338 ENTOMO1,OC;IA SINICA Volume 3 , Number 4, 1996, pp. 338-344

PRELIMINARY OBSERVATION OF TOXICOLOGICAL EFFECTS OF RHODOJAPONINS AND TOOSENDANIN ON NEUROMUSCULAR TRANSMISSION OF LARVAL DROSOPHZLA MELANGASTER MEIGEN

Jianhua Xu Institute of Biophysics and Biochemistry, Huazhong L’niversity of Science and Technology, Wuhan 430074, China

Anxi Liu” and Zhizhen Shang2’ Department of Biology” and Institute o f Elemento-Organic Chemistry” , Nankai University, Tianjin 300071, China (Received Feb. 15, 1996; accepted Apr. 8 , 1996)

Abstract With microeletrode intracellular recording technique, the three rhodojaponins and toosen- danin, which had been extracted and purified from two insecticidal plants, Rhododendron molle and Melia toosendan, respectively, were examined for their toxicological effects on the neuromuscular excita- tory junction potentials (EJPS) in the ventralateral longitudinal muscle cells of the third instar larval Drosophrlu melungaster Meigen. All of them were found to inhibit and eventually block the EJPS, and the time for blockage decreased with higher concentrations. Comparison among the three rhodojaponins showed an inhibitory activity sequence of rhdojaponin B >rhodojaponin 1 >rhodojaponin V . This pre- liminary observation suggested that the disruption of neuromuscular transmission might contribute to the fatal actions of these botanic insecticides.

Key words Meigen

rhodojaponins , toosendanin, neuromuscular transmission, Drosophila melungaster

1 INTRODUCTION

Flowers and leaves of Rhododendron molle G. and Don (Ericaceae) have long been used in southern China, and were reported to be strongly toxic against many insect pests including brown plant hopper , rice yellow stem borer, cabbage worm, the ear-cutting caterpillar, soil insect pests, aphids and mites (Shang et al. 1990). From these plant ma- terials, three insecticidal diterpenes, i. e. , rhodojaponin 1 (R- H ) rhodojaponin IU ( R-

BI ) , and rhodojaponin V (R- V 1 , have been extracted and purified (Shang et al. 1990). These results were confirmed by Klocke et al. (1 991 ) . The rhodojaponins were found to be both contact/stomach toxic and antifeedant for larvae of Chilo suppressalis , Spodoptwa frugiperda and the Colorado potato beetle , Leptinotarsa decemlineata (Shang et al. 1990, Klocke et al. 1991). For the mode of action of these botanic insecticides, however , no studies has been made yet.

Jianhua Xu et a1 : Rhodojaponins and toosendanin on neuromuscular transmission 339

On the other hand, a greater amount of knowledge has accumulated about the toxi- cology of toosendanin, a triterpene which has been extracted from Melia toosendan Sieb et Zec . Toosendanin was found to be stomach toxic , growth - regulating and antifeedant against many important Lepidopterous pests, such as Ostriniu furnacdis and Leucania separatu (Chiu and Zhang 1987 ), Electrophysiolagical studies have demonstrated that toosendanin could inhibit neuromuscular transmission in rat diaphgram nerve-diaphgram muscle (Shi et al. 1985) as well as inhibit synaptic transmission in cockroach abdominal M ganglion (Zhou et al. 1987). In both preparations, acetylcholine (ACh) is utilized as the excitatory transmitter.

With a view to provide suggestions to the mode of action of these four botanic insecti- cides, in this preliminary study we report their effects on the neuromuscular transmission in the ventralateral longitudinal muscle of larval Drosophila melungaster Meigen.

2 MATERIALS AND METHODS

Insect The larvae of Drosophilu melangaster Meigen were obtained from laboratory- reared stocks. For electrophysiological experiment, the third instar larvae were used. Chemicals Rhodojaponins were extracted from flowers and leaves of Rhododendron molle and kindly provided by Prof. Zhun Liu in Institute of Elemento-Organic Chemistry, Nankai University. Toosendanin is kindly provided by Prof. Guoxing Shu in Military Medical Academy of China.

Before testing, rhodojaponins were dissolved in the physiological saline of D. melan- gaster and prepared for requested concentrations. Toosendanin was first dissolved in ethanol (less than 1% in the final solution) and then prepared with the saline. Electrophysiology For electrophysiological investigations on the ventralateral longitu- dinal fibre of the third instar larvae of D. melungaster, specimen preparation and intracel- lular microelectrode recording were performed as described by Jan and Jan (1976 a , b > . Recording glass microelectrodes were about 12MR in resistance and 0. 5 pm in inner dia- meter at the tip. Excitatory junction potentials (EJPS) thus recorded were demonstrated on a VC6020 digital storage oscilloscope (Hitachi, Japan 1 after a 10 X microelectrode preamplifier. When normal EJPS signals were induced by electrical pulse(s> of 1 V X 1 s , the tested chemical was added to the bathing saline and its effects observed and occasional- ly photoed if necessary.

3 RESULTS

When the glass microelectrode was inserted into the vicinity of neuromuscular junc- tion in ventralateral longitudinal muscles, the resting potential measured was - 38. 9 f

340 ENTOMOLOGIA SINICA Volume 3 , Number 4 , 1996

Fig. 1 Excitatory neuroniuscular junction poten- tial (ejp) in the ventralateral longitudinal muscle of the third instar larvae of Drosophila melungaster evoked by a single presynaptic depolarization. Let- ters A and B indicate the fast and slow components of the potential, respectively. The arrow indicates the artifact of the electric stimulus.

7. 40 mV (n=49). If the motor nerve was stimulated with a single electrical pulse ( l V , Is) added to the suction stimulating electrode, a normal exitatory junction PO- tential (Fig. 1 ) will be recorded in the cor- respondingly innervated longitudinal mus- cles. The potential consisted of both a fast component with a greater amplitude and a slow one with longer duration. Usually, for the purpose of judging the effect of chemical in a particular preparation , the amplitudes of both components were chosen

as the index, because the down phase of the fast component fused into the rise phase of the slow component and this made it difficult to compare potential durations observed,at different times,

All of the chemicals were found to decrease the amplitudes of EJPS after their appli- cation in the bath solution . Such inhibitory process would lead to eventual blokage of EJPS (Fig. 2 ) . It seemed that the slow component was inhibited more quickly, while the fast one gradually suppressed and the recorded EJPS became smaller and narrower.

Fig. 2 In situ inhibition of excitatory neuromuscular junction potential (EJPS) in ventralateral longitu- dinal muscle of the third instar larvae of Drosophdu melungucter by rhodojaponin 1 .

a. pretreatment, b. 4min, c. 17min after application of 0.08 mg/mL rhodojaponin 1 . Interestingly, after discharges and repetitive firings of EJPS, which are bursts of PO-

tentials either evoked by a single depolarization or produced spontaneously, respectively , were observed with R- 1 and R- V during their inhibitory courses (Fig. 3 ) . Such en- hanced spontaneous firings appeared shortly after the chemical's application and would di- minish and disappear with time proceeding. Repetitive firings of EJPS were also found in toosendanin-treated preparations (Fig. 4). But in the case of rhodojaponin 1 , similar over-excitation of neuromuscular transmission was not detected, probably due to its quick inhibition of ETPS.

Jianhua Xu et uf : Rhcdojaponins and toosendanin on neuromuscular transmission 34 1

I 10 mv C

1 -

20 ms

Fig. 3 Repetitive firings(a-c) and after discharges ( d y e ) of EJPS in the ventralateral longitudinal mus- cle of the third instar larvae of Drosophila melungustrr during the inhibition course of 0. 08 mg/mL rho-

dojaponin K . a. 12min, b. 16min. c. 20min1 d. 3min, e. 7min after application of 0.08 mg/mI, rhodo-

japonin U .

C d

-1 - I ‘ O m ” 20 ms

e

Fig. 4 Inhibitory e f f e c t s b d ) and elevated spontaneous rrpetitive firings(e , f ) of toosendanin (0.08 mg/mL) on EJPS of the ventralateral longitudinal muscle of the third instar larvae of Drosophifa

melungaster. a. pretreatment, b. 5min, c. 27min, d. 3jmin after application of 0.08 mg/mL toosendanin.

To evaluate the activities of these compounds, the time between the application of chemical and the eventual blockage of EJPS (blockage time) was calculated and compared (Table 1). Toosendanin appeared to be a weak inhibitor because it took a mean blockage time of 32. 2 s even at high concentration (0. 08 mg/mI,). Among the three rhcdo- japonins, R- IU possessed the strongest inhibitory activity, and took least time to block EJPS. For example, at 0. 08 mg/mL, its blockage time was 1 7 . 4 s with 21. 9 s for R- 1

34 2 EKTOMO1,OGIA SINICA Volume 3, Number 4 , 1996

and 32. 8 s for R-V, respectively.

Table 1 melanguster with different concentrations of rhodojaponins (n= 10)

Blockage time for ejps in the ventralateral longitudinal muscle of the third instar larvae of D.

Chemical Concentration Blockage time (sec. )

(mg/ml,) (mean1S . M. )

Rhodojaponin 1

Rhodojaponin 1

Rhodojaponin V

Toosendanin

0.008 0. 08 0. 16 0. 32

0.008 0. 08 0. 16 0. 32

0. 08

0. 08

33. 8f 4. 5 2 1 . 9 1 3 . 2 18. 6+2. 3 10. l f 2 . 8

33. 5k2. 4 17. 4 f 1. 9 9. 4 1 1 . 7 1. 5 1 0 . 3

32. 81-4. 2

3 2 . 2 f 4 . 9

A negative relationship was observed between the applied concentrations and the blockage time (Fig. 5) . In other words, when applied at higher concentration, the chemical would inhibit and block the EJPS more quickly.

40 -

.r - . 5 . 0 ' . " . . . . . . . . . . . a

0. 04 0. 08 0. 12 0. 16 0. 2 0. 24 0. 28 0. 32

concentration ( s )

Fig. 5 in the ventralateral longitudinal muscles of the third instar larvae of Drosophila melangaster.

Il'egative relationship between concentrations of rhcdojaponins and blockage time for EJPS

4 DISCUSSION In the previous studies, the above three rhodojaponins were found to cause a progres-

Jianhua X u t t al : Rhodojaponins and toosendanin on neuromuscular transmission 343

sive series of poisoning symptoms typical for neuro-toxin in treated intact insects (Shang et ul. 1990, Klocke et ul. 1991). We have also observed the poisoned adult cockroach Periplanetu americanu L. and larval Drosophila melanguster Meigen. Shortly after rhodo- japonins applied topically on their bodies, both insects appeared to be hyperactive, and proceeded into ataxia, convultions, and paralysis which led to their eventual deaths. A certain correlationship could be inferred between these symptoms and their inhibitory effects on neuromuscular EJPS. For example hyperactivity and ataxia of intact insects correspond with the repetitive firings of EJPS which will lead to over excitation of mus- cles; and paralysis may be attributed to a certain inhibition of EJPS. Moreover, our ob- servations and other authors' results agreed in that R- 1 was the most effective insecticide among the three. And the known insecticidal activities of rhodojaponins in bioassays are corresponding to their inhibitory efficiencies in the present electrophysiological study , i. e. , R- !I >R- 1 >R- V . Therefore, it may be suggested that inhibition on neuromuscu- lar excitatory junction transmission may be at least one factor relevant to the eventual death of poisoned intact insects.

both with rat diaphgram muscle preparations (Shi et al. 1985) and with cockroach ab- dominal M ganglion preparations (Zhou et al. 1986). In the present study, toosendanin was found to inhibit evoked EJPS in the ventral longitudinal muscle of the fruit fly larvae, without an early facilitory action which was observed by Shi et al. (1985). Instead, ele- vated spontaneous EJPS were observed during the early time. This may suggest a facili- tory effect in the motor nerve endings, too. Recently, with rat diaphgram neuromuscular preparation toosendanin was reported to inhibit fast potassium ion channels and enhance the influx of extracellular calcium which would induce an increased release of transmitter (ACh) in the presynaptic nerve membrane ( X u and Shi 1993). Whether or not similar mechanism occurs in the present preparation remains obscure until further investigations demonstrate presynaptic events, because these three preparations differ from each other in transmitters (ACh or Glu) and types of synaptic transmisSZon (neuromuscular or in- terneuron transmission).

On the other hand, to make further elucidation of the mechanism underlying elevated EJPS, it is interesting to mention here similar observations of Adams et al. about the mode of action of pyrethroids on the longitudinal ventralateral muscle of larval housefly. They reported that pyrethroids could inhibit EJPS in this preparation , along with the occurrence of repetitive firing and after discharge which they attributed to overexcited mc- tor nerves (Adams and Miller 1979).

Lastly, the authors would like to repeat a known proposal that it is more valuable to

Inhibitory effects of toosendanin on synaptic transmission have been reported before

344 ENTOMOLOGIA SINICA Volume 3 , Number 4 , 1996

find a chemical possessing toxicological effects on insect neuromuscular transmission in- stead of actions on its central nervous system (like most of the synthetic insecticides in present use). Because (acetylcholine) is explored in the CNSs both of insects and mam- malians as well as in the neuromuscular synapses of mammalians, while glutamate is demonstrated to be the major transmitter of insect neuromuscular excitatory junctions (Jan and Jan 1976a,b), Therefore, an insecticide effective selectively on insect neuromus- cular transmission would be safer in use than one active on the central nervous system.

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