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Action of the venom of Microbracon hebetor say on larvae and adults of Philosamia cynthia hübn
Comp. Biochem. Physiol., 1969, Vol. 28, pp. 603 to 618. Pergamon Press. Printed in Great Britain
ACTION OF THE VENOM OF M I C R O B R A C O N H E B E T O R SAY ON LARVAE AND ADULTS OF P H I L O S A M I A C Y N T H I A HUBN.* T. P I E K and E. E N G E L S Pharmacological Laboratory, University of Amsterdam, Polderweg 104, Amsterdam (O), The Netherlands
(Received 19June 1968) A b s t r a c t - - 1 . The venom of Microbracon hebetor affects the frequency of the spontaneous miniature potentials in flight muscle fibres of Philosamia cynthia. The amplitude of the miniature potentials is decreased to a much smaller extent. 2. After administration of Microbracon venom paralysis sets in earlier at a high level of neuromuscular activity than at a low level of activity. 3. The results of these experiments indicate that Microbracon venom may block the neuromuscular transmission at a presynaptic site.
INTRODUCTION THE VENOM produced by the braconid wasp Microbracon hebetor Say ( = Habrobracon juglandis Ashmead) causes paralysis of its natural prey, the larvae of the Lepidopterans Galleria mellonella, Ephestia kuhni~lla and Plodia interpunctella (Beard, 1952). As has been shown previously (Pick, 1966), larvae of Philosamia cynthia are also susceptible to Microbracon venom. Since nervous conduction is not abolished in paralysed parts of Philosamia larvae, and nerve action potentials can still be recorded in totally paralysed larvae of Galleria (Beard, 1952) and of Philosamia (Pick, 1966), it may be assumed that Microbracon venom has its paralysing effect either on the muscle fibres or on the neuromuscular junction. T h e fact that in paralysed larvae a contraction of the muscles can still be evoked by field stimulation with strong stimuli may indicate that the muscle fibres themselves remain reactive and that the paralysis is caused by an effect of Microbracon venom on the neuromuscular junction (Beard, 1952; Pick, 1966). T h e present paper describes further attempts to elucidate the mechanism of action of Microbracon venom. New experiments are presented to establish the absence of an effect on the muscle fibre membrane. T h e effect of the venom at the neuromuscular junction has been studied by an investigation of the action of the venom on the spontaneous miniature excitatory post-synaptic potentials (MEPSP's). In a previous paper (Pick, 1966) the results of studies of Microbracon venom on larvae of Philosamia cynthia were presented. Since the integumental muscles * Supported in part by the European Research Office, United States Army, Frankfurtam-Main, Germany. 603
604
T. PIEK AND E. ENGELS
of these larvae are easily d a m a g e d d u r i n g dissection, t h e m a j o r i t y of t h e experim e n t s d e s c r i b e d i n t h e p r e s e n t p a p e r were p e r f o r m e d o n the flight m u s c l e s of a d u l t m o t h s . T h e l o n g i t u d i n a l flight m u s c l e s are m o r e suitable t h a n t h e vertical m u s c l e s since t h e y are less easily d a m a g e d d u r i n g p r e p a r a t i o n . T h e flight m u s c l e s of l e p i d o p t e r a n s are of the s y n c h r o n o u s t y p e a n d are therefore c o m p a r a b l e to other i n t e g u m e n t a l muscles. MATERIALS AND METHODS Dried venom was prepared as described previously (Pick, 1966). T h e venom was dissolved in a K-rich insect saline containing per litre: NaC1, 9 m M ; KC1, 32 raM; MgC12, 60 raM; CaCI,, 5 m M ; and NaHCO3, 1 m M (modified after Clark & Harvey, 1965). Philosamia cynthia moths were bred in a greenhouse at 25°C and 60 per cent r.h. The larvae were fed on Ligustrum and Prunus leaves. For the experiments described in this paper, pupae were collected in November and December and stored at 5°C until they were needed in order to obtain adult moths. Larvae and moths reared in the autumn and winter were smaller than larvae and moths reared in spring and summer. Venom solutions were injected either into intact larvae or into the thorax of intact moths. I n dissected animals, the venom was added to the bathing saline which had an average flow rate of about 100/zl/min. For electrical stimulation of intact moths platinum electrodes, 250/z in diameter, were stuck laterally into the thorax. I n dissected moths similar electrodes were placed directly on the longitudinal flight muscle. The electrodes were isolated from ground. Dissection of the mesothorax, even from the ventral side of the insect, interferes with the system of antagonistic muscles controlling the wing beat. Nevertheless stimulation of the combined meso- and metathoracic ganglion results in a powerful wing beat and a visible contraction of the flight muscles. Stimulation of nerve II N I (nomenclature according to Ntiesch, 1957) results in a contraction of the large mesothoracic longitudinal flight-muscle only. Movements of the muscles were either noted or recorded. In order to record the muscular contractions a small pin, carrying a triangular piece of paper, was stuck into the muscle. Contractions of the muscle displaced the triangle in a light beam. The light beam was directed on a photodiode from which the changes in light intensity could be recorded on an oscilloscope. The electrical activity of single flight muscle fibres was recorded using intracellular capillary glass microelectrodes filled with 3 M KCI, with a resistance of 10-30 M~). In some experiments a positive (depolarizing) or negative (hyperpolarizing) pulse was passed across the muscle membrane, between one channel of a double-barrelled microelectrode and a bath electrode. The current through this channel was monitored on one trace of a dual-beam oscilloscope, while the resulting change in membrane potential was recorded from the second channel on the second beam (del Castillo et al., 1953). T h e voltage drop across a 50-k£) resistance was used to monitor the intraceIIularly applied stimulating currents. Intracellular spontaneous miniature postsynaptic potentials (MEPSP's) of flight muscle cells were recorded using a Tektronix 3A61 Amplifier containing a low pass filter with an upper 3 dB frequency limit at 60 c/s and an attenuation of 6 dB per octave. Under these circumstances the noise was about 10/xV. A 50 c/s artifact of about 20/xV was left. The MEPSP's could be distinguished easily from the 50 c/s by their shape. The amplitude of the miniature potentials could not be recorded accurately, since a filter was used and errors that may have resulted from attenuation of the potentials by the filter have not been compensated for. The amplitudes of the miniature potentials were measured from the point where they started from the resting phase to the peak voltage.
605
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The miniature activity was recorded for periods of 1 rain at different times before and after administration of Microbracon venom, RESULTS
a. General effects of Microbracon venom W h e n Philosamia cynthia larvae are injected with increasing concentrations of Microbracon venom, the time between the m o m e n t of injection and the appearance of total paralysis of the larvae decreases. T h e relation found between log venom concentration and log paralysis time is shown in Fig. 1. T h e lower time limit of
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FIG. 1. Dose-effect relation of Microbracon venom in larvae of Philosamia weighing about 3 g. The mean + S.E.M. of ten determinations is presented. P.U. = one Philosamia unit. The dose in number of venom organs/g body wt. as well as the paralysis time are plotted on a logarithmic scale. about 9 min probably represents the time necessary for mixing of the venom with the h e m o l y m p h and its penetration into the muscles. One Philosamia unit (1 P.U.) of Microbracon venom has been defined as the dose, injected per g body wt. that paralyses a Philosamia larva of about 3 g in 30 min at 25°C. T h i s unit is equivalent to about three venom organs obtained from recently killed wasps. In intact Philosamia moths the spontaneous wing beat is abolished within 30 rain after an intrathoracic injection of one P.U. of Microbracon venom. T h e
606
T. Ping AND E. ENGELS
susceptibility of the adults is about equal to that of the larvae. The venom appears to have no effect on the heart beat of either the larva or the adult moth. b. The effect of Microbracon venom on the mechanical responses of the flight muscles of intact moths and of dissected moths on extracellular stimulation Experiments were started using intact moths, in which the wing movements were noted after field stimulation of the thorax (see Materials and Methods). The flight muscles respond mechanically to stimulation at 1-1000 c/s. The rheobase of the longitudinal flight muscles (downward stroke of the wings) is about 50 per cent higher than that of the vertical flight muscles (upward stroke of the wings). In non-paralysed animals the chronaxie for both groups of muscles was 0.150.20 msec. In moths, paralysed by Microbracon venom, wing movements occurred only when a voltage was used for the field stimulation two to three times higher than that necessary for the maximal effect in non-paralysed muscles. At 1 c/s as well as at 100 c/s the chronaxie for the vertical muscles now varied from 1.1 to 1-3 msec and that for the longitudinal muscles from 2-9 to 3-4 msec. This markedly increased chronaxie may indicate that in paralysed moths the wing muscles no longer respond to indirect stimulation but still respond to direct stimulation. Further experiments, this time on dissected moths, showed that in nonparalysed preparations electrical stimulation of the longitudinal flight muscles results in a visible contraction, with a chronaxie of 0.2-0-6 msec. After administration of a high dose (20-30 P.U. per ml) of Microbracon venom a chronaxie of 2.9-3.1 msec was found. These data are in agreement with those observed in intact animals. c. The effect of Microbracon venom on the electrical responses of the muscle fibres to intracellular stimulation If long-lasting depolarizing pulses (100-150 msec) are applied intracellularly, the majority of the fibres in a given muscle do not produce an active response. However, a small number of fibres respond after a lag time that decreases with increasing depolarization (Fig. 2a). On repetition of the stimulation the reactivity of the membrane decreases while the response now often consists of a number of oscillations which fade rapidly (Fig. 2b). Similar reactions have been found by a number of authors in orthopterans (Cerf et aL 1959; Hill & Usherwood, 1961; Usherwood, 1962). The response of the Philosamia flight muscle fibres is always graded in amplitude, depending on the voltage applied. Inward current pulses hyperpolarize the muscle fibre and an approximately linear relationship between voltage and current is found. After changing the bathing fluid from normal saline to saline containing 20-30 P.U. of Microbracon venom/ml, the excitability of the fibres remains unchanged, up to at least 50 min after administration of the venom (Fig. 2c).
FIG. 2. T h e effect of Microbracon v e n o m on electrical responses of muscle fibres to long-lasting positive (depolarizing) pulses, a. R e s p o n s e of a fibre showing one single g r a d e d response, b. R e s p o n s e of the same fibre to a second series of stimulations. T h e response is n o w a series of u n d u l a t i o n s , c. R e s p o n s e of the same fibre 50 m i n after a d m i n i s t r a t i o n of Microbracon v e n o m (20 P . U . / m l ) . T h e zero potential line is m a r k e d with*. C a l i b r a t i o n : 20 m V a n d 1 0 m s e c per m a j o r division.
FIG. 4. S p o n t a n e o u s m i n i a t u r e p o t e n t i a l activity of a Philosamia flight muscle fibre. I, before, a n d I I - V , respectively, 2, 7, 15 a n d 25 m i n after a d m i n i s t r a t i o n of Microbracon v e n o m 30 P . U . / m l . N o t e the increase in f r e q u e n c y in I I a n d III, followed b y a decrease in IV a n d V, a n d also the s o m e w h a t decreased a m p l i t u d e in I V a n d V. C a l i b r a t i o n : 100/zV a n d 100 msec respectively.
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Figure 3 shows a representative example of the voltage-current relation of a single Philosamia flight muscle fibre before, during and after venom treatment. It
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Fro. 3. Membrane voltage-current diagrams for Philosamia flight muscle fibres. l , Before venom treatment; O, 30 min after administration of a Microbracon venom solution containing 20 P.U. per ml; &, 60 min after washing with saline. is apparent that administration of 20 P.U. of Microbracon venom did not affect the membrane resistance. T h e s e experiments confirm the notion that Microbracon venom does not affect the excitability of Philosamia flight muscle fibres.
608
T. Pink ANDE. ENGELS
d. The effect of Microbracon venom on the spontaneous miniature potentials in flight muscle fibres Most fibres of the Philosamia flight muscle have an effective membrane resistance of about 0.1 Mfl. In such fibres the resting potentials vary from 30 to 50 mV. Miniature excitatory postsynaptic potentials (MEPSP's) were observed in all normal resting muscle fibres (see Fig. 4 I). In all fibres studied, the maximum amplitude was about 150/~V. The lowest amplitude that could be measured with some accuracy was 12/~V. The median of the amplitudes over several periods of 1 min was determined and was found to vary from 30 to 50/~V. These MEPSP's are very small in comparison to those measured by Usherwood (1963) in orthopterans. In order to compare our method with that used by Usherwood, the MEPSP's of the flight muscles of the orthopteran Schistocerca gregaria were recorded. Amplitudes from 20 up to 1200/~V were observed, which is in agreement with the findings of Usherwood (1963). After the experiments described in the present paper were completed it was found that the amplitudes of the MEPSP's of the longitudinal flight muscles of Philosamia cynthia reared in summer were about twice as high as the amplitudes of moths reared in winter. The frequency distribution of the amplitudes (Fig. 5A) is similar to that found in other insects (Usherwood, 1963) and in frogs (Fatt& Katz, 1952). The effect of Microbracon venom on the frequency and amplitude of the MEPSP's was studied in twelve resting preparations. Figure 4 presents representative parts of records of one such experiment. These results are also presented as histograms in Figs. 5A, B, C and D. Figures 4 III and 5B show an initial increase in frequency at 7-8 min after administration of Microbracon venom (30 P.U. per ml). Moreover, a shift to somewhat lower amplitudes seems to have occurred, which is especially notable by the disappearance of potentials of more than 60/~V. About 15 rain after administration of the venom the frequency starts to decrease (Figs. 4 IV and 5C) and after 25 min the frequency has fallen below the control value (Figs. 4 V and 5D). In Fig. 6A the results of the same experiment are presented in a different way. In this figure the frequency as well as the median of the amplitudes have been plotted against time. Apart from the marked changes in frequency (an initial increase followed by a decrease), this figure also shows a small decrease in amplitude. In the experiment described in Fig. 6A the initial frequency was relatively high (about 300 potentials/min). In different experiments the initial frequencies may vary from about 50 to 500 potentials/min. Figure 6B shows an experiment in which the initial frequency was relatively low (70 potentials/min). Although Fig. 6B also shows the initial increase in frequency followed by a decrease, the time course of these frequency changes is slower. The relation between the initial MEPSP frequency and the time course of the changes in frequency caused by Microbracon venom is demonstrated more clearly by the six experiments presented in Table 1. In these experiments the moment of maximal frequency and the moment at which the frequency again had fallen to its initial value have
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F I G . 5. Histograms of miniature potentials recorded in one muscle fibre, before and after changing the bathing saline for a saline containing Microbracon venom 30 P.U./ml. A, Before treatment; B, 7-8 min after administration of venom, note the increase in frequency; C, 15-16 rain after administration of venom; D, 25-26 rain after administration of venom, note the decrease in frequency.
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FIG. 6. Effect of the venom of Microbracon (30 P.U./ml) on the frequency and the amplitude of spontaneous miniature potentials in Philosamia flight muscles. T h e m e d i a n of the amplitude (/tV) and the frequency of the potentials (No./min) has been plotted against time. A. A fibre with a relatively high initial frequency. B. A fibre with a relatively low initial frequency.
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been determined in a number of muscle fibres with different MEPSP frequencies. In the first four experiments the venom concentration was 30 P.U./ml, and in the last two experiments 60 P.U./ml. TABLE 1--THE COURSE
OF
RELATION BETWEEN THE I N I T I A L FREQUENCY OF THE
THE
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Experiment No.
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Venom Control value of Moment of Moment of return concentration MEPSP frequency maximal frequency to control value (P.U./ml) (No./min) (min) (rain)
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60-70 70-80 100-120 250-280 110-130 420-450
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36-39 26-28 19-23 18-22 10-11 3-4
Table 2, presenting three experiments on fibres with approximately identical frequencies, shows that the time course of the frequency changes can also be accelerated by increasing the venom concentration. TABLE 2--EFFECT
OF THE CONCENTRATION OF
Microbracon VENOM ON MEPSP's
THE T I M E COURSE OF
THE CHANGES I N FREQUENCY OF THE
Experiment No. 1 2 3
Venom Control value of Moment of Moment of return concentration MEPSP frequency maximal frequency to control value (P.U./ml) (No./min) (rain) (rain) 30 60 120
100-120 110-130 100-130
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20-22 10-11 7-8
It can be concluded from the experiments presented in Figs. 4, 5 and 6 that the main effect of Microbracon venom in resting muscles is an increase in frequency of the MEPSP's, followed by a decrease. An increase in frequency of MEPSP's can be caused by a number of unspecific effects among which are changes in tonicity and ionic composition of the bathing saline (Usherwood, 1961). Since the initial increase in frequency of MEPSP's, seen in the present experiments, might be caused by such an unspecific effect, not related to the paralysing action of the venom, the experiments were repeated with a "venom" inactivated by keeping the solution at about 20°C during 20 hr. By this, in itself very mild treatment, the paralysing potency of the venom was decreased from 30 P.U./ml to less than 0.01 P.U./ml. Table 3 shows that this inactivated venom had no effect on the frequency of the MEPSP's, whereas untreated venom administered to the same preparation caused the expected
612
T . P I E K AND E . ENGELS
increase in frequency followed by a gradual decline to zero. Venom inactivated by heating at 90°C during 10 rain also remained without effect on the frequency of the MEPSP's. In addition it was shown that changing the salt concentration by a factor of 2 did not affect the frequency of the MEPSP's. TABLE
3--EFFECT OF INACTIVATED Microbracon VENOM ON THE FREQUENCY OF MINIATURE POTENTIALS IN A LONGITUDINAL F L I G H T MUSCLE FIBRE OF Philosamia
Time
Frequency
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Bathing fluid Saline Inactivated venom
112 113 119 105 115 Untreated venom solution 158 154 65 36 13 0
Treatment of a venom solution of 30 P.U./ml for 20 hr at 20°C reduced its activity to less than 0.01 P.U./ml. Frequency in number of potentials/min. Time in rain.
e. The effect of Microbracon venom on the MEPSP-frequency, the EPSP-amplitude and the muscular contraction in indirectly stimulated flight muscles The effect of stimulation of the flight muscles on the time course of the frequency changes of the MEPSP's was at first studied in four preparations in which one flight muscle was stimulated at 1 c/s while the other was not stimulated. Both muscles were treated with 20 P.U. venom/ml saline. In a representative experiment the contractions of the stimulated muscle were abolished after 15 min, but the fibre still showed some miniature activity for a further 5 min. When the miniature activity had stopped completely, the microelectrode was placed in a fibre of the non-stimulated muscle. In this muscle miniature activity was still present at that moment but stopped about 26 min after administration of the venom, i.e. 6 min later than in the stimulated muscle. In the three other experiments miniature potential activity also persisted longer in the resting muscle than in the stimulated muscle. In six other preparations the amplitude of the EPSP's and the frequency of the MEPSP's, both recorded from the same muscle fibre, were recorded simultaneously with the contraction of the muscle. After a control period of 0.5 hr, in which the preparations were tested every 3-5 min for constancy of contraction, height, EPSP-amplitude and MEPSP-
VENOM OF M I C R O B R A C O N
613
frequency, the experiment was started. All experiments in which these three parameters were not constant within about 20 per cent during the control period were discarded. After this control period, the measurements were continued for a further 8 min with stimulation every 2-3 min after which the bathing saline was changed to a saline containing 20, 30 or 50 P.U. of Microbracon venom per ml respectively in the different experiments. Figure 7 (lower curves) shows three of the six experi250
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FIG. 7. Upper graphs: effect of three different Microbracon venom concentrations (20, 30 and 50 P.U./ml) on the MEPSP frequency in Philosarnia flight muscle fibres. T h e muscles were not stimulated. Lower graphs: effect of the same venom concentrations on the MEPSP-frequency, EPSP-amplitude and muscle contraction height. T h e muscle was stimulated indirectly every 2-3 rain, after a control period of 0"5 hr during which the muscle was stimulated every 3-5 rain.
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T . P I E K AND E . ENGELS
ments at the three different venom concentrations. The other three experiments showed similar results. At all three venom concentrations the contraction height of the muscle, the EPSP-amplitude and the MEPSP-frequency start to decrease at about the same moment and fall at about the same rate. However, in these experiments practically no initial increase in frequency of the MEPSP's was observed in contrast to the observations presented in Figs. 4, 5 and 6. The difference between the former experiments and the experiments presented in Fig. 7 is that in the latter experiments the muscles have been stimulated at regular intervals (every 3-5 min) during at least 0.5 hr, whereas in the former experiments the muscles were not stimulated at all. The upper part of Fig. 7 shows the results of three out of six other experiments in which the muscles were not stimulated and were treated with respectively 20, 30 or 50 P.U. of Microbracon venom/ml. Now the initial increase in frequency of the MEPSP's is present again. Moreover, it is apparent that the complete disappearance of the MEPSP's occurs later in the non-stimulated than in the stimulated muscles in agreement with the experiments mentioned before.
f. The effect of different levels o/functional activity on the time course of the paralysis caused by Microbracon venom 1. The effect of activity on the paralysis of flight muscles. In five dissected preparations the left and the right longitudinal flight muscles of Philosamia moths were stimulated at different frequencies via nerve II N1 (Ntiesch, 1957). The right muscle was stimulated at 1 c/s, the left muscle at 1 c/min. During this stimulation the bathing saline was replaced by saline containing either 30, 10, 3 or 1 P.U. of Microbracon venom/ml. A control preparation with saline alone showed no change in contraction height during 2 hr. In the preparations treated with venom, the right muscle, stimulated at 1 c/s, was paralysed considerably earlier than the left muscle, stimulated at 1 c/min. This was found at all four venom concentrations used (see Table 4). T A B L E 4----EFFECT OF INDIRECT STIMULATION OF THE LONGITUDINAL F L I G H T MUSCLES OF
Philosamia ON THE RATE OF PARALYSIS CAUSED BY DIFFERENT CONCENTRATIONS OF Microbracon VENOM
Time till total paralysis (min) Venom concentration (P.U./ml)
Right muscle (1 c/s)
Left muscle (1 c/min)
30 10 3 1 0
10 16 22 34
22 30 36 54 No paralysis
The right flight muscle is stimulated at 1 c/s, the left muscle at 1 e/min.
V E N O M OF .VIICROBRACON
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2. Effect of activity on the onset of paralysis of intact larvae. Three groups of ten larvae of Philosamia cynthia were used for this experiment. The first group was injected with 15/~1 saline/g body wt. During the first 15 min after the injection, the larvae were kept immobilized by means of a mixture of 80% C O ~ + 2 0 % 0 3. The larvae were then allowed to recover in air. Normal muscular activity returned about 5 min later and the animals showed a normal behaviour for at least a number of days. The second group was injected with 1 P.U. Microbracon venom in 50/zl saline/g body wt. This group was also immobilized by the C O ~ + O 2 mixture during the first 15 min after the injection. Normal muscular activity was also observed 5 min after exposure to air. Subsequently the larvae became paralysed, and paralysis was complete at 45.2 + 1.7 (S.E.M.) rain after the injection. The third group was also injected with 1 P.U. Microbracon venom in 50/zl. saline/g body wt. but was not immobilized. These animals were paralysed completely in 31-6 + 2.1 (S.E.M.) min after the injection. Therefore, the onset of paralysis was markedly retarded by immobilization of the larvae. DISCUSSION The observations that Galleria larvae paralysed by Microbracon venom show normal nerve action potentials (Beard, 1952) and that the conduction in the nervous system of paralysed larvae of Philosamia is not affected by this venom (Piek, 1966) indicate that the paralysis by Microbracon venom is not caused by a block of the nervous conduction. The observation that the muscles of paralysed larvae of Ephestia (Beard, 1952) as well as Galleria and Philosamia (Piek, 1966) are able to contract at high-voltage stimulation indicates that the excitability of the muscle fibres is not seriously blocked by Microbracon venom. Experiments described in the present paper show that Microbracon venom has no effect on either the effective membrane resistance or the response of the muscle fibre membrane to depolarizing pulses. This confirms the notion that the main effect of Microbracon venom is on the neuromuscular transmission. In order to examine the site of action in the neuromuscular junction, the effect of the venom on the MEPSP's was studied. A marked effect was observed on the frequency of the miniature potentials in flight muscle fibres of Philosamia, whereas the amplitude of these potentials was affected to a much smaller extent. Spontaneous miniature potentials were observed first in vertebrates (see Fatt & Katz, 1952) and afterwards in crustaceans (Dudel & Orkand, 1960) and Orthoptera (Usherwood, 1961). Experiments in vertebrates (Fatt & Katz, 1952; Del Castillo & Katz, 1954; Boyd & Martin, 1956) and in crustaceans (Dudel & Kuffler, 1961; Takeuehi & Takeuchi, 1966) show that the end-plate potentials are composed of a large number of smaller potentials presumably corresponding with the release of transmitter substance in a large number of quanta. The miniature end-plate
616
T. PIEKANDE. ENGELS
potentials presumably are the direct post-synaptic results of the spontaneous release of one or a few of these quanta of transmitter substance. Similar extensive arguments in support of the relation of spontaneous miniature potentials to the release of transmitter substance failin arthropods. Nevertheless, it is generally assumed that the miniature potentials in insects are indeed M E P S P ' s and are caused by spontaneous release of small amounts of transmitter substance (Aidley, 1967). Microbr~on venom predominantly affects the frequency of the MEPSP's. This indicates a presynaptic effect (Katz, 1962). Since the venom has much less effect on the amplitude of the MEPSP's, it is unlikely that the venom has a marked postsynaptic effect. It is remarkable that in resting muscles Mfcro~acon venom at firstincreases and later decreases the frequency of MEPSP's. Such an effect might be explained by assuming that the venom reduces the binding of the transmitter substance in the stores. This could result in an increased release of already-bound transmitter substance and thus explain the initialincrease in frequency of the MEPSP's. At the same time the storage of newly synthesized transmitter substance would be hampered, resulting eventually in a decreased release, thus explaining the subsequent fall in frequency of the MEPSP's. The fact that the time course of the changes in frequency of the MEPSP's is speeded up by activity of the neuromuscular system is in agreement with this assumption. The fact that the initial increase in frequency of the MEPSP's is absent in muscles that have been stimulated indirectly at regular intervals might possibly be explained by the following assumption. During rest transmitter substance accumulates and a certain proportion of the accumulated transmitter is present in a very labile binding and is therefore easily released by Microbracon venom. In the stimulated muscle this hypothetic excess store of transmitter is exhausted and therefore Microbracon venom is unable to release noticeable quantities of transmitter. Its action is now restricted to blocking the storage of newly synthesized transmitter substance at its appropriate site and thereby hampering a subsequent transmitter release. Obviously this is a highly speculative hypothesis. The notion that the effect of Microbracon venom on the MEPSP's is relevant to the paralysing action of this venom is strengthened by the observation that the onset of paralysis of the muscles is also speeded up by activity and especially by the observation that the decrease in frequency of the MEPSP's runs parallel to the decrease in amplitude of the EPSP and to the decrease in contraction height of the muscle. It was shown that a venom solution, inactivated under mild conditions, no longer affected the frequency of the MEPSP's. This may be an indication that the initial increase in frequency as well as the subsequent decrease is caused by a specific effect of the venom. Although increases in frequency have been observed to be caused by a number of unspecific factors in locusts (Usherwood, 1961), an increase of the salt concentration in the saline by a factor of 2 had no effect on the frequency of the MEPSP's in the Philosamia muscles.
V E N O M OF M 1 C R O B R A C O N
617
If it is nevertheless assumed that the increase in frequency is an unspecific effect and that the specific effect of the venom consists only of a decrease in frequency of the M E P S P ' s , the fact that activity of the neuromuscular system speeds up the decrease in frequency of the M E P S P ' s argues in favour of an inhibition of transmitter synthesis. However, an effect on the release mechanism cannot be excluded with certainty. One might consider the possibility that in resting muscles the decrease in frequency of the M E P S P ' s could be simply the result of an exhaustion of transmitter substance caused by the initial increase in frequency. However, this is unlikely since the quantity of transmitter substance responsible for the increased MEPSP-activity presumably is very small compared to the quantity of transmitter substance necessary for the formation of a normal EPSP. Since during stimulation with 1 c/min, a venom concentration of 30 P.U./ml gives rise to complete paralysis after about 20 min (see Table 4), the relatively small differences in time course shown in Fig. 6 cannot be explained by the threefold increase in frequency of the M E P S P ' s of relatively short duration. Moreover, in preparations that have been stimulated indirectly for some time, a decrease in frequency of the M E P S P ' s occurs without a preceding increase. At present the only conclusion that can be drawn with some confidence is that the venom of Microbracon hebetor Say probably exerts its action mainly on the presynaptic part of the neuromuscular transmission process.
Acknowledgements--I am indebted to Professor Dr. C. van der Meer for his many suggestions, his encouragement and criticism. I thank Dr. R. T. Simon Thomas and collaborators for the rearing of the insects used in this investigation and the preparation of venom gland extracts. REFERENCES AIDLEY D. J. (1967) The excitation of insect skeletal muscles. Adv. Insect Physiol. 4, 1-31. BEARDR. L. (1952) The toxicology of Habrobracon venom: a study of a natural insecticide. Bull. agric. Exp. Sta. No. 562. BoYD J. A. & MARTIN A. R. (1956) The end-plate potential in mammalian muscle. O7. Physiol. 132, 74-91. CERF J., GRUND~STH., HOYLEG. & McCAr~ F. V. (1959) The mechanism of dual responsiveness in muscle fibres of the grasshopper Romalea microptera. O7. gen. Physiol. 43, 377-395. CLARK R. M. & HARVEYW. R. (1965) Cellular membrane formation by plasmatocytes of diapausing Cecropia pupae, o7. Insect Physiol. 12, 561-568. DEL CASTILLOJ., H o ~ E G. & MACHNEX. (1953) Neuromuscular transmission in a locust. O7. Physiol. 121, 539-547. Dp.L CASTILLOJ. & KATZ B. (1954) Quantal components of the end-plate potential. O7. Physiol. 124, 560-573. DUDEL J. & KU~a~LF.RS. W. (1961) The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction. O7. Physiol. 155, 514-529. DUDEL J. & ORK~D R. K. (1960) Spontaneous potential changes at crayfish neuromuscular junctions. Nature, Lond. 186, 476-477. FATa" P. & KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. .7. Physiol. 117, 109-128.
618
T. PIEKAND E. ENGELS
HILL R. B. & USHERWOOD P. N. R. (1961) T h e action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria..7. Physiol. 157, 393-401. KATZ B. (1962) T h e transmission of impulse from nerve to muscle, and the subcellular unit of synaptic action. Proc. R. Soc. B 155, 455-479. NOESCI-I H. (1957) Die Morphologie des Thorax yon Telea polyphemus Cr. ( L e p i d . ) - - I I . Nervensystem. Zool. Jb. (Anat.) 75, 615-642. PIEK T. (1966) Site of action of venom of Microbracon hebetor Say (Braconidae, Hymenoptera), ft. Insect Physiol. 12, 561-568. TAKEOCHI A. & TAKEUCHI N. (1966) A study of the inhibitory action of y-amino butyric acid on neuromuscular transmission in the crayfish. J. Physiol. 183, 418-432. USHImWOOD P. N. R. (1961) Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. USHERWOOD P. N. R. (1962) T h e action of the alkaloid ryanodine on insect skeletal muscle. Comp. Biochem. Physiol. 6, 181-199. USHERWOOO P. N. R. (1963) Spontaneous miniature potentials from insect muscle fibres. J. Physiol. 196, 149-160.
ACTION OF THE VENOM OF M I C R O B R A C O N H E B E T O R SAY ON LARVAE AND ADULTS OF P H I L O S A M I A C Y N T H I A HUBN.* T. P I E K and E. E N G E L S Pharmacological Laboratory, University of Amsterdam, Polderweg 104, Amsterdam (O), The Netherlands
(Received 19June 1968) A b s t r a c t - - 1 . The venom of Microbracon hebetor affects the frequency of the spontaneous miniature potentials in flight muscle fibres of Philosamia cynthia. The amplitude of the miniature potentials is decreased to a much smaller extent. 2. After administration of Microbracon venom paralysis sets in earlier at a high level of neuromuscular activity than at a low level of activity. 3. The results of these experiments indicate that Microbracon venom may block the neuromuscular transmission at a presynaptic site.
INTRODUCTION THE VENOM produced by the braconid wasp Microbracon hebetor Say ( = Habrobracon juglandis Ashmead) causes paralysis of its natural prey, the larvae of the Lepidopterans Galleria mellonella, Ephestia kuhni~lla and Plodia interpunctella (Beard, 1952). As has been shown previously (Pick, 1966), larvae of Philosamia cynthia are also susceptible to Microbracon venom. Since nervous conduction is not abolished in paralysed parts of Philosamia larvae, and nerve action potentials can still be recorded in totally paralysed larvae of Galleria (Beard, 1952) and of Philosamia (Pick, 1966), it may be assumed that Microbracon venom has its paralysing effect either on the muscle fibres or on the neuromuscular junction. T h e fact that in paralysed larvae a contraction of the muscles can still be evoked by field stimulation with strong stimuli may indicate that the muscle fibres themselves remain reactive and that the paralysis is caused by an effect of Microbracon venom on the neuromuscular junction (Beard, 1952; Pick, 1966). T h e present paper describes further attempts to elucidate the mechanism of action of Microbracon venom. New experiments are presented to establish the absence of an effect on the muscle fibre membrane. T h e effect of the venom at the neuromuscular junction has been studied by an investigation of the action of the venom on the spontaneous miniature excitatory post-synaptic potentials (MEPSP's). In a previous paper (Pick, 1966) the results of studies of Microbracon venom on larvae of Philosamia cynthia were presented. Since the integumental muscles * Supported in part by the European Research Office, United States Army, Frankfurtam-Main, Germany. 603
604
T. PIEK AND E. ENGELS
of these larvae are easily d a m a g e d d u r i n g dissection, t h e m a j o r i t y of t h e experim e n t s d e s c r i b e d i n t h e p r e s e n t p a p e r were p e r f o r m e d o n the flight m u s c l e s of a d u l t m o t h s . T h e l o n g i t u d i n a l flight m u s c l e s are m o r e suitable t h a n t h e vertical m u s c l e s since t h e y are less easily d a m a g e d d u r i n g p r e p a r a t i o n . T h e flight m u s c l e s of l e p i d o p t e r a n s are of the s y n c h r o n o u s t y p e a n d are therefore c o m p a r a b l e to other i n t e g u m e n t a l muscles. MATERIALS AND METHODS Dried venom was prepared as described previously (Pick, 1966). T h e venom was dissolved in a K-rich insect saline containing per litre: NaC1, 9 m M ; KC1, 32 raM; MgC12, 60 raM; CaCI,, 5 m M ; and NaHCO3, 1 m M (modified after Clark & Harvey, 1965). Philosamia cynthia moths were bred in a greenhouse at 25°C and 60 per cent r.h. The larvae were fed on Ligustrum and Prunus leaves. For the experiments described in this paper, pupae were collected in November and December and stored at 5°C until they were needed in order to obtain adult moths. Larvae and moths reared in the autumn and winter were smaller than larvae and moths reared in spring and summer. Venom solutions were injected either into intact larvae or into the thorax of intact moths. I n dissected animals, the venom was added to the bathing saline which had an average flow rate of about 100/zl/min. For electrical stimulation of intact moths platinum electrodes, 250/z in diameter, were stuck laterally into the thorax. I n dissected moths similar electrodes were placed directly on the longitudinal flight muscle. The electrodes were isolated from ground. Dissection of the mesothorax, even from the ventral side of the insect, interferes with the system of antagonistic muscles controlling the wing beat. Nevertheless stimulation of the combined meso- and metathoracic ganglion results in a powerful wing beat and a visible contraction of the flight muscles. Stimulation of nerve II N I (nomenclature according to Ntiesch, 1957) results in a contraction of the large mesothoracic longitudinal flight-muscle only. Movements of the muscles were either noted or recorded. In order to record the muscular contractions a small pin, carrying a triangular piece of paper, was stuck into the muscle. Contractions of the muscle displaced the triangle in a light beam. The light beam was directed on a photodiode from which the changes in light intensity could be recorded on an oscilloscope. The electrical activity of single flight muscle fibres was recorded using intracellular capillary glass microelectrodes filled with 3 M KCI, with a resistance of 10-30 M~). In some experiments a positive (depolarizing) or negative (hyperpolarizing) pulse was passed across the muscle membrane, between one channel of a double-barrelled microelectrode and a bath electrode. The current through this channel was monitored on one trace of a dual-beam oscilloscope, while the resulting change in membrane potential was recorded from the second channel on the second beam (del Castillo et al., 1953). T h e voltage drop across a 50-k£) resistance was used to monitor the intraceIIularly applied stimulating currents. Intracellular spontaneous miniature postsynaptic potentials (MEPSP's) of flight muscle cells were recorded using a Tektronix 3A61 Amplifier containing a low pass filter with an upper 3 dB frequency limit at 60 c/s and an attenuation of 6 dB per octave. Under these circumstances the noise was about 10/xV. A 50 c/s artifact of about 20/xV was left. The MEPSP's could be distinguished easily from the 50 c/s by their shape. The amplitude of the miniature potentials could not be recorded accurately, since a filter was used and errors that may have resulted from attenuation of the potentials by the filter have not been compensated for. The amplitudes of the miniature potentials were measured from the point where they started from the resting phase to the peak voltage.
605
VENOM OF ~VIICROBRACON
The miniature activity was recorded for periods of 1 rain at different times before and after administration of Microbracon venom, RESULTS
a. General effects of Microbracon venom W h e n Philosamia cynthia larvae are injected with increasing concentrations of Microbracon venom, the time between the m o m e n t of injection and the appearance of total paralysis of the larvae decreases. T h e relation found between log venom concentration and log paralysis time is shown in Fig. 1. T h e lower time limit of
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FIG. 1. Dose-effect relation of Microbracon venom in larvae of Philosamia weighing about 3 g. The mean + S.E.M. of ten determinations is presented. P.U. = one Philosamia unit. The dose in number of venom organs/g body wt. as well as the paralysis time are plotted on a logarithmic scale. about 9 min probably represents the time necessary for mixing of the venom with the h e m o l y m p h and its penetration into the muscles. One Philosamia unit (1 P.U.) of Microbracon venom has been defined as the dose, injected per g body wt. that paralyses a Philosamia larva of about 3 g in 30 min at 25°C. T h i s unit is equivalent to about three venom organs obtained from recently killed wasps. In intact Philosamia moths the spontaneous wing beat is abolished within 30 rain after an intrathoracic injection of one P.U. of Microbracon venom. T h e
606
T. Ping AND E. ENGELS
susceptibility of the adults is about equal to that of the larvae. The venom appears to have no effect on the heart beat of either the larva or the adult moth. b. The effect of Microbracon venom on the mechanical responses of the flight muscles of intact moths and of dissected moths on extracellular stimulation Experiments were started using intact moths, in which the wing movements were noted after field stimulation of the thorax (see Materials and Methods). The flight muscles respond mechanically to stimulation at 1-1000 c/s. The rheobase of the longitudinal flight muscles (downward stroke of the wings) is about 50 per cent higher than that of the vertical flight muscles (upward stroke of the wings). In non-paralysed animals the chronaxie for both groups of muscles was 0.150.20 msec. In moths, paralysed by Microbracon venom, wing movements occurred only when a voltage was used for the field stimulation two to three times higher than that necessary for the maximal effect in non-paralysed muscles. At 1 c/s as well as at 100 c/s the chronaxie for the vertical muscles now varied from 1.1 to 1-3 msec and that for the longitudinal muscles from 2-9 to 3-4 msec. This markedly increased chronaxie may indicate that in paralysed moths the wing muscles no longer respond to indirect stimulation but still respond to direct stimulation. Further experiments, this time on dissected moths, showed that in nonparalysed preparations electrical stimulation of the longitudinal flight muscles results in a visible contraction, with a chronaxie of 0.2-0-6 msec. After administration of a high dose (20-30 P.U. per ml) of Microbracon venom a chronaxie of 2.9-3.1 msec was found. These data are in agreement with those observed in intact animals. c. The effect of Microbracon venom on the electrical responses of the muscle fibres to intracellular stimulation If long-lasting depolarizing pulses (100-150 msec) are applied intracellularly, the majority of the fibres in a given muscle do not produce an active response. However, a small number of fibres respond after a lag time that decreases with increasing depolarization (Fig. 2a). On repetition of the stimulation the reactivity of the membrane decreases while the response now often consists of a number of oscillations which fade rapidly (Fig. 2b). Similar reactions have been found by a number of authors in orthopterans (Cerf et aL 1959; Hill & Usherwood, 1961; Usherwood, 1962). The response of the Philosamia flight muscle fibres is always graded in amplitude, depending on the voltage applied. Inward current pulses hyperpolarize the muscle fibre and an approximately linear relationship between voltage and current is found. After changing the bathing fluid from normal saline to saline containing 20-30 P.U. of Microbracon venom/ml, the excitability of the fibres remains unchanged, up to at least 50 min after administration of the venom (Fig. 2c).
FIG. 2. T h e effect of Microbracon v e n o m on electrical responses of muscle fibres to long-lasting positive (depolarizing) pulses, a. R e s p o n s e of a fibre showing one single g r a d e d response, b. R e s p o n s e of the same fibre to a second series of stimulations. T h e response is n o w a series of u n d u l a t i o n s , c. R e s p o n s e of the same fibre 50 m i n after a d m i n i s t r a t i o n of Microbracon v e n o m (20 P . U . / m l ) . T h e zero potential line is m a r k e d with*. C a l i b r a t i o n : 20 m V a n d 1 0 m s e c per m a j o r division.
FIG. 4. S p o n t a n e o u s m i n i a t u r e p o t e n t i a l activity of a Philosamia flight muscle fibre. I, before, a n d I I - V , respectively, 2, 7, 15 a n d 25 m i n after a d m i n i s t r a t i o n of Microbracon v e n o m 30 P . U . / m l . N o t e the increase in f r e q u e n c y in I I a n d III, followed b y a decrease in IV a n d V, a n d also the s o m e w h a t decreased a m p l i t u d e in I V a n d V. C a l i b r a t i o n : 100/zV a n d 100 msec respectively.
607
VENOM OF M I C R O B R A C O N
Figure 3 shows a representative example of the voltage-current relation of a single Philosamia flight muscle fibre before, during and after venom treatment. It
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608
T. Pink ANDE. ENGELS
d. The effect of Microbracon venom on the spontaneous miniature potentials in flight muscle fibres Most fibres of the Philosamia flight muscle have an effective membrane resistance of about 0.1 Mfl. In such fibres the resting potentials vary from 30 to 50 mV. Miniature excitatory postsynaptic potentials (MEPSP's) were observed in all normal resting muscle fibres (see Fig. 4 I). In all fibres studied, the maximum amplitude was about 150/~V. The lowest amplitude that could be measured with some accuracy was 12/~V. The median of the amplitudes over several periods of 1 min was determined and was found to vary from 30 to 50/~V. These MEPSP's are very small in comparison to those measured by Usherwood (1963) in orthopterans. In order to compare our method with that used by Usherwood, the MEPSP's of the flight muscles of the orthopteran Schistocerca gregaria were recorded. Amplitudes from 20 up to 1200/~V were observed, which is in agreement with the findings of Usherwood (1963). After the experiments described in the present paper were completed it was found that the amplitudes of the MEPSP's of the longitudinal flight muscles of Philosamia cynthia reared in summer were about twice as high as the amplitudes of moths reared in winter. The frequency distribution of the amplitudes (Fig. 5A) is similar to that found in other insects (Usherwood, 1963) and in frogs (Fatt& Katz, 1952). The effect of Microbracon venom on the frequency and amplitude of the MEPSP's was studied in twelve resting preparations. Figure 4 presents representative parts of records of one such experiment. These results are also presented as histograms in Figs. 5A, B, C and D. Figures 4 III and 5B show an initial increase in frequency at 7-8 min after administration of Microbracon venom (30 P.U. per ml). Moreover, a shift to somewhat lower amplitudes seems to have occurred, which is especially notable by the disappearance of potentials of more than 60/~V. About 15 rain after administration of the venom the frequency starts to decrease (Figs. 4 IV and 5C) and after 25 min the frequency has fallen below the control value (Figs. 4 V and 5D). In Fig. 6A the results of the same experiment are presented in a different way. In this figure the frequency as well as the median of the amplitudes have been plotted against time. Apart from the marked changes in frequency (an initial increase followed by a decrease), this figure also shows a small decrease in amplitude. In the experiment described in Fig. 6A the initial frequency was relatively high (about 300 potentials/min). In different experiments the initial frequencies may vary from about 50 to 500 potentials/min. Figure 6B shows an experiment in which the initial frequency was relatively low (70 potentials/min). Although Fig. 6B also shows the initial increase in frequency followed by a decrease, the time course of these frequency changes is slower. The relation between the initial MEPSP frequency and the time course of the changes in frequency caused by Microbracon venom is demonstrated more clearly by the six experiments presented in Table 1. In these experiments the moment of maximal frequency and the moment at which the frequency again had fallen to its initial value have
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FIG. 6. Effect of the venom of Microbracon (30 P.U./ml) on the frequency and the amplitude of spontaneous miniature potentials in Philosamia flight muscles. T h e m e d i a n of the amplitude (/tV) and the frequency of the potentials (No./min) has been plotted against time. A. A fibre with a relatively high initial frequency. B. A fibre with a relatively low initial frequency.
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611
VENOM OF M I C R O B R A C O N
been determined in a number of muscle fibres with different MEPSP frequencies. In the first four experiments the venom concentration was 30 P.U./ml, and in the last two experiments 60 P.U./ml. TABLE 1--THE COURSE
OF
RELATION BETWEEN THE I N I T I A L FREQUENCY OF THE
THE
CHANGES
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FREQUENCY
AFTER
CONCENTRATIONS OF
Experiment No.
MEPSP's
ADMINISTRATION
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VENOM
Venom Control value of Moment of Moment of return concentration MEPSP frequency maximal frequency to control value (P.U./ml) (No./min) (min) (rain)
1 2 3 4 5 6
30 30 30 30 60 60
60-70 70-80 100-120 250-280 110-130 420-450
15-16 18-22 11-15 9-10 9-10 ?
36-39 26-28 19-23 18-22 10-11 3-4
Table 2, presenting three experiments on fibres with approximately identical frequencies, shows that the time course of the frequency changes can also be accelerated by increasing the venom concentration. TABLE 2--EFFECT
OF THE CONCENTRATION OF
Microbracon VENOM ON MEPSP's
THE T I M E COURSE OF
THE CHANGES I N FREQUENCY OF THE
Experiment No. 1 2 3
Venom Control value of Moment of Moment of return concentration MEPSP frequency maximal frequency to control value (P.U./ml) (No./min) (rain) (rain) 30 60 120
100-120 110-130 100-130
14-15 9-10 5-6
20-22 10-11 7-8
It can be concluded from the experiments presented in Figs. 4, 5 and 6 that the main effect of Microbracon venom in resting muscles is an increase in frequency of the MEPSP's, followed by a decrease. An increase in frequency of MEPSP's can be caused by a number of unspecific effects among which are changes in tonicity and ionic composition of the bathing saline (Usherwood, 1961). Since the initial increase in frequency of MEPSP's, seen in the present experiments, might be caused by such an unspecific effect, not related to the paralysing action of the venom, the experiments were repeated with a "venom" inactivated by keeping the solution at about 20°C during 20 hr. By this, in itself very mild treatment, the paralysing potency of the venom was decreased from 30 P.U./ml to less than 0.01 P.U./ml. Table 3 shows that this inactivated venom had no effect on the frequency of the MEPSP's, whereas untreated venom administered to the same preparation caused the expected
612
T . P I E K AND E . ENGELS
increase in frequency followed by a gradual decline to zero. Venom inactivated by heating at 90°C during 10 rain also remained without effect on the frequency of the MEPSP's. In addition it was shown that changing the salt concentration by a factor of 2 did not affect the frequency of the MEPSP's. TABLE
3--EFFECT OF INACTIVATED Microbracon VENOM ON THE FREQUENCY OF MINIATURE POTENTIALS IN A LONGITUDINAL F L I G H T MUSCLE FIBRE OF Philosamia
Time
Frequency
-5 0 5 10 15 20 25 30 35 40 45 50 60 70
111
THE
Bathing fluid Saline Inactivated venom
112 113 119 105 115 Untreated venom solution 158 154 65 36 13 0
Treatment of a venom solution of 30 P.U./ml for 20 hr at 20°C reduced its activity to less than 0.01 P.U./ml. Frequency in number of potentials/min. Time in rain.
e. The effect of Microbracon venom on the MEPSP-frequency, the EPSP-amplitude and the muscular contraction in indirectly stimulated flight muscles The effect of stimulation of the flight muscles on the time course of the frequency changes of the MEPSP's was at first studied in four preparations in which one flight muscle was stimulated at 1 c/s while the other was not stimulated. Both muscles were treated with 20 P.U. venom/ml saline. In a representative experiment the contractions of the stimulated muscle were abolished after 15 min, but the fibre still showed some miniature activity for a further 5 min. When the miniature activity had stopped completely, the microelectrode was placed in a fibre of the non-stimulated muscle. In this muscle miniature activity was still present at that moment but stopped about 26 min after administration of the venom, i.e. 6 min later than in the stimulated muscle. In the three other experiments miniature potential activity also persisted longer in the resting muscle than in the stimulated muscle. In six other preparations the amplitude of the EPSP's and the frequency of the MEPSP's, both recorded from the same muscle fibre, were recorded simultaneously with the contraction of the muscle. After a control period of 0.5 hr, in which the preparations were tested every 3-5 min for constancy of contraction, height, EPSP-amplitude and MEPSP-
VENOM OF M I C R O B R A C O N
613
frequency, the experiment was started. All experiments in which these three parameters were not constant within about 20 per cent during the control period were discarded. After this control period, the measurements were continued for a further 8 min with stimulation every 2-3 min after which the bathing saline was changed to a saline containing 20, 30 or 50 P.U. of Microbracon venom per ml respectively in the different experiments. Figure 7 (lower curves) shows three of the six experi250
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30
,, 40
o
FIG. 7. Upper graphs: effect of three different Microbracon venom concentrations (20, 30 and 50 P.U./ml) on the MEPSP frequency in Philosarnia flight muscle fibres. T h e muscles were not stimulated. Lower graphs: effect of the same venom concentrations on the MEPSP-frequency, EPSP-amplitude and muscle contraction height. T h e muscle was stimulated indirectly every 2-3 rain, after a control period of 0"5 hr during which the muscle was stimulated every 3-5 rain.
IO
2omin,
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T . P I E K AND E . ENGELS
ments at the three different venom concentrations. The other three experiments showed similar results. At all three venom concentrations the contraction height of the muscle, the EPSP-amplitude and the MEPSP-frequency start to decrease at about the same moment and fall at about the same rate. However, in these experiments practically no initial increase in frequency of the MEPSP's was observed in contrast to the observations presented in Figs. 4, 5 and 6. The difference between the former experiments and the experiments presented in Fig. 7 is that in the latter experiments the muscles have been stimulated at regular intervals (every 3-5 min) during at least 0.5 hr, whereas in the former experiments the muscles were not stimulated at all. The upper part of Fig. 7 shows the results of three out of six other experiments in which the muscles were not stimulated and were treated with respectively 20, 30 or 50 P.U. of Microbracon venom/ml. Now the initial increase in frequency of the MEPSP's is present again. Moreover, it is apparent that the complete disappearance of the MEPSP's occurs later in the non-stimulated than in the stimulated muscles in agreement with the experiments mentioned before.
f. The effect of different levels o/functional activity on the time course of the paralysis caused by Microbracon venom 1. The effect of activity on the paralysis of flight muscles. In five dissected preparations the left and the right longitudinal flight muscles of Philosamia moths were stimulated at different frequencies via nerve II N1 (Ntiesch, 1957). The right muscle was stimulated at 1 c/s, the left muscle at 1 c/min. During this stimulation the bathing saline was replaced by saline containing either 30, 10, 3 or 1 P.U. of Microbracon venom/ml. A control preparation with saline alone showed no change in contraction height during 2 hr. In the preparations treated with venom, the right muscle, stimulated at 1 c/s, was paralysed considerably earlier than the left muscle, stimulated at 1 c/min. This was found at all four venom concentrations used (see Table 4). T A B L E 4----EFFECT OF INDIRECT STIMULATION OF THE LONGITUDINAL F L I G H T MUSCLES OF
Philosamia ON THE RATE OF PARALYSIS CAUSED BY DIFFERENT CONCENTRATIONS OF Microbracon VENOM
Time till total paralysis (min) Venom concentration (P.U./ml)
Right muscle (1 c/s)
Left muscle (1 c/min)
30 10 3 1 0
10 16 22 34
22 30 36 54 No paralysis
The right flight muscle is stimulated at 1 c/s, the left muscle at 1 e/min.
V E N O M OF .VIICROBRACON
615
2. Effect of activity on the onset of paralysis of intact larvae. Three groups of ten larvae of Philosamia cynthia were used for this experiment. The first group was injected with 15/~1 saline/g body wt. During the first 15 min after the injection, the larvae were kept immobilized by means of a mixture of 80% C O ~ + 2 0 % 0 3. The larvae were then allowed to recover in air. Normal muscular activity returned about 5 min later and the animals showed a normal behaviour for at least a number of days. The second group was injected with 1 P.U. Microbracon venom in 50/zl saline/g body wt. This group was also immobilized by the C O ~ + O 2 mixture during the first 15 min after the injection. Normal muscular activity was also observed 5 min after exposure to air. Subsequently the larvae became paralysed, and paralysis was complete at 45.2 + 1.7 (S.E.M.) rain after the injection. The third group was also injected with 1 P.U. Microbracon venom in 50/zl. saline/g body wt. but was not immobilized. These animals were paralysed completely in 31-6 + 2.1 (S.E.M.) min after the injection. Therefore, the onset of paralysis was markedly retarded by immobilization of the larvae. DISCUSSION The observations that Galleria larvae paralysed by Microbracon venom show normal nerve action potentials (Beard, 1952) and that the conduction in the nervous system of paralysed larvae of Philosamia is not affected by this venom (Piek, 1966) indicate that the paralysis by Microbracon venom is not caused by a block of the nervous conduction. The observation that the muscles of paralysed larvae of Ephestia (Beard, 1952) as well as Galleria and Philosamia (Piek, 1966) are able to contract at high-voltage stimulation indicates that the excitability of the muscle fibres is not seriously blocked by Microbracon venom. Experiments described in the present paper show that Microbracon venom has no effect on either the effective membrane resistance or the response of the muscle fibre membrane to depolarizing pulses. This confirms the notion that the main effect of Microbracon venom is on the neuromuscular transmission. In order to examine the site of action in the neuromuscular junction, the effect of the venom on the MEPSP's was studied. A marked effect was observed on the frequency of the miniature potentials in flight muscle fibres of Philosamia, whereas the amplitude of these potentials was affected to a much smaller extent. Spontaneous miniature potentials were observed first in vertebrates (see Fatt & Katz, 1952) and afterwards in crustaceans (Dudel & Orkand, 1960) and Orthoptera (Usherwood, 1961). Experiments in vertebrates (Fatt & Katz, 1952; Del Castillo & Katz, 1954; Boyd & Martin, 1956) and in crustaceans (Dudel & Kuffler, 1961; Takeuehi & Takeuchi, 1966) show that the end-plate potentials are composed of a large number of smaller potentials presumably corresponding with the release of transmitter substance in a large number of quanta. The miniature end-plate
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potentials presumably are the direct post-synaptic results of the spontaneous release of one or a few of these quanta of transmitter substance. Similar extensive arguments in support of the relation of spontaneous miniature potentials to the release of transmitter substance failin arthropods. Nevertheless, it is generally assumed that the miniature potentials in insects are indeed M E P S P ' s and are caused by spontaneous release of small amounts of transmitter substance (Aidley, 1967). Microbr~on venom predominantly affects the frequency of the MEPSP's. This indicates a presynaptic effect (Katz, 1962). Since the venom has much less effect on the amplitude of the MEPSP's, it is unlikely that the venom has a marked postsynaptic effect. It is remarkable that in resting muscles Mfcro~acon venom at firstincreases and later decreases the frequency of MEPSP's. Such an effect might be explained by assuming that the venom reduces the binding of the transmitter substance in the stores. This could result in an increased release of already-bound transmitter substance and thus explain the initialincrease in frequency of the MEPSP's. At the same time the storage of newly synthesized transmitter substance would be hampered, resulting eventually in a decreased release, thus explaining the subsequent fall in frequency of the MEPSP's. The fact that the time course of the changes in frequency of the MEPSP's is speeded up by activity of the neuromuscular system is in agreement with this assumption. The fact that the initial increase in frequency of the MEPSP's is absent in muscles that have been stimulated indirectly at regular intervals might possibly be explained by the following assumption. During rest transmitter substance accumulates and a certain proportion of the accumulated transmitter is present in a very labile binding and is therefore easily released by Microbracon venom. In the stimulated muscle this hypothetic excess store of transmitter is exhausted and therefore Microbracon venom is unable to release noticeable quantities of transmitter. Its action is now restricted to blocking the storage of newly synthesized transmitter substance at its appropriate site and thereby hampering a subsequent transmitter release. Obviously this is a highly speculative hypothesis. The notion that the effect of Microbracon venom on the MEPSP's is relevant to the paralysing action of this venom is strengthened by the observation that the onset of paralysis of the muscles is also speeded up by activity and especially by the observation that the decrease in frequency of the MEPSP's runs parallel to the decrease in amplitude of the EPSP and to the decrease in contraction height of the muscle. It was shown that a venom solution, inactivated under mild conditions, no longer affected the frequency of the MEPSP's. This may be an indication that the initial increase in frequency as well as the subsequent decrease is caused by a specific effect of the venom. Although increases in frequency have been observed to be caused by a number of unspecific factors in locusts (Usherwood, 1961), an increase of the salt concentration in the saline by a factor of 2 had no effect on the frequency of the MEPSP's in the Philosamia muscles.
V E N O M OF M 1 C R O B R A C O N
617
If it is nevertheless assumed that the increase in frequency is an unspecific effect and that the specific effect of the venom consists only of a decrease in frequency of the M E P S P ' s , the fact that activity of the neuromuscular system speeds up the decrease in frequency of the M E P S P ' s argues in favour of an inhibition of transmitter synthesis. However, an effect on the release mechanism cannot be excluded with certainty. One might consider the possibility that in resting muscles the decrease in frequency of the M E P S P ' s could be simply the result of an exhaustion of transmitter substance caused by the initial increase in frequency. However, this is unlikely since the quantity of transmitter substance responsible for the increased MEPSP-activity presumably is very small compared to the quantity of transmitter substance necessary for the formation of a normal EPSP. Since during stimulation with 1 c/min, a venom concentration of 30 P.U./ml gives rise to complete paralysis after about 20 min (see Table 4), the relatively small differences in time course shown in Fig. 6 cannot be explained by the threefold increase in frequency of the M E P S P ' s of relatively short duration. Moreover, in preparations that have been stimulated indirectly for some time, a decrease in frequency of the M E P S P ' s occurs without a preceding increase. At present the only conclusion that can be drawn with some confidence is that the venom of Microbracon hebetor Say probably exerts its action mainly on the presynaptic part of the neuromuscular transmission process.
Acknowledgements--I am indebted to Professor Dr. C. van der Meer for his many suggestions, his encouragement and criticism. I thank Dr. R. T. Simon Thomas and collaborators for the rearing of the insects used in this investigation and the preparation of venom gland extracts. REFERENCES AIDLEY D. J. (1967) The excitation of insect skeletal muscles. Adv. Insect Physiol. 4, 1-31. BEARDR. L. (1952) The toxicology of Habrobracon venom: a study of a natural insecticide. Bull. agric. Exp. Sta. No. 562. BoYD J. A. & MARTIN A. R. (1956) The end-plate potential in mammalian muscle. O7. Physiol. 132, 74-91. CERF J., GRUND~STH., HOYLEG. & McCAr~ F. V. (1959) The mechanism of dual responsiveness in muscle fibres of the grasshopper Romalea microptera. O7. gen. Physiol. 43, 377-395. CLARK R. M. & HARVEYW. R. (1965) Cellular membrane formation by plasmatocytes of diapausing Cecropia pupae, o7. Insect Physiol. 12, 561-568. DEL CASTILLOJ., H o ~ E G. & MACHNEX. (1953) Neuromuscular transmission in a locust. O7. Physiol. 121, 539-547. Dp.L CASTILLOJ. & KATZ B. (1954) Quantal components of the end-plate potential. O7. Physiol. 124, 560-573. DUDEL J. & KU~a~LF.RS. W. (1961) The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction. O7. Physiol. 155, 514-529. DUDEL J. & ORK~D R. K. (1960) Spontaneous potential changes at crayfish neuromuscular junctions. Nature, Lond. 186, 476-477. FATa" P. & KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. .7. Physiol. 117, 109-128.
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HILL R. B. & USHERWOOD P. N. R. (1961) T h e action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria..7. Physiol. 157, 393-401. KATZ B. (1962) T h e transmission of impulse from nerve to muscle, and the subcellular unit of synaptic action. Proc. R. Soc. B 155, 455-479. NOESCI-I H. (1957) Die Morphologie des Thorax yon Telea polyphemus Cr. ( L e p i d . ) - - I I . Nervensystem. Zool. Jb. (Anat.) 75, 615-642. PIEK T. (1966) Site of action of venom of Microbracon hebetor Say (Braconidae, Hymenoptera), ft. Insect Physiol. 12, 561-568. TAKEOCHI A. & TAKEUCHI N. (1966) A study of the inhibitory action of y-amino butyric acid on neuromuscular transmission in the crayfish. J. Physiol. 183, 418-432. USHImWOOD P. N. R. (1961) Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. USHERWOOD P. N. R. (1962) T h e action of the alkaloid ryanodine on insect skeletal muscle. Comp. Biochem. Physiol. 6, 181-199. USHERWOOO P. N. R. (1963) Spontaneous miniature potentials from insect muscle fibres. J. Physiol. 196, 149-160.