PHYLOGENETIC STUDIES ON MINIATURE ELECTRICAL OSCILLATION IN MUSCLES

TSUTOMU WAKABAYASHI ANDKAZUO IKEDA* Depertmentof Physiology,School of Medicine,University of ,Tokyo

Previously, Wakabayashi and Hagiwara (1) reported that a spontaneous irregular electrical oscillation of small size was often observed in the sound muscle of at rest and they supposed that it might be due to the injury caused by inserted electrodes. Hagiwara (2) reported again this irregular oscil- lation (miniature electrical oscillation) as injury discharges . In the first part of the present study an experiment was carried out to decide its nature. Fur- thermore, the oscillation was investigated in the thoracic and tymbal muscle of various , and the results were considered from phylogenetic point of view.

MATERIALSAND METHOD

Since it had been found by a preliminary experiment that miniature electrical oscillation was not observable in a muscle which does not contract so rapid as tymbal muscle, for example leg muscle, so in this study thoracic muscles (in- direct wing-muscles) of various of insects and tymbal muscles of Cicadidae were adopted. The materials used were the several species of various orders of insect which could be caught in the suburb of Tokyo. The following species were used for the electrical activity of thoracic muscle: Odonata: Platicnemis foliacea sasakii, Calopterix virgo japonica, Mnais stri- gata, Epiophrebia sperstes, Gomphus melampus, Antogaster sie- boldii, Aeschna juncea, Anax palthenope julius, Orthetrum albitylum sPeciosum, Orthetrum triangulare melania, Orthetrum japonicum japonicum, Sympetrum darwinianum, Sympetrum frequens, Rhyo- themis foliginosa. Neuroptera: Chrysopa intima, Hagenomyia micans. Orthoptera: Tettix jaPonicus, Euprepocnemis shiraki, Locusta migratoria danica, Paratenodera sinensis. : Terpnosia vacua, Platypleura kaepheri, Graptopsaltria nigrofuscata, Cryptotympana japonensis, japonensis, Oncotympana macu- laticollis, Meimna opalifera. : Pyrgus maculatus, Pieris rapae crucivora, Pieris melete, Eurema hecabe mandarina, Colias hyale poliographus, Neope goshkevitschii, Polygonia c-aureum, Parnassius glatiaris, Menelaides alcinosis, Received for publication June20, 1957. *若 林 勳 池田和夫

222 ELECTRICAL ACTIVITY IN INSECT MUSCLES 223

Papilio machaon, Papilio prothenor, Paj,ilio xuthus. Coleoptera: Mallambyx raddei, Melolontha japonica, Xylotrupes dichotomus, Anomala cuPrea. Hymenoptera: Ophion pungenus, Apis indica jaPonica, Vespa mandarinia, Polistes fadwigae, Parapolybia varia. Diptera: Bibiocephara infuscata, Eristalomyia tenax, Volucella jeddona, Musca domestica, Sarcophaga melanura.

The following species were used for the activity of the tymbal muscle: Terpnosia vacua, Platypleura kaempheri, Graptopsaltria nigrofuscata, CryP- totym _pana jaPonensis, , Oncotympana maculaticollis, Meimna opalifera. The experiment was performed with an antero-posterior muscle, a dorso- ventral muscle of thorax and a tymbal muscle. In order to attach or insert the recording electordes into a muscle, a pore was made on mesothoracic, meta- thoracic notum, pleuron or abdominal segment in accordance with the structure of each species. The apparatus for extracellular recording consisted of a pair of fine silver-wire electrodes, resistance-condenser-coupled amplifier, an electro- magnetic oscillograph and a cathode-ray oscilloscope. Often for the purpose of intracellular recording, a capillary microelectrode filled with3M KCL, a direct- coupled amplifier with a cathode-follower imput and a cathode-ray oscilloscope were employed. After the extracellular recording of miniature electrical oscil- lation with a muscle innervated by thoracic ganglion, the recording by the same means was performed with the muscle which was set free from thoracic ganglion by crashing it. Moreover, with the material in which the existence of miniature electrical oscillation had already been recognized by extracellular recording, intracellular recording was made. After each recording, it was ascertained that miniature electrical oscillation was extinguished by applying alcohol to the muscle, to make sure that the miniature electrical oscillation was free from artefact.

RESULTS 1. Extracellular recording of potentials of an innervated muscle Among the species which were investigated here, no miniature electrical oscillation was observed in thoracic muscles of Odonata (except Antogaster), Neurofitera, Orthofitera, Hemiptera, Lepidoptera and Coleoptera. But small elec- trical oscillation was observed, as shown in fig.1, in the dorso-ventral muscle of Antogaster. Usually it began after the cessation of an ordinary wing-beat and increased its amplitude in several seconds. It appeared, moreover, only after the wing-beat and dissappeared soon, so it looked like an afterpotential, but its form was very similar to the miniature electrical oscillation which will be described later. In some species of Diptera and Hymenoptera, the miniature electrical oscil- lation of the thoracic muscle was observed. In Diptera, miniature electrical oscillation was observed in Eristalomyia, Volucella and Musca but not in the other species. The amplitude of the minia- 224 T. WAKM3AYASHI AND K. IKEDA

FIG.1.•@ Antogaster sieboldii (dragon-fly)-Thoracic muscle.

Long lasting after-discharges following after action potential. Extracellular record- ing. After cessation of the natural wing-beat (left of A), train of discharges was initiated gradually (A) and grew in amplitude till regular discharge continued for a while (B and C). Temp.: 27•Ž. August24, 1954.

FIG.2

FIG.3

FIG.2.•@ Eristalomyia tenax (gad-fly )-Thoracic muscle. Extrcellular recording of miniature electrical oscillation: examples of various activity. Time-scale: 50c/s. Temp.: 20•Ž. November5, 1954.

FIG.3.•@ Volucella jeddona (gad-fly )-Thoracic muscle. Extracellular recording of miniature electrical oscillation: examples of various activity. Time-scale: 50c/s. Temp.: 14•Ž. November27, 1954. ELECTRICAL ACTIVITY IN INSECT MUSCLES 225 ture electrical oscillation which was recorded ranged from200to300pV. The discharge pattern characteristic of each species was rather irregular. In Hymenoptera, miniature electrical oscillation was observed in Vespa, Apis and Parapolybia but not in the other species. In general, the amplitude of the miniature electrical oscillation which was recorded in Hymenoptera attained up to800pV and was higher than that in Diptera. The discharge pattern of the species in this order was alike and, was similar to that of the tymbal muscle of Cicadidae. In the tymbal muscle of Cicadidae, miniature electrical oscillation was ob- tained in all species examined. It was somewhat superior to that of the thoracic muscle of Hymenoptera in amplitude and frequency.

2. Extracellular recording of potentials of a denervated muscle In the thoracic muscle of Eristalomyia, Volucella, Musca, Apis, Vespa and

FIG.4

FIG.5

FIG.4.•@ Parapolybia varia (wasp)-Thoracic muscle. Extracellular recording of miniature electrical oscillation: Examples of various activity. Time-scale: 50c/s. Temp.: 19•Ž. November2, 1954. FIG.5.•@ Apis indica japonica (honey bee)-Thoracic muscle. Extracellular recording of miniature electrical oscillation: Examples of various activity. Time-scale: 50c/s. Temp.: 18•Ž. November1, 1954. 226 T. WAKABAYASHI AND K. IKEDA

FIG.6

FIG.7

FIG.6.•@ Vespa mandarinia ( wasp )-Thoracic muscle. Extracellular recording of miniature electrical oscillation: spontaneous fructuation of regularity. Time-scale: 50c/s. Temp.: 15•Ž. November12 , 1954. FIG.7.•@ Vespa mandarinia (wasp)-Thoracic muscle. Extracellular recording of miniature electrical oscillation: regular discharges and fructuation of intervals. Time-scale: 50c/s. Temp.: 15•Ž. November12 , 1954.

Parapolybia and tymbal muscle of Terpnosia, Platypleura, Graptopsaltria, Cryp- totympana, Tanna, Oncotympana and Meimna, the miniature electrical osillation was observed not only with the innervated preparation but also with the pre- paration the ganglion of which was crashed. No difference of the discharge pattern was obtained between the two cases. The miniature electrical oscillations of Diptera are shown in figs.2and3; they consisted of lower amplitude than that of Hymenoptera and Cicadidae. The miniature electrical oscillations of Hymenoptera are shown in figs.4, 5, 6and7. As shown in these figures the discharge pattern of Parapolybia borders on that of Diptera. Sometimes the irregular oscillation was transformed gradually into the passable regular one concerning amplitude and frequency. Figs.6 and 7show the transformation of irregular oscillation into regular one. In most cases, this regular wave lasted for20-60seconds until it turned gradually into irregular oscillation again. Snch regular oscillations were scarcely observed in the thoracic muscle of Diptera, while they were generally observed in the ELECTRICAL ACTIVITY IN INSECT MUSCLES 227 thoracic muscle of Apis and Vespa and in the tymbal muscle of Cicadidae. During the continuation of trains of discharges it was noted that the frequency of discharges increased or decreased, and in extreme cases discharge stopped for a while (fig.7). But the cause of it could not be made clear. The miniature electrical oscillation of the tymbal muscle of Cicadidae showed

FIG.8.•@ Graptopsaltria nigrofuscata ()-Tymbal muscle. Extracellular recording of miniature electrical oscillation: spontaneous transforma- tion of the pattern. Time-scale: 50c/s. Temp.: 28•Ž. August27, 1954.

FIG.9.•@ Intracellular recording of miniature electrical oscillation.

A: Eristalomyia tenax (gad-fly) D: Parapolybia varia (wasp) B: Volucella jeddona (gad-fly) E and F: Apis indica japonica (honey bee) C: Polistes fadwigae (wasp) G and H: Vespa mandarinia (wasp) November1-27, 1954. 228 T. WAKABAYASHI AND K. IKEDA

often a remarkable change of pattern, as shown in fig.8, spontaneously in the course of time. In one and the same preparation, irregular oscillation shown in fig.8-A transformed into a beat-like oscillation shown in fig.8-B which changed into another beat-like one as shown in fig.8-C. Details of it will be reported in the future.

3. Intracellular recording of miniature electrical oscillation In the species whose miniature electrical oscillation had been found by means of extracellular electrodes, the oscillation could be observed also by in- tracellular micro-electrode. In the case of intracellular recording, the irregular oscillation the pattern of which was characteristic in individual species lasted through scores of minutes and gradually decayed. In most cases, oscillations were irregular and composed of potential waves of various amplitudes and durations, but in Vespa and Cica- didae they sometimes became regular: its general feature was similar as in the case of the extracellular recording. The amplitude of the miniature electrical oscillation was generally at most 20 mV, while the resting potential was60-80 mV. Fig.9 shows intracellular recordings of the oscillation in Diptera, Hyme- noptera (thoracic muscle) and Cicadidae (tymbal muscle).

DISCUSSIONAND CONCLUSION According to the results of the investigation on the electrical activity of resting thoracic muscles, insects could be classified into two groups. The minia- ture electrical oscillation of thoracic muscle was observed in Diptera and Hyme- noptera but not in Odonata, Neuroptera, Orthoptera, Hemiptera and Coleoptera. Exceptionally in the case of Antogaster in which the oscillation was found only after the cessation of its wing-beat. Therefore phylogenetically the miniature electrical oscillation is developed only in the higher orders of insect and not in those of the lower orders. From the phylogenetic point of view, a thoracic muscle which generates miniature electrical oscillation have developed presu- mably from one which generates no oscillation. The miniature electrical oscillation will be discussed below from a view point of phylogenesis.

A) The miniature electrical oscillation and the thoracic mechanism of wing- beat The problem of the phylogenetic development of thoracic muscle should be discussed in consideration of the phylogenetic development of wing-beat mecha- nism. As to the wing-beat mechanism of insects, the indirect system of muscle- thoracic exoskeleton-wing have developed from the direct system of muscle- wing. The most complete form of the former system can be seen in Diptera and Hymenoptera. The phylogenetic development of wing-beat mechanism may be found in well developed elastic property of the thoracic exoskeleton and in the alteration of the mode of muscle contraction from isotonic to isometric type. Furthermore, it can be phylogenetically suggested that a parallelism really exists between the development of wing-beat mechanism and the taxonomic distribution ELECTRICAL ACTIVITY IN INSECT MUSCLES 229 of miniature electrical oscillation in the thoracic muscle. In fact, miniature electrical oscillation was not observed in the thoracic muscle of Odonata, Neu- roptera, Orthoptera, Lepidoptera and Coleoptera which have the thoracic ex- oskeleton incompletely developed. And muscles cannot contract isometrically in them. On the other hand miniature electrical oscillation could always be recorded in the thoracic muscle of Diptera and Hymenoptera in which the isometric con- traction could be expected due to its elastic exoskeleton. Furthermore, the miniature electrical oscillation in Hymenoptera was superior to that in Diptera in amplitude and frequency, and the discharge pattern in Parapolybia was re- garded as an intermediate type between Hymenoptera and Diptera. No minia- ture electrical oscillation was usually recorded in the resting thoracic muscle of Odonata but after-discharge following a wing-beat was observed in Antogaster. It might possibly be doubted if the amplitude of miniature electrical oscillation in an insect of lower order is too small to be detected, but anything definite cannot be said about it.

B) The miniature electrical oscillation and the rapidity of the muscle It was recognized that there is an interesting correlation between the ap- pearance of miniature electrical oscillation and the rapidity of muscle contraction. Every thoracic muscle in which a miniature electrical oscillation was recorded contracted usually in frequency of more than60per second. In Hemiptera, miniature electrical oscillation was observed in its tymbal muscle but not in thoracic muscle. And the former muscle contracts with frequency of80-120per second, while the latter20-30per second. After all, the parallelism can be pointed out between the generation of miniature electrical oscillation and the rapidity of muscle twitch as well as the parallelism between miniature electrical oscillation and the phylogenesis of wing-beat mechanism.

C) Electrical oscillating phenomena described by other authors According to Hagiwara (2), the irregular oscillation, which was observed by means of extracellular electrode in the resting tymbal muscle of Cicadidae, was the summation of action potentials perhaps of simple form which were generated due to injury. In the present study where the intracellular recording of poten- tials with the thoracic muscle of Diptera and Hymenoptera and the tymbal muscle of Cicadidae was made, similar miniature electrical oscillations were obtained as the extracellular recording, though of somewhat simpler form. Moreover, miniature electrical oscillations were found to be myogenic, since they appeared even without innervation. In addition, the miniature electrical oscillation con- tinued for more than scores of minutes unlike the injury discharge which ap- peared by inserting the intracellular electrode of large diameter into insect muscle. From these facts, it may most probably be claimed at present that the miniature electrical oscillation is not such as an injury discharge, but is myogenic in nature. In the leg muscle of some arthropodes, irregular discharges have been re- ported by Rijlant (3). It may probably be the action potential of its tonic fibre and not the miniature electrical oscillation as obtained by present authors, since 230 T. WAKABAYASHI AND K. IKEDA from the result of the present study such a slow muscle as tibialis cannot be expected to generate the miniature electrical oscillation at rest. Roeder (4) has observed such oscillation as in the present study, but he regarded this "low voltage sinusoidal wave" as an artefact caused by the move- ment of muscle. Alvarez (5) obtained rhythmic action potentials from a circular muscle strip of ileum of the dog and rabbit after making it immovable by painting with adrenaline. In the heart of Rana temPoraria, Mines (6) obtained strong regular action potentials of "same general form as the ordinary electrogram and no smaller" in a condition in which no movement was perceptible by perfusing it with a calcium-free solution. Whether they are of the same nature as the miniature electrical oscillation cannot be decided and should be reserved for future investigation.

D) The miniature electrical oscillation and the myogenic rhythm of wing-beat Reviewing over the species on which experiments have been performed hitherto concerning the myogenic determination of wing-beat frequency, it could be accepted that the taxonomic distribution of the miniature electrical oscillation corresponds to that of muscles which is believed to have myogenic rhythm. Since the importance of load in wing-beat mechanism had been pointed out by Heidermanns (7), Kraemer (8) and Cremer (9), studies of the mechanism of high frequency wing-beat have been focussed on the special property of tension in thoracic muscle. Experiments performed from this point of view with Cal- liphora (4) and (10), Drosophila (11), eight species of Diptera (12), Lucilia (4), Eristalis (4), Vespa (4), Sarcophaga (13), Chironomus (14) and Forcipomyia (14), have revealed that the rapidity of wing-beat depends on an unique myogenic mechanism. It is interesting to note that every species on which miniature electrical oscillation was observed belongs to these groups of insects (Diptera and Hymenoptera) on which the rapidity of wing-beat have been interpreted by so-called myogenic rhythm theory. So it appears that there are good correlation from phylogenetic point of view.

E) The miniature electrical oscillation and the lack of correlation between action potential and movement It is already known that in some species of insect the movement of thoracic exoskeleton or tymbal shows no one-to-one relation to the action potential of thoracic or tymbal muscle. This phenomenon has been observed in the thoracic muscle of CalliPhora (4) and (10), Lucilia (4), Eristalis (4), Vespa (4), eight species of Diptera (12), Sarcophaga (13), fly (15) and fly (16) and the tymbal muscle of Platypleura (17), (18) and (19): these are the thoracic muscle of Diptera and Hymenoptera and the tymbal muscle of Hemiptera in which the miniature electrical oscillation was observed in the present study. This is ano- ther correlative point to be added to those already described which are (a) the structure of thoracic exoskeleton, (b) the type of contraction (isotonic or iso- metric), (c) the rapidity of contraction and (d) the myogenic determination of the frequency of contraction. ELECTRICAL ACTIVITY IN INSECT MUSCLES 231

SUMMARY

1. Miniature electrical oscillation of insect muscle which had been found by Wakabayashi and Hagiwara was investigated phylogenetically. As materials the thoracic muscle of Odonata, Neuroptera, Orthoptera, Hemiptera, Lepidoptera, Coleoptera, Hymenoptera and Diptera and the tymbal muscle of Hemiptera were used. 2. Miniature electrical oscillation was obtained in the thoracic muscles of Diptera and Hymenoptera and the tymbal muscle of Hemiptera, but not in Odo- nata, Neuroptera, Orthoptera, Lepidoptera and Coleoptera. 3. Similar oscillation was observed with extra- and intracellular electrodes. Most probably the oscillation is not injury discharge and is myogenic in nature. 4. The oscillation is obtained in the rapid muscle of insects of higher order, so it can possibly be supposed to appear with the phylogenetic progress of the insect. 5. The correlations of the appearance of the oscillation to the development of exoskeleton, the type of contraction, the rapidity of contraction and the myo- genic determin ation of contraction were also indicated. It is the fact of tax- onomic significance.

We wish to express our thanks to Professor K. Uchizono for his advice and criticism during the work, when he was studying in our laboratory. This work was aided by a grant from the Ministry of Education.

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