained by centrifugation was then lyophilized in a serum bottle with a Virtis dry ice freeze-dryer. This lyophilized material, henceforth referred to as venom, was weighed and then was stored in a dessicator kept at -1 ~5° in a deep freeze . Kt~Ax et al. [3] have reported that there is seasonal variation in the lethality of L. mactans mactans venom. In order to eliminate this variable, all of the venoms used were collected during the Summer of 1963 . Also, all of the spiders used, with the exception of the L. macrons tredecimguttatus which were obtained from Israel, came from the same geographical area, the state of Florida. The method of Lrrcl~.n and WILCOXON [4] was used to determine the LDao of each venom in mice. Groups of six 20-30 g white mice of a single strain obtained from a commercial source were injected intraperitoneally with varying doses of venom dissolved in a constant volume (0~2 mI) of physiological saline . Death or survival at the end of 48 hr was considered the endpoint. In this study 48 hr was used for the following reason. The ütijected mice passed through two fairly distinct crises. The fast occurnd within 201
J. D. McCRONE
202
6-8 hr after injection, the other 188 hr after injection. Autopsy of the animals that died within 6-8 hr revealed no gross pathological changes, although the lungs were pale and inüated. Those animals which died later, however, showed marked gross pathological changes. The lungs had a reddish marbled appearance with numerous petechiae and there was often extensive hemorrhaging in the abdominal cavity . If an animal survived these two crises and lived for 48 hr it would eventually recover in most cases. RESULTS
The average amount of venom obtained per spider for each species and the LD6o for each venom are given in Table l . The average amount obtained per spider was calculated TAaLE
1.
AVaIeAa$ AlSOUNT
Species L. mactans t~tatus L. gonmetricus
OF
VBNOM
No . of spiders used 30 255
OH'rAINBD
AND LETHALITY
DATA FOR SEVERAL La1rOdLCINS
Avera®e Amount of Mouse LD,O in mg/kg venom obtained (95 ~ Confidence (mg/spider) interval)
SP8CIES .
Lethality factor (LD, o s/avera®e amount of venom obtained)
0238
0~59 (0~33-1 ~06)
0~404
00970
043 (031-0~59)
0226
L. tiarblus
74
0254
1 ~80 (120-2 ~70)
0l41
L. maetans mactans
49
0l90
1 ~30 (120-2 ~70)
0106
L. bfshopi
70
0l57
220 (129-3~74)
0~071
by dividing the total amount ofvenom extracted from each species by the number of spiders used of that species. Since the effectiveness of a venom is a function of both the amount of venom obtained and the venom lethality, a lethality factor similar to the toxicity factor used by WfnzTEMOxs and IRAN [5] was calculated for each species (Table 1). This value represents the number of LD 6o per average amount of venom obtained . DISCUSSION
The presence of gland material in the venom gland extract probably results in a very conservative estimate of the actual lethality of the venoms studied ; however, the LD 6o values reported here do give a fairly accurate estimation of the relative lethality of the venoms . It is difficult and probably meaningless to compare my lethality values with those previously published by other workers because of the marked differences in experimental conditions. An exception is the LD6o value given by BEITINI and Tosctll-1ÏtONTALI [6] for the L. mactans trea!ecimguttattts found in Italy. Using slightly different methods, they obtained an LD6o value for mice of 0 ~9 mg per kg while I obtained a value of 0"59 mg
Comparative Lethality of Severnl latrodectus Venons
203
per kg. The small difference may be due to one or more .of the following factors. First, the spiders canoe from different geographical areas. Secoàd, the difference could just be the result of experimental error since their value falls within my 95 per cent confidence interval. Third, they used a shorter assay period which would tend to raise their value. The most surprising result of this study is the finding that the brown widow L. geometricus has one of the most lethal of the Latrodectus venous. It has never been considered a particularly dangerous species although there have been reports of people being bitten with fairly severe results [7, 8j. Two factors probably account for the fact that more cases have not been reported . One is that the average amount ofvenom obtained per spider is very low, the other is that L. geometricus is much less aggmssive and irritable than any of the other species studied. SUMMARY
The average amount of venom obtained per spider, the LDso vahu itt mice of the venom, and a lethality factor was determined for Latrodectus mactats macrons, L. macrons tredecimguttatus, L. variolus, L. bishopi and L. geometricus using lyophilized venom gland extract. (2). All have very potent venons, but there are species differences in the three attributes that were measured . (1).
Ackrrowledgmuus-I wish to thank Mr. M. L. Nsrzt oFt+ for txhnical aid. Dr. H. W. Lsvl of the Museum of Comparative Zoology, Harvard University and Dr. A. Ssvtov of the Hebrew University, Jerusalem kindly helped me obtain ogg sacs of L mactau tredecirnquttatw. This study was supported by Public Health Service Cirant C3M 11206-01 from the National Institute of (leaeral Medical 3cd~oes. RBFBRENCBS [1] Lsvr, H. W., The spider genus Latrodectur (Amusas, Theridüdae) . 7Yars. Amer. nier. Soc., T1, 7,1959. [2] McCx~oxs, J. D. and LBVr, H. W., North American widow epideas of theLatrodatrv cmwcavlensis group (Araneae : Theridüdae). Psyche (in press). [3] IC~aex, H. L., W ei and H>m~v, R A., Seasonal variation in venom of black widow spiders. Amer.1. Tlrop. Med. Hyg., 10, 477, 1960. [4] Lrrc~~n, J. T. and Wu eoxox, F., A simplified method of evaluating dosaeffect experiments. 1. Pharnmcol., 96, 99, 1949. [S] Wi~rr>~oaB, F. W. and IC~a.~x, H. L., Medtaaify Emportant scorpions fie tira Pacffrc Aras in Yerromoar and Poisonous Animais and Noxious Piaule of the Pacf/rc Region, H. L. IC~~ sud W. V. MxcF~eur~, Eds. New York : Macneillan, p. 107, 1963 . [6] lisrzau, S. and Taecm-Fnoxrus, N., Sioch l and Toxicological aspects of Iatrodectw tredecinr guttatus venom. XI. Iru. Songr. F. Ertom. Wetn Werh. B., 3, 1960. [7] Bra, W. 7., The black widow and five other venomous spiders in the United States . Ark. Agr. exp. Sta. Bui., 608, 1959 . [8l Mt ePx~esox, J. W. E., A poisonous spider. 1. mad. Assoc. S. ~., 3, 691, 1929 .