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5-Hydroxytryptamine content of some arthropod venoms and venom-containing parts
Toxican, 1963, Vol, l, pp . 165-173.
Perganon Prees Ltd., Printed in Grcat Britain
5-HYDROXYTRYPTAMINE CONTENT OF SOME ARTHROPOD VENOMS AND VENOM-CONTAINING PARTS . JoxN H. WELSH and CAROLYN S. BATTY Biological Laboratories, Harvard University, Cambridge, Massachusetts Absfraá-The venom or venom-containing parts of 30 species of arthropods from North and South America were examined for their content of 5-hydroxytryptamine (5-HT) . The characteristic fluorescence of 5-HT was used in its identification and quantitative estimation . No S-HT was detected in venom or telsoes of 7 species of scorpions. Venoms of S spiders were found to contain 5-HT . No 5-HT was detected in the stinging apparatus of two species of ants and 3 species of solitary wasps. Relatively large amounts of 5-HT were found in the sting apparatus of all social wasps that were examined . Most S-HT was found in those wasps known to produce very painful stings. INTRODUCTION
S-HYDROXYTRYPÎ'AMINE (5-HT, serotonin, enteramine) has been found in the venom or venom apparatus of certain scorpions and social wasps and, in small amount, in bee venom (Table 1). The venom sacs of the hornet, Vespa crabro, have a higher content of 5-HT than any other known natural source [7]. 5-HT has been shown not to contribute to the toxcity of the venom of the scorpion, Leiurus quinquestriatus [1 ], but, when present in venoms, it may serve a defensive role due to its ability to produce severe pain [4, 8, 9]. It is also possible that 5-HT facilitates the uptake of the toxic components) of a venom by increasing the TABLE I .
SOME EARLIER OBSERVATIONS ON THE OCCURRENCE OF S-HT IN ARTHROPOD VENOMS AND VENOMCONTAININC PARTS
:species ArlclInoidea Lciurus quinquestriatus Buthutus minax Ha~i~"urus arizonensis Vejolis spirrigerus "V~:juvis sp ." Insceta Polislesgallica Vesper vulgaris Vesyrr crabro Apis ,~rrellijera >,
Venom or part venom venom telson
5-HT --wet ; d ^dry) Fig/g . (w-up to 40004 30-404 none detected SO~and 1384
venom apparatus ' venom ~ venom sacs (3 sets) venom apparatus
165
0 "7-0" 8/v .a . 3204 7,5004 14,7004 19,0004 21w 0"0005/v .a .
Ref. [l, 2, 3] [3] [4] [4] [4] [5] [6] I71 [4] [5]
16 6
JOHN H. WELSH and CAROLYN S. RATTY
permeability of cells, and increasing the rate of blood flow at the site of puncture and venom injection. Since a more complete knowledge of the distribution of 5-HT in arthropod venoms might permit more definite conclusions to be made concerning its possible roles, further observations appeared desirable. A visit to Brazil, by way of Trinidad, provided an opportunity for collecting venoms and venom containing parts of a variety of arthropods, including scorpions, spiders and wasps. These were returned to Harvard University for assay for their 5-HT content. The results, along with a few additional observations on North American species, will be reported in this paper. MATERIALS and METHODS
The species studied, and the regions from which they came, are given in Table 2. Those from Trinidad were collected in the vicinity of Simla, the field station of the New York Zoological Society, while those from Brazil were collected in the vicinity of Sao Paulo, or obtained from Butantan Institute. The venoms were obtained through the kind co-operation of Dr. W. Büc1~RL and his assistants at Butantan . They were released by electrical stimulation onto slips of preweighed aluminium foil and then dried under reduced. pressure. The slips of foil were TABLE
2.
HYDROXYTRYPTAMINE CONTENT OF VENOS, VENOM APPARATUS AND SOME OTHER PARTS OF VARIOUS ARTHROPODS
Species and Locality'
Venom or Part
DryWeight mg.
i`g. 5-HT per sample or part
5-HTisg./g.
Class Arachnoidea SCORPIONS
Tityus bahiensis
(B) (B) (T) (B) (B) (F) (A)
3 telsons (female 3 telsons (males) 3 telsons 2 telsons venom (9 animals)t venom (l animal) 1 telson 2 telsons
17 32 l7 <1 <1 5 8
<0~014/tel . <0 "014/tel. <0~014/tel . <0 "048/tel. <02/sample <0 "04/sample -
<3 <1 "5 ~3 < 12 <2
Tityus serrulatus Tityus trinitatus Tityus trivittatus Opisthocanthus cavaporun: Centruroides gracilis Vejovis spinigerus
(B) (B) (B) (B) (B)
venom (1 spider) venom (1 spider) venom (1 spider)t venom (3 spider)t venom (1 spider)
7 5 18 20 3
0"4/spi. 0 "7 spi. <0 " 8/spi. <0 " 16/spí . 0" l/spi .
58 140 33
(B)
venom (glands) (6 pr .)
13
0 "03/pr.
14
(B)
chelicerae (1 pr.) venom (3 spiders)t fi
4 3 5 6 5 S
2 "5/pr. 0"7/spi. 0 "8/spi. 2 "4/spi. -
520 880 1300 1540 2270 2650
SPIDERS
Family Therophcsidae Acanthoscurria atrox Acanthoscurria sternalis Lasiodora klugü Pamphobeteus roseus Pterinopelma vellutinunt Family Lycosidae Lycosa erythrognatlut Family Ctenidae Phoneutria fera ..
venom (many spiders)
5-Hyd~oxytryptamine Content of Some Arthropo3 Venoms and Venom-Containing Parts
Class Myriapode C~~$ Sonlopendra vlridlcornis Class Inxcta STtr~aznvc~ Arrrs Odontomachus haematodes Sttnomyrmex emarglnatus SOIITARY W~srs Family Mutt7lidae Darymutilla sp. Family Sphecidae Bembix texana Stictia Carolina
Soctu. W~srs Family Vespidae Polybia rejects Polybia occldentalis scutellaris Polistes juscatus Polistes verslcolor Polistes versicolor Polistes versicolor vulgates Vespa maculate Synoeca swinama Bu~Le H~ Bombus sp.
8 "7
(B)
chelicerae (1 pr.)
172
(T) (T')
22 sting apparatus 22 abdomens$ 28 whole ants$
-
<0-003/s .a. <0-035/abd . <0 "008/ant
(F)
10 sting apparatus
-
<0 "012/s.a.
(F) (F)
18 sting apparatus 16 sting apparatus
-
<0 "008/s.a. <0 "016/s.a.
C~
55 sting apparatus SS larvae$ 55 pupae$ SS adults$
-
0082/s.a . <0-03/larva 0-043/pupa 0-08/adult
-
(B)
SO pupal abdomens 50 adult abdomens 34 sting apparatus 18 sting apparatus 181arvae$ 18 pupae$ 11 sting apparatus 11 whole adults
204 138 16 625
0"004/abd. 0"28/abd. 0"81/s.a . 1 ~2/s.a . 0 "17/larva 0"16/pupa 1 "04/s .a. 0 "87/adult
0"8 103 712 1S
(NE) (I')
20 sting apparatus 40 heads 7 sting apparatus 15 sting apparatus$ 15 sting apparatus 45 heads$
-
1 "94/s .a. 0 "28/head 1 "29/s .a. 2 "74/s .a. 0"97/s .a. 0"03/head
1370§ -
(NE)
36 sting apparatus
-
0~02/s.a.
spines
41
-
(NE) ('I) (B) (B)
S"tuvcuaa C~~r~xPna.nx Automeris (ilustrls ~ (B)
1 "5/pr .
167
235
' Localities : (A)=Arizona: (B)=Brazil ; (i~=Florida ; (NE)=New England; ('T)=Trinidad, B.W.I. t Venom dissolved in 3 ml . 0"1N HCl ; 1 ml. conc. HCl added and S-HT estimated with spectofluorometer. $ Sample ptrserved in 95 per cent ETOH ; alcohol removed under reduced pressure ; sample and residue extracted in usual manner. § Estimated, assuming dry weight of one sting apparatus=2 mg.
170
JOHN H. WELSH and CAROLYN S. BATTY
ADAM and W>?1sS [1] extracted 5-HT from Leiurus venom by washing with 95 per cent acetone . The toxicity of the venom was unaffected. Since the sting of scorpions is usually very painful, those lacking 5-HT must contain some other pain-producing substance(s) . Spiders . With two exceptions, 5-HT was found in the venom, venom glands, or chelicerae of eight species of spiders. The two exceptions were Lasiodora klugü and Pamphobeteus roseus . The venoms of these two species were not extracted in the usual manner . Instead, they were dissolved in 3 ml. of 0 ~1 N HC1 and 1 ml. conc. HC1 was added. Large amounts of interfering substances) obscured the excitation and fluorescence peaks that characterize 5-HT. The possible presence of 5-HT in the venoms of L, klugü and P. roseus can be ascertained only by further study of their venoms. Ofmuch interest is the large amount of 5-HT that was found in the venom ofPhoneutria fera, the most poisonous of the Brazilian spiders [12]. The first two values (520 and 880 !~g 5-HT/gm . venom) were obtained by dissolving the venom in HC1 and omitting the extraction procedure. The last three values (1540, 2270 and 2650 fig . 5-HT/g.) were obtained at different times using samples of the same lot of pooled venom . The extent to which the variation is due to "experimental error" or to a lack of homogeneity within this batch of venom is not known. An adequate supply of venom of P. fera permitted us to confirm the presence of 5-HT by paper chromatography. Venom was spotted on paper and run with 5-HT and bufotenine in three different solvents : (a) n-butanol : acetic acid : H80 (60 :15 :25) ; (b) n-butanol : HC1 : HEO (100 :20 conc. HCl : Hs0 to saturation) ; (c) 95 parts of 95 ~ ethanol : 5 parts conc. ammonia . The developed chromatograms were sprayed with p-dimethylaminocinnamaldehyde . In each of the solvents, the venom gave only one indole spot which, in color and Rf value, was identical with 5-HT (Fig. 2). By comparing the size and color intensity of the venom spots with those of different concentrations of known 5-HT it was estimated that the pooled sample of P. fera contained approximately 2000 Egg 5-HT/g. This agrees well with the average for the last three values given in Table 2 which were obtained with the same lot of venom. Centipede . One specimen of the large Brazilian centipede, Scolopendra viridicornis, was available for study . Its chelicerae were removed, dried, and subsequently extracted . The very heavily chitinized jaws and exoskeleton of the chelicerae account for a large fraction of their dry weight. Therefore the actual 5-HT content of the venom glands must be much higher than that given for the whole chelicerae . Stinging ants and solitary wasps. Two Trinidad ants that produce very painful stings are Odontomachus haematodes and Stenomyrmex emarginatus. The first of these is a relatively large species and the sting apparatus could be readily removed from anaesthetized speci mens. Twenty-two of these were removed and dried, while entire abdomens, containing the sting apparatus, were removed from an equal number and placed in ethanol . No 5-HT was detectable in either sample. A sample of intact specimens of Stenomyrmex, preserved in ethanol, likewise yielded no detectable 5-HT. Solitary wasps and ants are closely related . It was therefore of interest to examine the sting apparatus of some solitary wasps. Material from three species was kindly provided by Dr. HOWARD EVANS. Extracts of all three species failed to show evidence for the presence of 5-HT using the fluorescence method .
sr
Bufot .
Ir
Bufot.
Img P Fero
sr
SHT
Ir SHT
FIG. Z. PAPER CHROMATOGRAM OF I mg. OF VENOM OF PI7071C1III'1ü fClü RUN WITH I AND S fig . OF BUFOTENINE AND OF S-HT. DEVELOPED FOR 6 ht'. IN BUTANOL - HCI - H,O.
5-Hydroxytryptamine Content of Some Arthropod Venoms and Venom-Containing Parts
171
Social wasps . Seven species or subspecies of social wasps were studied. From six of these, representing four genera, the sting apparatus was removed and examined for 5-HT content. Of the four genera, members of the genus Polybia are the smallest and are reported to give the least painful sting. The species of Polistes studied were larger than Polybia and their stings are sometimes quite painful . Vespa maculate and Synoeeca surinama are large wasps and their stings are known to produce severe pain. In fact, Synoeca is said to be the most feared of all of the wasps found in Trinidad. Unfortunately the weights of the sting apparatus of several of the species were not obtained . However, the 5-HT values per sting apparatus (or per abdomen) are low for Polybia, higher for Polistes, and that for the sting apparatus of Synoeca, preserved in ethanol, is the highest of all . Thus, there is an apparent correlation between the painfulness of the sting of these social wasps and the 5-HT content of the venom apparatus . The hornet, Vespa crabro, produces a very painful sting and its venom contains a record amount of 5-HT [7] . Since larvae, pupae, and adults were present in the nests of Polybia rejecta and Polistes versicolor, collections of each were made and assayed for their 5-HT content . As the nervous systems of arthropods had earlier been shown to contain very low levels of 5-HT [4], it would be not unreasonable to assume that any 5-HT found in entire individuals came from the venom glands and sacs. No 5-HT was detected in larvae ofP. rejecta and, ifpresent, its level is less than 003 P.g/larva. Pupae of this species yielded 0~043wg/pupa, and adults 0 ~08 hg/individual . This value for adults corresponds almost exactly with the value obtained for the sting apparatus of this species (0 082 hg/s .a.). Larvae and pupae of Polistes versicolor yielded equivalent amounts of 5-HT, while for adults a value of 0 ~87 wg/individual was obtained. This is to be compared with 1 ~04 Fig/sting apparatus for individuals collected in Brazil and 1 ~2 Wg/sting apparatus for individuals collected in Trinidad. Abdomens of adult Polybia occidentalis scutellatis yielded over 100 times as much 5-HT as did those of pupae of the same species. From these observations, it appears that adult wasps have a higher content of 5-HT than either of the immature stages, which is, perhaps, to be expected . The finding of a relatively large amount of 5-HT in heads of Polistes versicolor vulgates and of a much smaller amount in heads of Synoeca surinama seems clearly to indicate that the sting apparatus is not the sole source of 5-HT in these wasps. Bumble Bee and Stinging Caterpillar . The sting apparatus of an undetermined species of Bombes, a bumble bee, was found to contain less 5-HT than any of the wasps. The caterpillars of Automeris, a genus of moth, are known to produce a painful reaction when they come in contact with the skin. Automeris io is the most familiar offender in North America. One specimen of a Brazilian species of Automeris (probably ilustris) was available for study. Its branching spines were cut off close to the body and dried. On extraction and assay a very considerable amount of 5-HT was obtained. Since a large fraction of the total weight of these spines is represented by cuticle, the 5-HT content of the urticating fluid must be very high . DISCUSSION
Enough information concerning the occurrence of 5-HT in arthropod venoms is now available to permit a few tentative generalizations . It is present in venoms or venom containing parts of relatively few ofthe scorpions that have been examined. It appears that it may occur rather commonly in spider venoms. It has not been detected in the sting apparatus of certain ants and solitary wasps. All social wasps, thus far examined, have
172
JOHN H . WELSH and CAROLYN S . BATiY
been shown to have relatively large amounts of 5-HT in their venoms or venom containing parts. For a summary of the literature on the distribution of 5-HT in venoms of animals other than arthropods, and in urticant plants, one may refer to the review of ERSPAMER [13] . While 5-HT may be an important pain-producing factor when present in venoms and. thereby, play a defensive role, it is not the only such factor found in venoms, since acetylcholine, histamine, and kinins also are commonly present [6, 7]. All four substances have been shown to occur in the venom of Vespa crabro [7] . in a popular account of venoms and the causes of pain, KEELS [9] has said, "If 1 had been asked to concoct a really potent pain-producing brew consisting of substances of low molecular weight and of animal origin, the mixture found in wasp and hornet venom would have been at the top of my list." Since pain is produced by the depolarization of certain sensory nerve endings as the result of changes in permeability and ion movements, a more fundamental role of 5-HT, acetylcholine, histamine, and the kinins of venoms might be to increase the rate of absorp tion into the tissues and body fluids of the truly toxic components. That these are usually proteins is becoming increasingly clear, and many of them act by rapid immobilization of the animal receiving the venom . From the viewpoint of the venomous predator that feeds on paralyzed prey the rapid spread of the paralyzing factors) is obviously an advantage . We conclude, therefore, that 5-HT in arthropod venoms plays at least two possible roles, one in defence and the other in facilitating the distribution and penetration of the paralyzing components) to vulnerable sites. SUMMARY Venoms and venom-containing parts of a variety of American scorpions, spiders, ants. wasps (solitary and social), a poisonous centipede, and an urticant caterpillar were collected and dried or preserved in ethanol . 5-Hydroxytryptamine (5-HT) was extracted by the method developed at the National Institutes of Health and quantitatively estimated by the fluorescence method . Venoms or telsons of seven species of scorpions failed to yield amounts of 5-HT that could be detected by the method employed. Venoms or chelicerae ~of five species of spiders that were extracted in the usual manner all yielded appreciable amounts of 5-HT. Venom of Phoneutria fera gave the highest values . ranging up to about 2~5 mg/g dry venom. 5-HT was found in the chelicerae of the poisonous centipede, Scolopendra viridicornis . The stinging apparatus of two species of ants and three of solitary wasps failed to yield detectable amounts of 5-HT. The stinging apparatus of six species of social wasps all yielded relatively large amounts of 5-HT. Those wasps known to produce very painful stings gave the highest values per sting apparatus. The urticant spines of a caterpillar of the genus Automeris were found to contain considerable 5-HT. These results are discussed in relation to earlier observations on 5-HT in arthropod venoms and there are speculations concerning the possible roles of 5-HT in ven~m~ . especially a defensive role as a producer of pain .
5-Hydroxytryptamine Content of Some Arthropod Venoms and Venom-Containing Parts
173
Acknowledgements-This study was supported by a Fulbright Research Fellowship at the University of Sao Paulo, Brazil to J.H .W . and by Grant NB-00623 from the Institute of Neurological Diseases and Blindness, L1 .S. Public Health Service. One of us (J .H .W.) is greatly indebted to Prof. PAULO SAWAYA and his staffof the Department of Physiology, University of Sao Paulo, for the facilities and assistance that were provided . Collections of Trinidad material were made while J .H .W. enjoyed the hospitality of the field station of the New York Zoological Society at Sinlla . Dr. W. BticaeRL and his assistants at Butantan Institute kindly provided the venoms from Brazilian scorpions and spiders. Material from solitary wasps was collected by Dr . HOWARD EVANS. Identifications of wasps were made by Dr. J . C. BEQuAERr and of ants by Dr . W. L. BROWN. REFERENCES
ADAM, [i] lb;. R. and WEISS, C., Nature, 178, 421, 1956. [2] ADAM, b~. R. and WEISS, C., J. Exp. Bial. 35, 39, 1958 . [3] ADAM, F:. R. and WEISS, C., Nature, 183, 1398, 1959. [4] WEISit, ;I . H. and MOORHERD, M., J. Neurochen) ., 6, 146, 1960 . [5] ERSPAMER, V., Pharnracol. Rev ., 6, 425, 1954 . [6] JACQUES, R. and SCHACIirER, M., Brit . J. Pharn)acol., 9, 53, 1954 . [7] BHOOLA, K. D., CALLE, J. D. and SCHACH7ER, M., J. Physiol., 159, 167, 1961 . [8] ARMSrRONG, D., DRY, R. M. L., KEELE, C. A. and MARKHAM, J. W., J. Physiol., 120, 326, 1953 . (9] KEELI:, C;. A., New Scientist, 17, 396, 1963 . (10] BDGDANl3KI, D. F., PLECSCHER, A., BRODLE, B. B, and UDENFRIEND, $. ,J. Pharn)acol. exp. Therap., 117, 82, 1956 . [1l] BüCHERL, W., Arzneimit. - Forsch ., 5, 68, 1955 . [12] BücHFJtt ., W ., Arznein)it. - Forsch ., 6, 293, 1956 . [13] ERSPAMI:R, V., Prog. in Drug Research, 3, 151, 1961 .
Perganon Prees Ltd., Printed in Grcat Britain
5-HYDROXYTRYPTAMINE CONTENT OF SOME ARTHROPOD VENOMS AND VENOM-CONTAINING PARTS . JoxN H. WELSH and CAROLYN S. BATTY Biological Laboratories, Harvard University, Cambridge, Massachusetts Absfraá-The venom or venom-containing parts of 30 species of arthropods from North and South America were examined for their content of 5-hydroxytryptamine (5-HT) . The characteristic fluorescence of 5-HT was used in its identification and quantitative estimation . No S-HT was detected in venom or telsoes of 7 species of scorpions. Venoms of S spiders were found to contain 5-HT . No 5-HT was detected in the stinging apparatus of two species of ants and 3 species of solitary wasps. Relatively large amounts of 5-HT were found in the sting apparatus of all social wasps that were examined . Most S-HT was found in those wasps known to produce very painful stings. INTRODUCTION
S-HYDROXYTRYPÎ'AMINE (5-HT, serotonin, enteramine) has been found in the venom or venom apparatus of certain scorpions and social wasps and, in small amount, in bee venom (Table 1). The venom sacs of the hornet, Vespa crabro, have a higher content of 5-HT than any other known natural source [7]. 5-HT has been shown not to contribute to the toxcity of the venom of the scorpion, Leiurus quinquestriatus [1 ], but, when present in venoms, it may serve a defensive role due to its ability to produce severe pain [4, 8, 9]. It is also possible that 5-HT facilitates the uptake of the toxic components) of a venom by increasing the TABLE I .
SOME EARLIER OBSERVATIONS ON THE OCCURRENCE OF S-HT IN ARTHROPOD VENOMS AND VENOMCONTAININC PARTS
:species ArlclInoidea Lciurus quinquestriatus Buthutus minax Ha~i~"urus arizonensis Vejolis spirrigerus "V~:juvis sp ." Insceta Polislesgallica Vesper vulgaris Vesyrr crabro Apis ,~rrellijera >,
Venom or part venom venom telson
5-HT --wet ; d ^dry) Fig/g . (w-up to 40004 30-404 none detected SO~and 1384
venom apparatus ' venom ~ venom sacs (3 sets) venom apparatus
165
0 "7-0" 8/v .a . 3204 7,5004 14,7004 19,0004 21w 0"0005/v .a .
Ref. [l, 2, 3] [3] [4] [4] [4] [5] [6] I71 [4] [5]
16 6
JOHN H. WELSH and CAROLYN S. RATTY
permeability of cells, and increasing the rate of blood flow at the site of puncture and venom injection. Since a more complete knowledge of the distribution of 5-HT in arthropod venoms might permit more definite conclusions to be made concerning its possible roles, further observations appeared desirable. A visit to Brazil, by way of Trinidad, provided an opportunity for collecting venoms and venom containing parts of a variety of arthropods, including scorpions, spiders and wasps. These were returned to Harvard University for assay for their 5-HT content. The results, along with a few additional observations on North American species, will be reported in this paper. MATERIALS and METHODS
The species studied, and the regions from which they came, are given in Table 2. Those from Trinidad were collected in the vicinity of Simla, the field station of the New York Zoological Society, while those from Brazil were collected in the vicinity of Sao Paulo, or obtained from Butantan Institute. The venoms were obtained through the kind co-operation of Dr. W. Büc1~RL and his assistants at Butantan . They were released by electrical stimulation onto slips of preweighed aluminium foil and then dried under reduced. pressure. The slips of foil were TABLE
2.
HYDROXYTRYPTAMINE CONTENT OF VENOS, VENOM APPARATUS AND SOME OTHER PARTS OF VARIOUS ARTHROPODS
Species and Locality'
Venom or Part
DryWeight mg.
i`g. 5-HT per sample or part
5-HTisg./g.
Class Arachnoidea SCORPIONS
Tityus bahiensis
(B) (B) (T) (B) (B) (F) (A)
3 telsons (female 3 telsons (males) 3 telsons 2 telsons venom (9 animals)t venom (l animal) 1 telson 2 telsons
17 32 l7 <1 <1 5 8
<0~014/tel . <0 "014/tel. <0~014/tel . <0 "048/tel. <02/sample <0 "04/sample -
<3 <1 "5 ~3 < 12 <2
Tityus serrulatus Tityus trinitatus Tityus trivittatus Opisthocanthus cavaporun: Centruroides gracilis Vejovis spinigerus
(B) (B) (B) (B) (B)
venom (1 spider) venom (1 spider) venom (1 spider)t venom (3 spider)t venom (1 spider)
7 5 18 20 3
0"4/spi. 0 "7 spi. <0 " 8/spi. <0 " 16/spí . 0" l/spi .
58 140 33
(B)
venom (glands) (6 pr .)
13
0 "03/pr.
14
(B)
chelicerae (1 pr.) venom (3 spiders)t fi
4 3 5 6 5 S
2 "5/pr. 0"7/spi. 0 "8/spi. 2 "4/spi. -
520 880 1300 1540 2270 2650
SPIDERS
Family Therophcsidae Acanthoscurria atrox Acanthoscurria sternalis Lasiodora klugü Pamphobeteus roseus Pterinopelma vellutinunt Family Lycosidae Lycosa erythrognatlut Family Ctenidae Phoneutria fera ..
venom (many spiders)
5-Hyd~oxytryptamine Content of Some Arthropo3 Venoms and Venom-Containing Parts
Class Myriapode C~~$ Sonlopendra vlridlcornis Class Inxcta STtr~aznvc~ Arrrs Odontomachus haematodes Sttnomyrmex emarglnatus SOIITARY W~srs Family Mutt7lidae Darymutilla sp. Family Sphecidae Bembix texana Stictia Carolina
Soctu. W~srs Family Vespidae Polybia rejects Polybia occldentalis scutellaris Polistes juscatus Polistes verslcolor Polistes versicolor Polistes versicolor vulgates Vespa maculate Synoeca swinama Bu~Le H~ Bombus sp.
8 "7
(B)
chelicerae (1 pr.)
172
(T) (T')
22 sting apparatus 22 abdomens$ 28 whole ants$
-
<0-003/s .a. <0-035/abd . <0 "008/ant
(F)
10 sting apparatus
-
<0 "012/s.a.
(F) (F)
18 sting apparatus 16 sting apparatus
-
<0 "008/s.a. <0 "016/s.a.
C~
55 sting apparatus SS larvae$ 55 pupae$ SS adults$
-
0082/s.a . <0-03/larva 0-043/pupa 0-08/adult
-
(B)
SO pupal abdomens 50 adult abdomens 34 sting apparatus 18 sting apparatus 181arvae$ 18 pupae$ 11 sting apparatus 11 whole adults
204 138 16 625
0"004/abd. 0"28/abd. 0"81/s.a . 1 ~2/s.a . 0 "17/larva 0"16/pupa 1 "04/s .a. 0 "87/adult
0"8 103 712 1S
(NE) (I')
20 sting apparatus 40 heads 7 sting apparatus 15 sting apparatus$ 15 sting apparatus 45 heads$
-
1 "94/s .a. 0 "28/head 1 "29/s .a. 2 "74/s .a. 0"97/s .a. 0"03/head
1370§ -
(NE)
36 sting apparatus
-
0~02/s.a.
spines
41
-
(NE) ('I) (B) (B)
S"tuvcuaa C~~r~xPna.nx Automeris (ilustrls ~ (B)
1 "5/pr .
167
235
' Localities : (A)=Arizona: (B)=Brazil ; (i~=Florida ; (NE)=New England; ('T)=Trinidad, B.W.I. t Venom dissolved in 3 ml . 0"1N HCl ; 1 ml. conc. HCl added and S-HT estimated with spectofluorometer. $ Sample ptrserved in 95 per cent ETOH ; alcohol removed under reduced pressure ; sample and residue extracted in usual manner. § Estimated, assuming dry weight of one sting apparatus=2 mg.
170
JOHN H. WELSH and CAROLYN S. BATTY
ADAM and W>?1sS [1] extracted 5-HT from Leiurus venom by washing with 95 per cent acetone . The toxicity of the venom was unaffected. Since the sting of scorpions is usually very painful, those lacking 5-HT must contain some other pain-producing substance(s) . Spiders . With two exceptions, 5-HT was found in the venom, venom glands, or chelicerae of eight species of spiders. The two exceptions were Lasiodora klugü and Pamphobeteus roseus . The venoms of these two species were not extracted in the usual manner . Instead, they were dissolved in 3 ml. of 0 ~1 N HC1 and 1 ml. conc. HC1 was added. Large amounts of interfering substances) obscured the excitation and fluorescence peaks that characterize 5-HT. The possible presence of 5-HT in the venoms of L, klugü and P. roseus can be ascertained only by further study of their venoms. Ofmuch interest is the large amount of 5-HT that was found in the venom ofPhoneutria fera, the most poisonous of the Brazilian spiders [12]. The first two values (520 and 880 !~g 5-HT/gm . venom) were obtained by dissolving the venom in HC1 and omitting the extraction procedure. The last three values (1540, 2270 and 2650 fig . 5-HT/g.) were obtained at different times using samples of the same lot of pooled venom . The extent to which the variation is due to "experimental error" or to a lack of homogeneity within this batch of venom is not known. An adequate supply of venom of P. fera permitted us to confirm the presence of 5-HT by paper chromatography. Venom was spotted on paper and run with 5-HT and bufotenine in three different solvents : (a) n-butanol : acetic acid : H80 (60 :15 :25) ; (b) n-butanol : HC1 : HEO (100 :20 conc. HCl : Hs0 to saturation) ; (c) 95 parts of 95 ~ ethanol : 5 parts conc. ammonia . The developed chromatograms were sprayed with p-dimethylaminocinnamaldehyde . In each of the solvents, the venom gave only one indole spot which, in color and Rf value, was identical with 5-HT (Fig. 2). By comparing the size and color intensity of the venom spots with those of different concentrations of known 5-HT it was estimated that the pooled sample of P. fera contained approximately 2000 Egg 5-HT/g. This agrees well with the average for the last three values given in Table 2 which were obtained with the same lot of venom. Centipede . One specimen of the large Brazilian centipede, Scolopendra viridicornis, was available for study . Its chelicerae were removed, dried, and subsequently extracted . The very heavily chitinized jaws and exoskeleton of the chelicerae account for a large fraction of their dry weight. Therefore the actual 5-HT content of the venom glands must be much higher than that given for the whole chelicerae . Stinging ants and solitary wasps. Two Trinidad ants that produce very painful stings are Odontomachus haematodes and Stenomyrmex emarginatus. The first of these is a relatively large species and the sting apparatus could be readily removed from anaesthetized speci mens. Twenty-two of these were removed and dried, while entire abdomens, containing the sting apparatus, were removed from an equal number and placed in ethanol . No 5-HT was detectable in either sample. A sample of intact specimens of Stenomyrmex, preserved in ethanol, likewise yielded no detectable 5-HT. Solitary wasps and ants are closely related . It was therefore of interest to examine the sting apparatus of some solitary wasps. Material from three species was kindly provided by Dr. HOWARD EVANS. Extracts of all three species failed to show evidence for the presence of 5-HT using the fluorescence method .
sr
Bufot .
Ir
Bufot.
Img P Fero
sr
SHT
Ir SHT
FIG. Z. PAPER CHROMATOGRAM OF I mg. OF VENOM OF PI7071C1III'1ü fClü RUN WITH I AND S fig . OF BUFOTENINE AND OF S-HT. DEVELOPED FOR 6 ht'. IN BUTANOL - HCI - H,O.
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Social wasps . Seven species or subspecies of social wasps were studied. From six of these, representing four genera, the sting apparatus was removed and examined for 5-HT content. Of the four genera, members of the genus Polybia are the smallest and are reported to give the least painful sting. The species of Polistes studied were larger than Polybia and their stings are sometimes quite painful . Vespa maculate and Synoeeca surinama are large wasps and their stings are known to produce severe pain. In fact, Synoeca is said to be the most feared of all of the wasps found in Trinidad. Unfortunately the weights of the sting apparatus of several of the species were not obtained . However, the 5-HT values per sting apparatus (or per abdomen) are low for Polybia, higher for Polistes, and that for the sting apparatus of Synoeca, preserved in ethanol, is the highest of all . Thus, there is an apparent correlation between the painfulness of the sting of these social wasps and the 5-HT content of the venom apparatus . The hornet, Vespa crabro, produces a very painful sting and its venom contains a record amount of 5-HT [7] . Since larvae, pupae, and adults were present in the nests of Polybia rejecta and Polistes versicolor, collections of each were made and assayed for their 5-HT content . As the nervous systems of arthropods had earlier been shown to contain very low levels of 5-HT [4], it would be not unreasonable to assume that any 5-HT found in entire individuals came from the venom glands and sacs. No 5-HT was detected in larvae ofP. rejecta and, ifpresent, its level is less than 003 P.g/larva. Pupae of this species yielded 0~043wg/pupa, and adults 0 ~08 hg/individual . This value for adults corresponds almost exactly with the value obtained for the sting apparatus of this species (0 082 hg/s .a.). Larvae and pupae of Polistes versicolor yielded equivalent amounts of 5-HT, while for adults a value of 0 ~87 wg/individual was obtained. This is to be compared with 1 ~04 Fig/sting apparatus for individuals collected in Brazil and 1 ~2 Wg/sting apparatus for individuals collected in Trinidad. Abdomens of adult Polybia occidentalis scutellatis yielded over 100 times as much 5-HT as did those of pupae of the same species. From these observations, it appears that adult wasps have a higher content of 5-HT than either of the immature stages, which is, perhaps, to be expected . The finding of a relatively large amount of 5-HT in heads of Polistes versicolor vulgates and of a much smaller amount in heads of Synoeca surinama seems clearly to indicate that the sting apparatus is not the sole source of 5-HT in these wasps. Bumble Bee and Stinging Caterpillar . The sting apparatus of an undetermined species of Bombes, a bumble bee, was found to contain less 5-HT than any of the wasps. The caterpillars of Automeris, a genus of moth, are known to produce a painful reaction when they come in contact with the skin. Automeris io is the most familiar offender in North America. One specimen of a Brazilian species of Automeris (probably ilustris) was available for study. Its branching spines were cut off close to the body and dried. On extraction and assay a very considerable amount of 5-HT was obtained. Since a large fraction of the total weight of these spines is represented by cuticle, the 5-HT content of the urticating fluid must be very high . DISCUSSION
Enough information concerning the occurrence of 5-HT in arthropod venoms is now available to permit a few tentative generalizations . It is present in venoms or venom containing parts of relatively few ofthe scorpions that have been examined. It appears that it may occur rather commonly in spider venoms. It has not been detected in the sting apparatus of certain ants and solitary wasps. All social wasps, thus far examined, have
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been shown to have relatively large amounts of 5-HT in their venoms or venom containing parts. For a summary of the literature on the distribution of 5-HT in venoms of animals other than arthropods, and in urticant plants, one may refer to the review of ERSPAMER [13] . While 5-HT may be an important pain-producing factor when present in venoms and. thereby, play a defensive role, it is not the only such factor found in venoms, since acetylcholine, histamine, and kinins also are commonly present [6, 7]. All four substances have been shown to occur in the venom of Vespa crabro [7] . in a popular account of venoms and the causes of pain, KEELS [9] has said, "If 1 had been asked to concoct a really potent pain-producing brew consisting of substances of low molecular weight and of animal origin, the mixture found in wasp and hornet venom would have been at the top of my list." Since pain is produced by the depolarization of certain sensory nerve endings as the result of changes in permeability and ion movements, a more fundamental role of 5-HT, acetylcholine, histamine, and the kinins of venoms might be to increase the rate of absorp tion into the tissues and body fluids of the truly toxic components. That these are usually proteins is becoming increasingly clear, and many of them act by rapid immobilization of the animal receiving the venom . From the viewpoint of the venomous predator that feeds on paralyzed prey the rapid spread of the paralyzing factors) is obviously an advantage . We conclude, therefore, that 5-HT in arthropod venoms plays at least two possible roles, one in defence and the other in facilitating the distribution and penetration of the paralyzing components) to vulnerable sites. SUMMARY Venoms and venom-containing parts of a variety of American scorpions, spiders, ants. wasps (solitary and social), a poisonous centipede, and an urticant caterpillar were collected and dried or preserved in ethanol . 5-Hydroxytryptamine (5-HT) was extracted by the method developed at the National Institutes of Health and quantitatively estimated by the fluorescence method . Venoms or telsons of seven species of scorpions failed to yield amounts of 5-HT that could be detected by the method employed. Venoms or chelicerae ~of five species of spiders that were extracted in the usual manner all yielded appreciable amounts of 5-HT. Venom of Phoneutria fera gave the highest values . ranging up to about 2~5 mg/g dry venom. 5-HT was found in the chelicerae of the poisonous centipede, Scolopendra viridicornis . The stinging apparatus of two species of ants and three of solitary wasps failed to yield detectable amounts of 5-HT. The stinging apparatus of six species of social wasps all yielded relatively large amounts of 5-HT. Those wasps known to produce very painful stings gave the highest values per sting apparatus. The urticant spines of a caterpillar of the genus Automeris were found to contain considerable 5-HT. These results are discussed in relation to earlier observations on 5-HT in arthropod venoms and there are speculations concerning the possible roles of 5-HT in ven~m~ . especially a defensive role as a producer of pain .
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Acknowledgements-This study was supported by a Fulbright Research Fellowship at the University of Sao Paulo, Brazil to J.H .W . and by Grant NB-00623 from the Institute of Neurological Diseases and Blindness, L1 .S. Public Health Service. One of us (J .H .W.) is greatly indebted to Prof. PAULO SAWAYA and his staffof the Department of Physiology, University of Sao Paulo, for the facilities and assistance that were provided . Collections of Trinidad material were made while J .H .W. enjoyed the hospitality of the field station of the New York Zoological Society at Sinlla . Dr. W. BticaeRL and his assistants at Butantan Institute kindly provided the venoms from Brazilian scorpions and spiders. Material from solitary wasps was collected by Dr . HOWARD EVANS. Identifications of wasps were made by Dr. J . C. BEQuAERr and of ants by Dr . W. L. BROWN. REFERENCES
ADAM, [i] lb;. R. and WEISS, C., Nature, 178, 421, 1956. [2] ADAM, b~. R. and WEISS, C., J. Exp. Bial. 35, 39, 1958 . [3] ADAM, F:. R. and WEISS, C., Nature, 183, 1398, 1959. [4] WEISit, ;I . H. and MOORHERD, M., J. Neurochen) ., 6, 146, 1960 . [5] ERSPAMER, V., Pharnracol. Rev ., 6, 425, 1954 . [6] JACQUES, R. and SCHACIirER, M., Brit . J. Pharn)acol., 9, 53, 1954 . [7] BHOOLA, K. D., CALLE, J. D. and SCHACH7ER, M., J. Physiol., 159, 167, 1961 . [8] ARMSrRONG, D., DRY, R. M. L., KEELE, C. A. and MARKHAM, J. W., J. Physiol., 120, 326, 1953 . (9] KEELI:, C;. A., New Scientist, 17, 396, 1963 . (10] BDGDANl3KI, D. F., PLECSCHER, A., BRODLE, B. B, and UDENFRIEND, $. ,J. Pharn)acol. exp. Therap., 117, 82, 1956 . [1l] BüCHERL, W., Arzneimit. - Forsch ., 5, 68, 1955 . [12] BücHFJtt ., W ., Arznein)it. - Forsch ., 6, 293, 1956 . [13] ERSPAMI:R, V., Prog. in Drug Research, 3, 151, 1961 .