The effect of scorpion venom and pure toxins on the cockroach central nervous system

Toxicon, 1972, Vol. 10, pp. 39904. Pergamon Ptem. Printed iu Great Britain

THE EFFECT OF SCORPION VENOM AND PURE TOXINS ON THE COCKROACH CENTRAL NERVOUS SYSTEM V . D'A7EIJ.0* , E. ZLOTKIN§, F . MIRANDAt, S . LISSITZKY~

and S .

BETTINI *

'Department of Parasitology, Istituto Superiors di Sanità, Rome, Italy. tLaboratoire de Biochimie, Faculté de Médecine, Section Nord, Marseille, France . $Laboratoire de Biochemie Médicale, Faculté de Médecine, Marseille, France. §On leave of absence from the Department of Entomology and Venomous Animals, The Hebrew University of Jerusalem, Israel . (Acceptedfor publication 131anaary 1972)

Abstract-The "insect toxin" separated from the venom of the scorpion Androctonus australis Hector causes block of the induced afferent transynaptic response at the sixth abdominal ganglion of the cockroach Perlplaneta amerkana . This effect is similar to that obtained with the scorpion crude venom. The "mammal toxin" II also separated from the same venom, however, does not affect synaptic transmission . It appears that the toxicity of the "insect" and "mammal" toxins of the above venom is based on their specific affinity to the nervous systems of different groups of animals .

Ir HAS been

INTRODUCTION

shown that the rapid paralyzing and lethal effect of the venom of the scorpion Androctonus australis Hector and of six other scorpion venoms to blowfly larvae, is due to toxic proteins different from those lethal for mice (ZLOTKIN et al., 1971x, b ; 1972c) . The fly-larvae toxic protein ("insect toxin") (I'I) has recently been isolated from the venom ofA. australis and purified as a low molecular weight single chained protein cross-linked by four disulfide bridges (ZLOTKIN et al., 1972x) . This toxin has a strong paralyzing activity when injected into insects belongingto several species from different orders (ZLOTKIN et a1.,1972b) . The "mammal toxins" (MT) of A. australis have been isolated previously (MIRANDA and LISSTTZKY, 1958 ; MIItANDA et al., 1964 ; ROCHAr et al., 1967) and, recently, three toxins (named I, II, III) highly toxic to mice have been purified (MIItANDA et al., 1970). These materials were found to be inactive when applied to several insect species as well as to other arthropods (ZI,orKIN et al., 1971b; 1972b) . The discrimination between the "insect" and "mammal" toxins was based only on mortality and on paralysis tests. It was, therefore, deemed useful to investigate the specific mode of action of the two toxins at a neurophysiological level. MATERIALS AND METHODS

Toxic materials Crude venom of A. australis was obtained according to the method of MIRANDA et al. (1970) . The method of purification of "mammal toxin" II as well as its chemical-structural properties have been described by MIRANDA et al. (1970) and ROCIiAr et al. (1970) . Being 42 times more toxic than the crude venom, this toxin is the most potent "mammal toxin" so far obtained from scorpion venoms . The pure "insect toxin" was obtained by gel filtration TOXICON I971 Vo1.10.

399

V. n'AJELLO, E. ZLOTKIN, F. MIRANDA, S. LISSITZKY and S. BETT1Ni

40 0

followed by two steps of ion exchange chromatography, using the "contraction-paralysis" of blowfly larvae (ZLOTKIN et al., 1971 a) as a biological test for purification control . The final product was 267 times more potent than the crude venom (ZLOTKIN et al ., 1972x) . Neurophysiological techniques Solutions ofvenom or pure toxins were injected into the ventral side of the hind abdomen of male cockroaches by means of a device previously described (ZLOTKIN et al., 1971 a). Ventral nerve cords of one-month-old adult males of Periplaneta Americana were dissected free and treated according to n'AJEt.LO et al. (1969) . The cord was placed in a bath on a set of silver-silver chloride electrodes as shown in Fig. 1 . The nerves were stimulated with a standard square pulse of 125 V and 02 msec. The external recordings were obtained with a "Tektronix" storage oscilloscope with incorporated differential preamplifier Type 2A61 . The crude venom and the toxins were dissolved in a suitable saline solution (RoeneR and WEIANT, 1958) and applied by immersing the whole cord for different periods of time in the solution . When high concentrations of the "insect toxin" were used, the toxic solution was applied as a drop on the 6th abdominal ganglion and/or on the connectives . Giant fibers of the central nervous system of P. Americana originate from the 6th abdominal ganglion and run without interruption up to the third thoracic ganglion . The afferent cereal fibers reach the 6th abdominal ganglion where they make junctions with the giant neurones (ROEDER, 1953) . The effect of the venom and pure toxins was assayed both on the transynaptically evoked response (StlRl, Fig . 1) and on the axonal conduction of connectives (StaRI, Fig. 1), and on the cereal nerves (St8-Rg, Fig. 1).

st, R., FIG. I . SCHEMATIC PRFSENTATION OF THE PREPARATION.

I-6,

abdominal ganglia of the ventral cord; St, stimulating electrodes ; G, ground ; CN, cereal nerves .

R,

recoiling electrodes;

RESULTS

Paralyzing e,~`ect ofcrude venom andpure toxins Groups of 4-5 male cockroaches were injected with different amounts of crude venom as well as of pure toxins and the motor effects were noted. As shown in Table 1, both crude venom and pure insect toxins produced an almost immediate motor paralysis while no effect was noted in cockroaches injected with pure "mammal toxin" II. E,~`ect on nerve cord E,~ect of crude venom. Incubation of the nerve cord in a solution of 5 mg/ml of crude venom resulted in a burst of spontaneous activity at a frequency of 160-300 spike/sec and an amplitude of 0~8-2~0 mV, as recorded at RI (Fig. 2D) . This excitatory phenomenon TOXICON 1971 Vol. 10 .

Scorpion venom on cockroach nervous system

401

TABLE I. EFFECTS ON MOTOR ACrIVTfY HY IN1ECfING CRUDE VENOM AND PURE TOXIN3

Toxic material

Crude venom

"Insect toxin"~ "Mammal toxin"t (2)

Amount injected (~

Time to reach immobility (min)

50 75 100 150

no effect 4-7 1-2 immediate

0~5 I~0 2~0 10 20 30

3-10 1-3 immediate no effect no effect no effect

"Increase in toxicity as compared to that of crude venom for fty larvae = 267 times (ZLOTKIN et at., 1972a). tIncrease in toxicity as compared to that of crude venom for mouse = 42 times (MIRANDA et at., 1970).

preceded a gradual decrease (Fig. 2E) of the induced response (Stl-RI) and the final block (Fig . 2H) (a preparation was considered as "blocked" when no response was obtained by a 10 .fold increase of threshold amplitude) . This block was accompanied by disappearance of the spontaneous activity (Fig. 2C, F, G,1) . On the other hand, the response to direct stimulation whether ofthe connectives (St.~-R~ or the cereal nerves (Sta-R~ was only slightly affected (Figs. 2J and 3). In eight preparations, the block of transynaptic response evoked by cereal stimulation (StlR~ was obtained in an average time of 19 (SD~6~5) min. Rinsings of the nerve cord for 10-20 min resulted only in a partial recovery (Fig. 2L) which was preceded and accompanied by low amplitude burst of spontaneous activity (Fig. 2K). Incubation of a nerve cord preparation in a solution of 1 mg/ml of crude venom for periods of 300 min resulted in short bursts of spontaneous discharges not exceeding an amplitude of 0~6-0~8 mV followed by approximately 50 per cent decrease in the amplitude ofthe evoked transynaptic response (Stl-Rl). The amplitude as measured in five preparations decreased from an average value of 9~0 (SD~1 ~5) mV to a level of 4~2 (SD f2~9) mV. The directly evoked axonal response at the level of cereal nerves as well as of connectives (StsRI, Ste-R~ was not affected . E,~ect of "insect toxin" . When the nerve cord was treated with a solution of 50 wg/ml of the A. australis "insect toxin", the same sequence of events as described for the crude venom was obtained, i .e. the decrease and block of transynaptic response was preceded by bursts of spontaneous activity . With "insect toxin" the average time required for transynaptic block (Stl-RI), as calculated from eight preparations, was 175 (SD ~ 7~5) min . Rinsings of the cord resulted only in partial recovery . However, application of 500 wg/ml of "insect toxin" in two preparations resulted in an immediate burst of strong spontaneous activity followed in 1-2 min by a complete block of the transynaptic response. When this solution was applied to the connectives for 20 min, axonal conduction was only slightly affected . E~`ect of "mammal toxin" II. Incubation of the nerve cord in solutions of 500 and 1000 TOXICON 1972 Yol. 10.

402

V . D'AJELLO, E . ZLOTKIN, F. MIRANDA, S. LISSTTZKY and S . BETTINI

!~g/ml of"mammal toxin" II for periods ranging from 30 to 60 min did not induce any of the alterations caused by "insect toxin" or by crude venom, except for a decrease in the evoked transynaptic response from the initial mean value of 9~6 (SD ~ 3~6) mV to a level of 7~6 (SD ~ 1 ~5) mV as measured in eight preparations . In Fig. 4 are reproduced the induced response and spontaneous activity of the nerve cord incubated with "mammal toxin" II at a concentration of 1000 Wg/ml followed by incubation with "insect toxin" at a concentration of 50 Fig/ml . Responses of the CNS isolatedfrom envenomated animals Nine adult males of P. americana were injected with 200 ~g of crude venom. The insects

showed immediate motor paralysis, and only occasional movements of the cerci or antennae could be observed during the first 10 hr following injection. At different time intervals from injection the nerve cords were dissected free, rinsed for 10-15 min, and the responses to the evoked standard stimuli as well as spontaneous activity were recorded (see Table 2). TABLE 2. AMPLTT'UDE OF IIYDUCED RFSPONSES AND OF SPONTANEOUS ACTIVITY IN NERVE CORDS DLSSECTED FROM INSECrS PARALYZED HY THE INJECTION OF 2OO {~g OF CRUDE VENOM Time following venom injection (hr)

Conduction response on connectives (mV)

Transynaptic response (mV)

Spontaneous activity (mV)*

9-10

16 15 10

1 ~5 3~5 5~0

0~2 02 0~3-0~4

15-16

11 11 0

0~0 4~0 0~0

0~2-0~3 0~2 0~1

22-23

0 0 0

0~0 0~0 0~0

below 0~1 below 0~1

below 0~1

*Normal value, approx . 0~2 mV.

The average amplitude of a transynaptic response evoked by cercal stimulation, as calculated from 22 untreated preparations derived from normal animals, was 10 (SD ~ 3~4) mV and the average response amplitude of the directly-induced response on the connectives was 12 (SD ~ 3-1) mV. Table 2 shows clearly that in the nerve cords of paralyzed insects at 10 hr from injection conduction response as well as spontaneous activity remained unaffected while transynaptic response was only partially inhibited. Thus, it is evident that the CNS of paralyzed insects was still functional. DISCUSSION

The data presented indicate that the CNS of P. Americana is highly resistant to the venom of the scorpion A. australis . This is demonstrated by the fact that the nerve cords derived from paralyzed animals were still functional. The results suggest, therefore, that the quick paralysis obtained by injecting crude venom or "insect toxin" into the live cockroach is due to a peripheral action . TOXICON 1972 YoL 10 .

Before After

0.2 ImV 'H

50 m ore

I D2 mV H

50

meet .

I2 mV 2 m sac

I2mV H 2 m sec

I02 mV H 50 m sec

02 mV

12 mV

Omse

2 m sec

H

2. RESPONSE OF THE NERVE CORD TO APPLICATION OF CRUDE VENOM . response induced by cereal stimulation (St,-R,) before venom; (B) response induced by direct stimulation of connectives (St,-R,) before venom. (Note the shorter delay as compared to A, due to the shorter pathway and absence of synaptic transmission); (C) spontaneous activity (recorded form R,) before venom ; (D) spontaneous activity following 20 min of incubation in a solution of 5 mg/ml of crude venom; (E) transynaptic response induced by cereal stimulation (St,-R,) 2 min after D ; (F) spontaneous activity, 30 sec after E ; (G) spontaneous activity, immediately after F ; (H) block of transynaptic (St,-R,) response 30 sec after G ; (I) spontaneous activity, 2 min after H ; (J) response induced by direct stimulation of connectives immediately after I ; (K) after 10 min of rinsing; bursts of spontaneous activity preceding recovery ; (L) partial recovery of transynaptic response, immediately after K . FIG .

(A) Transynaptic

roxlcorv r9n var. io .

f.p . 402

FIG . 3. RESPONSE OF THE NERVE CURD TO APPLICATION OF CRUDE VENOM.

Recorded after25 min of incubation in 5 mg/ml solution of crudevenom. Uppertrace-response induced by direct stimulation of cereal nerves (St,-R,) ; central trace-disappearance of transynaptic response induced by cereal stimulation (StlR,); lower trace-response induced by direct stimulation of connectives (St,-R, ).

I 2mV

2 m sec

I~ H Q

m~eeG

I0.2 mV 50 m sec

mV 12 H~ 2 m sec

FIG.4. RESPONSE OF THE NERVE CORD TO HIGH CONCENTRATION OF "MAMMAL TOXIN" II FOLLOWED BY APPLICATION OF "INSECT TOXIN" .

At 02 msec response induced by direct stimulation of connectives (St,-R,) and at 2~1 msec transynaptic response induced by cereal stimulation (St,-R,) . Both obtained after 40 min incubation of the nerve cord in a solution of 1 mg/ml of "mammal toxin" II ; (B) spontaneous activity immediately after A ; (C) burst of spontaneous activity after 10 min of incubation in a solution of "insect toxin" of 50 ~g/ml ; (D) transynaptic response induced by cereal stimulation (Stl-R,) 2 min after C. (E) Upper trace : disappearance of transynaptic response induced by cereal stimulation. Lower trace : response induced by direct stimulation of connectives, 2 min after D. (A)

71~XICON 1971 Vot. 70

Scorpion venom on cockroach nervous system

403

Since high concentrations of the venom in vitro only slightly affect the axonal conduction of the connectives and cereal nerves, it is evident that the block of the response induced by cereal stimulation is due to a direct effect on the synaptic transmission at the sixth abdominal ganglion . The strong correlation between the blockage of synaptic transmission and the immediately preceding bursts of strong spontaneous activity indicate that the excitatory and blocking phenomena might be causally related to the same factor. The present data are in accordance with previous findings suggesting the impermeability of intact nerves to scorpion venom (DEL Pozo and AIVGUIANO, 1947 ; ADAM and WEISS, 1959 ; PARNAS arid RUSSELL, 1967 ; ZLOTKiN et al., 1970) and the excitatory effect of the venom at the synapse, as was demonstrated at the neuromuscular junction (DEL Pozo et al., 1966 ; BRAZIL in ADAM and WESS, 1966 ; PARNAS and RUSSELL, 1967 ; PARNAS et al., 1970). However, according to a recent study (PARNAS et al., 1970) the crude venom of the scorpion Leiurus quinquestriatus blocks axonal conduction at the locust nerve cord connectives and of the crab leg nerve. The discrepancy between these results and those obtained with other scorpion venoms on different axonal preparations should be sought, we believe, in the different nature of the venoms or test preparations . The "insect toxin" when applied at a concentration of 100 times lower than the crude venom [which corresponds to 2~5 times the equivalent amount of crude venom as estimated on the basis of their paralysis activity in fly larva (ZLOTKIN et al., 1972x) ] caused a block of the synaptic transmission similar to that produced by the crude venom. On the other hand, the "mammal toxin" when applied at a concentration 20 times higher than the "insect toxin" [which corresponds to 8 times the equivalent amount of crude venom as estimated on the basis of their toxicity to mice (IVIIRANDA et al., 1970)] was inactive . The same ratio between the activity of crude venom and of "insect toxin" as based on their effect on the synaptic transmission, applies also, to theirparalyzing activity on the whole animal (Table 2). In spite of the fact that the above paralyzing activity is not related to the effect on the CNS it appears that the two actions of the crude venom are mainly due to its "insect toxin" component. The increased spontaneous activity with typical bursts of impulses may be due to stimulation of the neurons' soma-axonal "trigger zone" where the firing impulses originate, as suggested in the case of Latrodectus venom action on the same cockroach preparation (n'A .IELLO et al., 1971). The high potency, rapidity of action and the paralytic nature of the effect of scorpion venoms and their pure toxins to mammals (ROCHAT et al., 1967; ZLOTKIN and SHULOV, 1969) as well as to arthropods (ZLOTKIN et al., 1971x ; 19726) suggests that in both groups of animals the effects are due to neurotoxicity. It may be assumed that the mammal and insect toxins have the ability to discriminate between analagous loci of action in different species of animals . By the use of a very simple and common preparation like the CNS of Periplaneta (which, however, cannot be identified as the natural target organ of the scorpion venom) it was demonstrated that the specific ability of a toxic protein to paralyze and kill insects is correlated with a specific affinity to the nervous system of an insect, as contrasted to a different toxic protein, derived from the same venom, which is unable to produce either of these phenomena. REFERENCES Ant, K. R. and WSSS, C. (1959) Scorpion venom. Z. Tropenmed. Parasit. 10, 334. BRAZIL, V. Discussion of the paper by An.~, K. R. and W~ss, C. (1966) Some aspects of the pharmacology of the venoms of African scorpions. Mems. Inst. Batantan Simp . Inter . 33 (2), 603. T'OSICON 1971 Vol. !0.

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n'AIELLO, V., MwaNI, F. and BETTINI, S. (1971) The effect of the venom of the Black Widow Spider Latrodectus mactans tredecimguttatus on the giant neurones of Pepiplaneta americarra. Toxicon 9, 103. n'e4JeI.LO, V., Mwuxo, A. and BETTINI, S. (1969) Effect of the venom of the BWS Latrodectus macta»s tredecimguttatus, on evoked action potential in the isolated nerve cord ofPeriplaneta americatra . Toxicon 7, 139. DEL Pozo, E. C. and ANGUIANO, L. G. (1947) Acciones del veneno del alacrân (Centruroides noxins Hoffmann y C. suffuses Pocock) sobre la actividad motors del mlisculo estriado . Revta. Inst. Salubr. Erlfer»r. Trap . (Mex .) 8, 231 . DEL Pozo, E. C., $ALAS, M . and PACHELO, P. (1966) Effects of scorpion venom at neuromuscular junction . Meets. I»st . Batsman Simp . Internat . 33, (3), 961 . MIRANDA, F. and LrssrrzKY, S. (1958) Purification de la toxine du venin de scorpion (Androctonus australis L.) . Biochim. Biophys. Acts 30, 217. MmANnA, F., ROCHAT, H. and LISSITZKY, S. (1964) Purifications des neurotoxines (scorpamines) d'Arrdractonus australis (L .) et de Bathos occltanus (Am.). Toxicon 2, 51 . MmwNnA, F., KUPEYAN, C., RocHwr, H., RocHwr, C. and LISSITZKY, S. (1970) Purification of animal neurotoxins : Isolation and characterization of eleven neurotoxins from the venoms of the scorpion Artdroctonus australis Hector, Buthus occitanus trutetanus and Lieurus quinqueslriatus quinquestriatus. Ear. J. Biochem. 16, 514. PARNAS, I. and RUSSELL, F. E. (1967) Effects of venoms on nerve, muscle and neuromuscular junction . In Anlrnal Toxins, p. 401, (RussELL, F. E. and SAIJNDER3, P. R., Eds.). Oxford : Pergamon Press. PARNA3, L, AVGAR, D. and $HULOV, A. (1970) Physiological effects of venom of Leiurus qulnquestriatus on neuromuscular systems of locust and crab. Toxicon 8, 67. ROCHAT, C., ROCHAT, H., MIRANDw, F. and LLSSIrzxY, S. (1967) Purification and some properties of the neurotoxins of Androctonus australis Hector. Biochemistry 6, 578. ROCHAT, H., ROCHAT, C., KUPEYAN, C., MIRANDA, F., LISSITZKY, S. and EDMAN, P. (1970) Scorpion neurotoxins : A family of homologous proteins . Febs Letters 10, 349. RoEnEe, K. D. (1953) Electric activity in nerves and ganglia. 1»sect Physiol. 67, 423. ROEDER, K. D. and WEIANT, E. A. (1958) Methods ajTesting Che»ricals on Insects. Vol. I ., (SHEPARD, H., Ed .) . Minneapolis : Burgess . ZLOrxnv, E. and SHULOV, A. S. (1969) Recent studies on the mode of action of scorpion neurotoxins. A review. Toxicon 7, 217. ZLOTKIN, E., BLONDHEIM, S. A. and $HULOV, A. S. (1970) Effect of the venom of scorpion Leiurusquinquestriatus on the tympanic nerve of the locust Locusts migratoria migratariaides. Toxicon 8, 47 . ZLOrxnv, E., FRAENKEL, G., MrnwranA, F. and LISSIrzxv, S. (1971a) The effect of scorpion venom on blowAy larvae -a new method for the evaluation of scorpion venoms potency. Toxicon 9, 1 . ZLOrxnv, E., MIRANDA, F., KUPEYAN, C. and LrssrrzKV, S. (19716) A new toxic protein in the venom of the scorpion Androctonus australis Hector. Toxicon 9, 9. ZLOrKIN, E., RocHwr, H., KUPEYAN, C., MIRANDA, F. and LISSITZKY, S. (1972) Purification and properties of the "insect toxin" from the venom of the scorpion Androctonus australis Hector. In press. ZLOTKIN, E., MIRANDA, F. and LISSITZKY, S. (19726) A factor toxic to crustacean in the venom of the scorpion Androctonus australis Hector. Toxicon 10, 2l 1 . ZLOTKIN, E., MiRANnA, F. and LISSITZKY, S. (1972c) Proteins in scorpion venoms toxic to mammals and insects Toxico» 10,207.

TOXICON1971 Yol.la.