The Neuromuscular Mechanism of Stridulation in Crickets (Orthoptera: Gryllidae)

Study of the insect neuromuscular system appears very promising as a means of explaining behaviour in terms of cellular operation. The relatively small number of neurons, the ganglionic nature of the nervous system, the simplicity of the neuromuscular arrangement, and the repetitiveness of behavioural sequences all lend themselves to a solution of this problem. As a result, an increasing number of investigators have been turning their attention to insects and especially to the large orthopterans.

Recently, Ewing & Hoyle (1965) and Huber (1965) reported on muscle activity underlying sound production in crickets. The acoustic behaviour is well understood (Alexander, 1961) and in the genera Gryllus, Acheta and Gryllodes communication is mediated by three basic songs composed of three types of pulses. While working independently on this system at the University of Cologne (W.K.) and the University of Michigan (D.B.) using various Gryllus species, we found a number of basic differences between the muscle activity in our crickets and that reported by Ewing & Hoyle (1965) for Acheta domesticus. These two genera, Gryllus and Acheta, are so nearly identical that they are distinguished solely by differences in the male genitalia (Chopard, 1961). The present paper constitutes a survey of muscle activity patterns producing stridulation in four species of field crickets. Since the work of the two authors was carried out independently, non-overlapping experiments will be initialled to indicate the investigator.

Adult male field crickets of the following species were used in these experiments: Gryllus firmus, G. pennsylvanicus (from Michigan), G. veletis (from Michigan; D.B.) and G. campestris (W.K.) The first three species are North American and the last is European. The majority of the work was conducted upon freely moving chronic preparations. However, a series of experiments (D.B.) designed to establish the reliability of the chronic recordings and to aid in their interpretation should be examined first.

The mechanical structure of the mesothoracic exoskeleton was investigated to determine the relationship between muscle contraction and movement of the stridulatory apparatus. The tissue was cleaned away and the sclerites were moved at the points of muscle insertion to see how contraction of the muscles affected the forewings. In addition, 100 µ copper wires were fixed bilaterally to the exoskeleton at the insertions of twelve muscles: the subalars, basalars, first tergal promotors of coxa, and first tergal remotors of coxa (Fig. 1 ; the last two muscles along with the tergo-tro-chanter will be called the medial dorsoventrals). Pulling these in the direction of muscle contraction demonstrated the function of individual muscles and indicated how the contraction of one set of muscles affected operation of the others. Motion pictures were taken to clarify these effects.

In an acute preparation similar to that of Wilson & Weis-Fogh (1962), muscles were stimulated both directly and through their nerves to confirm results obtained in the mechanical experiments. The head and prothorax were removed and the pterothorax was waxed to a rigid support by the sternum. The thorax was perfused with Hoyle’s (1955a) locust perfusion fluid and could be kept functional for several hours. Individual muscles were stimulated by placing insulated 100 µ Nichrome electrodes at both ends and passing current pulses from a Grass S 5 Stimulator between them at rates from 1/sec. to 150/sec. The dorsal longitudinals, the second basalars, the subalars, the first tergal remotors and the first tergal promotors were stimulated in this manner.

In field crickets relatively long lengths of motor nerve trunks to muscles or synergistic groups of muscles can be isolated. In an acute preparation such nerves were dissected free and placed across two 50 µ silver wires 0·5 mm. apart. The nerves were lifted clear of the thorax into air or mineral oil and stimulated by single pulses or 5 sec. trains up to 150/sec. The effects of the ensuing muscle contraction upon the exoskeleton were observed. By stimulating at the surface of the perfusion fluid, before or after cutting the nerve, it was shown that the muscle contraction was due to neuronal activity. Except for the subalars, all of the muscles which had been stimulated directly were checked by stimulation through their nerves.

In fresh acute preparations, spontaneous wing raising and lowering or opening and closing frequently occurred. When this happened, it was possible to see which muscles were active and to take motion pictures of their activity.

The cricket wing, like other insect wings, has two equilibrium positions, one in which it is flattened and extended for flight and one in which it may stridulate or remain folded. In experiments with normal animals, acute preparations and isolated exoskeletons both wings were ‘popped’ into either position or one was placed in each position in order to determine the relationship between the upstroke-downstroke movement of flight and the closing-opening movements of stridulation.

Muscle action potentials were recorded in the thorax during flight (D.B.) and during normal stridulation. To accomplish this, a small hole was drilled through the cuticle, and a fine wire electrode insulated to the tip (40 µ stainless steel (W.K.) ; 100 µ Nichrome (D.B.)) was inserted into the muscle and fixed to the cuticle with adhesive wax. The indifferent electrode was a stainless-steel wire uninsulated for 1 mm. from the tip placed in the abdominal cavity (W.K.) or an uninsulated copper loop in the prothoracic haemolymph cavity (D.B.). The electrodes were clipped to long flexible leads allowing complete freedom of movement. Potentials were amplified by a Tektronix 122 pre-amplifier (W.K.) or a Grass A.C. pre-amplifier (D.B.), and displayed on a Tektronix 502A dual-beam oscilloscope. Sound pulses were picked up by microphone (Beyer M100 (W.K.); Ampex 802 (D.B.)) and displayed synchronously with the muscle potentials on the oscilloscope. Beam displacement was recorded on paper oscilloscope film.

In some animals, recording electrodes were placed in synergistic muscles (with a common indifferent electrode) to determine the degree of synchrony and order of firing (W.K.)

The electrode position was identified by both chemical and physical means. The tip could be fairly accurately located by careful dissection, especially when it was inserted just through the cuticle. After the end of an experiment, a pulse of current was passed between the electrodes, using the recording electrode as anode, and the cations were either stained with Prussian-blue (W.K.) or precipitated in a bright red nickel-dimethylglyoxime complex (D.B.). The latter method involved anaesthetizing the animal in CO₂, removing the thorax, evacuating it, and placing it for 12 hr. in a solution of 5% sodium acetate, 1% dimethylglyoxime, 7% ammonia, and 87% ethanol (47%). Current was passed after the thorax had been impregnated because a more concentrated precipitate formed and previous experiments had shown that there was no electrode shift during post-mortem manipulation.

Since recording occurred in a volume conductor with a number of concurrently active sources of e.m.f., experiments were carried out to determine the relationship of the electrode tip to potential sources (D.B.). In several animals, four electrodes were placed in the thoracic segments and potentials were recorded sequentially from all pairs (Fig. 2). Tips were marked and their positions and the nature of the tissue around them were determined.

The muscles observed could be placed into two groups according to their activity : those that fire during the opening of the forewings and those that fire during the closing or sound producing (Pierce, 1948) stroke of the forewings (Table 2). This is a chronological as opposed to a functional classification and an ‘opener’ might, for example, be maintaining the horizontal position of the forewings rather than moving them apart. We found that in our animals muscles fell into the opposite groups from those assigned by Ewing & Hoyle (1965) for A. domesticus.

Manipulation of the exoskeleton showed the critical effect of a muscle to be its movement of the exoskeleton with respect to the wing pivot or pleural apodeme. Lowering the tergum or dorsal sclerite of the mesothorax closes the wings ; tilting it forward and down raises them. Pushing the tergopleural arms backward medially and pulling them forward laterally spreads the wings. Lowering the subalar sclerite lowers and opens the wings. Pulling down on the basalar sclerite spreads and may lower the wings. Alternate pulling of wires attached to the medial dorsoventral insertion points and basalar or subalar sclerites produces normal-appearing stridulatory motion and position of the wings.

Direct stimulation, nerve stimulation, and spontaneous activity confirmed the muscle effects indicated by the wire experiments. Anterior medial dorsoventrals raise and close the wings; posterior medial dorsoventrals primarily close them. Dorsal longitudinals inserting on the tergopleural arm, subalars and basalars open the wings while the subalars have an especially strong lowering effect.

Observation of wing action in both equilibrium positions demonstrated clearly that the upstroke of flight is homologous with the closing stroke of stridulation and, conversely, the downstroke corresponds to the opening stroke.

Recording sequentially from pairs of electrodes showed first that some electrodes record and some do not. The non-recording electrodes were in fat bodies or tracheae or were covered with clotted hemolymph. Of recording electrodes, those completely inside a muscle were unlikely to record from units outside that muscle. If an electrode was on the surface of a muscle it picked up large potentials from that muscle but was subject to interference from other muscles. If an electrode was not directly on the surface of a muscle, it could pick up potentials from muscles some distance away in the thorax. One recording sequence is shown in Fig. 2. This animal had two indifferent electrodes in the prothoracic hemolymph cavity, one electrode inside the first tergal remotor of coxa and one electrode on the lateral surface of the subalar. No sizeable potentials were seen when recording between the indifferent electrodes. While recording between the remotor electrode and an indifferent electrode, large spikes were seen associated with closing activity and there was no interference from outside muscles (Fig. 2A). When recording from the subalar electrode and the same indifferent electrode, opening potentials were seen from the subalar but they were irregular, due to interference from synergists (Fig. 2B). Also, the activity of antagonists, such as the adjacent remotor, was seen as cross-talk during the intense closing pulses.

Fig. 2C was recorded between the two active electrodes. Potentials of opposite polarity appeared as the muscles were alternately active in the opening—closing pattern. In all recordings, spike patterns were consistent with the active electrode position.

Chronic recordings from well over a hundred animals support the conclusions outlined in Table 2. There were no inconsistent results, and patterns were confirmed independently in each laboratory. In the calling or aggressive pulse, closing is produced by the medial row of dorsoventral muscles. The first promotor fires just previous to and during the course of the sound pulse (Fig. 3A, G. camp. ; Fig. 4B, G. firmus). The first remotor fires in the same general pattern (Fig. 3B, G. camp.; Fig. 4C, G. penn.). In G. camp, the tergo-trochanter is not active during this type of pulse; in the North American species it is most active in flight and can fire occasionally when the wings are raised at the beginning of a chirp sequence or during an intense pulse (Fig. 4D, G. firmus). Also, in these species, the promotor can fire up to 9 times per pulse and is the most active muscle hitherto observed. Evidently, several of the muscles fire more frequently per pulse in the North American species than in the European species although this has not been critically measured.

Wing opening during the aggressive or calling pulse is associated with activity in the dorsal longitudinals inserted on the tergopleural arm, the subalars, and the basalars. The subalar can fire from one to several times beginning in the last phase of the sound pulse or just after its completion (Fig, 2B, G. firmus;, Fig. 3E, G. camp.;, Fig. 4E, G. penn.). The second basalar fires in a pattern very similar to that of the subalar (Fig. 3D, G. camp.) ; although the third basalar is very active in flight, it does not fire during stridulation except occasionally when the wings are raised and opened for the first time in a chirp sequence (Fig. 4A, G. penn.). Simultaneous recordings from the second basalar and the subalar show that although they fire in relative synchrony, the basalar always leads the subalar by a short interval (Fig. 5, G. camp.).

The activity of the muscles alters greatly during the courtship song which is composed of two phases: a series of soft pulses followed by a loud ‘tick’ at the end of the chirp. In all of the species examined, all of the medial dorsoventral muscles fire synchronously just before the ‘tick’ (Fig. 6A, B, G. camp;, Fig. 7A, G. veletis;, Fig. 7B, G. penn. ; Fig. 7C, G. firmus). There is no apparent opener activity during this phase but, in contrast to the closers, the openers are very active in the soft pulse phase. The subalars show prolonged firing during this part of the chirp (Fig. 6D, G. camp. ; Fig. 7F, G. penn.). In some cases there is rapid multiple firing of up to fifteen spikes which may produce the vibration of the wings in the open position seen in high-speed motion pictures (D.B.). Second basalar activity is again similar to that of the subalar in G. camp. In the North American species the third basalar is inactive.

Analysis of the dorsal longitudinals presents a difficult problem because electrodes fixed to the overlying tergum are moved during stridulation. Since electrodes are marked with the wings lowered, their positions during chirping are not clear. However, it appears that 81a and 81b are antagonistic. Both originate at the junction of the meso- and metathoracic terga but 81a inserts on the front margin of the mesothoracic tergum while 8iè inserts on the tergopleural arm. Judging from the anatomy the action of 81 b on the tergopleural arm spreads the wings but movement of the tergum itself by 81a may raise the wings or may adjust the rigidity of the thorax in some fashion. Opening activity of 81b is shown during the calling-type pulse (Figs. 3C, 6C, G. camp. ; Fig. 7E, G. firmus) and during the soft pulse phase of courtship (Fig.6C, G. camp. ; Fig. 7D, E, G. firmus). Fig. 7E, a continuation of the stridulation shown in Fig. 7D, illustrates the transition back to calling with the incorporation of both ‘ticks’ and calling-type pulses. 81a is seen firing prior to the ‘tick’ and during the closing phase of the calling pulse. A similar situation is found in Fig. 6C, although in this particular record 81b fires only during the opening prior to the ‘tick’.

It is difficult to make valid estimates of motor unit distribution. Fast-axon and slow-axon activity cannot be rigorously distinguished without recordings of intracellular or mechanical changes (Hoyle, 1955b). However, some units can be separated on the basis of size differences and summation effects. Fig. 8 shows both multiple firing of a single unit and varying degrees of summation of two units in the first tergal remotor of G. camp. Judged on these grounds, multiple units also occur in the subalar, the second basalar, the third basalar, both dorsal longitudinals, the tergal promotor, the tergal remotor and the tergo-trochanter. Most of the muscles appear to have at least two large units and one or more small. The small units may reflect slow-axon activity.

It has not been possible to correlate changes in pulse intensity directly with activity in any single muscle; increased intensity is usually associated with higher activity levels in all muscles. In some cases, force production can be related to a smaller set of muscles. For example, during the soft-pulse phase of courtship, the forewings are driven by the opener group.

If acoustic communication is examined with the intention of analysing behaviour in terms of its smallest functional parts, it is helpful to consider the system as a series of sequentially triggered units. ‘Output’ arises in supraoesophageal ganglion interneurons (Huber, 1960, 1962, 1965) and passes through premotor interneurons, motor neurons, muscle units, and skeletal movement to produce behaviour. The questions asked in this paper are how movements of the thoracic exoskeleton operate the stridulatory apparatus, how contraction of the muscles moves the mesothoracic exoskeleton, and how force production in the thorax is distributed in time and space by patterning of motor axon activity to produce and modulate stridulation.

Manipulation of the exoskeleton and direct or indirect stimulation of muscles yield relatively straightforward answers to the first two questions. Since muscle action potentials reflect fast-axon acitivity 1:1 in crickets, (Huber, 1965), chronic recording offers a fine tool for analysing ganglionic output patterns. It is worth examining its reliability critically. Chemical stains have been found to be precise indicators of electrode-tip position and, as shown by multiple electrode recordings, potentials can be assigned to muscles in which the tip is embedded. However, the time association of muscle action potentials and sound pulses remains to be fixed. If it can be assumed that the lag between the muscle action potential and the development of mechanical force is similar in the stridulatory muscles of the cricket and the flight muscles of the locust, the work of Neville & Weis-Fogh (1963) gives information on this period. There should be about 1 msec, from the muscle action potential to the onset of mechanical contraction and 15–20 msec, to the peak. By this criterion the cricket potentials lead by the proper interval to produce the assigned activity. If the single loud pulse in courtship and the synchronous dorsoventral spike preceding it can be causally related, this also gives the correct time-lag. Finally, if both opener and closer activity are being recorded (Fig. 2C, G. firmus), the spike that occurs last should be associated with the last wing movement in a chirp (disregarding the possibility of a completely elastic return). Direct observation of stridulation, high-speed motion pictures, and Walker’s (1962) analysis of calling show the last movement to be closing. Therefore, the final spike is from a ‘closer’ and again indicates the proper interval. This being determined, the final question is answered and one can be sure that potentials are being correctly interpreted.

The operation of the exoskeleton by the muscles during insect flight has been examined by a number of investigators. Wilson & Weis-Fogh (1962) have shown that the muscles involved in locust flight can be divided into groups producing upstroke and downstroke. The former is composed of the medial row of dorsoventral muscles, while downstroke is produced partly by elasticity and partly by the dorsal longitudinals and the lateral dorsoventral muscles, the subalars and basalars. Since these are the same two groups involved in stridulation and flight in the cricket, and since stridulation appears to have evolved from flight (Huber, 1962), if the homology between these two wing movements could be established it would indicate which group should be assigned to each stroke. Upstroke has now been shown to be homologous with closing and so one would expect the medial row of dorsoventral muscles to produce closing and the dorsal longitudinals, subalars and basalars to produce opening. Our studies have shown through several lines of evidence that this, excepting the bifunctional dorsal longitudinal, is the case. This conclusion is also supported by Huber’s (1965) recordings of activity in the subalar and remotor muscles of G. camp, during stridulationl

The critical parameters of behaviour, stridulatory patterns and musculature are either very similar or identical in A. domesticas and the Gryllus species studied by us. It appears highly unlikely that differences in muscle function of the magnitude outlined in Table 2 could occur. Preliminary recordings from A. domesticus substantiate this since subalar activity conforms to the gryllid pattern. Therefore, we find that our results do not confirm those of Ewing & Hoyle (1965) pertaining to the role of muscles in stridulation.

The authors would like to thank Drs R. D. Alexander, F. Huber and D. M. Maynard for critical reading of the manuscript. The work was supported by a National Science Foundation Cooperative Graduate Fellowship (D.B.) and by grants to Dr Huber from Die Deutsche Forschungsgemeinschaft (W.K.); some equipment was provided by National Institutes of Health Training Grant No. 5-TIGM 989 (D. B.).