Effects of Wing Campaniform Sensilla Lesions on Stridulation in Crickets

Schäffner & Koch (1987) showed that lesions of the cubital campaniform sensilla (CCS) cause severe alterations in the song of the male cricket, and that these ‘lesion songs’ are less attractive to the female. However, we did not consider which parts of the sound-generating system fail when the lesion songs occur. One possible explanation for missing syllables could be a lack of wing motion due to missing activity in the wing muscles. Another possibility could be that the wings slide past each other without touching and thus generate no sound, or that the wings ‘get stuck’ at the beginning of the motion. An investigation of these possibilities will help to explain why the CCS play such an important role in cricket sound production.

In this paper the function of the CCS is investigated further. To this end, a variety of experimental techniques was used: extracellular recordings from the cubital nerve, recordings of the wing motion and myograms of the large opener and closer muscles. The wing motion recordings were used to analyse the sliding or sticking motion, while the myograms were used to check if any abnormalities in the motor output pattern could be the cause of the observed disturbances.

Male Gryllus campestris were first selected as ‘good singers’ (Schäffner & Koch, 1987). The animal was tethered carefully using Plastilin. The wire used for the extracellular recordings was copper or steel wire (20 or 30 μm in diameter), insulated with varnish except at the tip.

Two recording sites were used. In a unipolar electrode arrangement, the active electrode was inserted into a small hole that had been pierced in the cubital vein near the CCS. The indifferent electrode was placed in the mesothorax or, in later experiments, between the fourth and fifth abdominal segments. In the differential electrode arrangement, two electrode wires were placed medial and lateral to the cubital vein. With this technique, lesions of the cubital nerve were avoided. In addition the cross-talk from the large wing muscles was substantially reduced.

The recording wires were usually fixed with ‘insect wax’ made from 66 % beeswax and 34 % colophonium. The wires were also fixed on the anal field in order to reduce the load on the insertion site. Finally, the wires were waxed to the pronotum and led to the preamplifiers. The wing motion was not hampered by the electrodes. The extracellular potentials, as well as the sound signal of the calling songs were stored on an instrumentation tape recorder (Racal Store 7DS). The data were later played back on a storage oscilloscope, or, at reduced speed, on a chart recorder (Schwarzer US266). Fifteen males were used for these recordings. The wing nerve recordings were made in collaboration with Dr G. Karnper, whose help and advice is gratefully acknowledged.

For precise wing motion recordings, the method of inductive angular measurements with miniature sensing coils was used (Koch, 1980). We also used the special technique of position stabilization and signal analysis by analogue computing as described in Koch & Elliott (1983) and Elliott & Koch (1983). This improved system of movement recording permits the direct display of the movement of the wings relative to each other and also the measurement of relative wing velocity. The position-sensing coils were l×2×0·2mm and consisted of 30 windings of copper wire (17 μm in diameter). Mechanograms of 12 male Gryllus campestris were made before and after lesions of the cubital nerve.

The males were carefully tethered with Plastilin leaving the pleural area of the mesothorax free. The electrodes were placed in M99 (remotor coxae) and M90 (subalar) following the method of Kutsch (1969). For each muscle, two electrode wires (steel, 30 um in diameter) were inserted into the upper third of the epimerum, and fixed with insect wax. The indifferent electrode was inserted into the abdomen between the fourth and fifth segments. Electromyogram (EMG) potentials and acoustic signals were amplified and stored on a tape recorder (Racal Store 7DS) and later played back at reduced speed on a chart recorder (Gould). For analysis of the timing, EMG potentials were analysed with a peak-detecting window discriminator, whereas the onset of the sound was measured using a level discriminator applied to the envelope signal. For selected 1-min sections of the recordings, these timing signals were stored on the RK05 disk of the PDP11/40 computer using the digital inputs of the LPS11 laboratory interface (Schäffner & Koch, 1987).

The crickets were kept in glass jars (8 cm in diameter, with a peat substrate) for the EMG and sound recordings. As described by Huber (1965), the males are not disturbed in their singing behaviour by the chronically implanted electrodes. Even the circadian rhythmicity of singing is maintained (see Wiedenmann & Loher, 1984). EMG recordings of four G. bimaculatus males with lesions of the cubital nerve on both sides were made.

In a large proportion of the recordings, especially when the indifferent electrode was placed in the thorax or abdomen, strong cross-talk from the main wing muscles was observed (see Fig. 2B). However, muscle potentials have considerably slower rise and fall times, and thus it was possible clearly to distinguish the fast nerve potentials. In addition, the muscle potentials helped in gaining precise time relationships between the nerve signals and the wing motion, together with the sound signal. With the electrodes inserted directly into the cubital vein, we observed the changes in the sound pattern described by Schäffner & Koch (1987). With electrodes inserted directly into both cubital veins, chirps with missing syllables were observed. Thus we assume that in these recordings the wire had damaged or destroyed the cubital nerve and/or the CCS.

In differential recordings, extracellular nerve potentials (durations less than 1 ms) were observed during sound production (Figs 1, 2). In five animals, bursts of up to 20 nerve impulses were registered, mostly correlated with the closing phase and also mostly correlated with a somewhat deformed syllable envelope (Fig. 1). As seen in Fig. 2A, we also recorded single nerve impulses during the second half of the closing stroke. These were sometimes followed by a series of smaller impulses, which can be interpreted as a series of repetitive discharges. Thus, it is possible to record sensory units from the region of the cubital nerve during singing, but further studies must show the precise origin and time relationship of such potentials. To explain these results, we assume that the units recorded here react only to strong force peaks.

These may occur if the movement of the wings is somewhat irregular, i.e. a sudden sticking of the wing could produce sharp transient force peaks (see also Discussion).

Fig. 3A shows wing position and velocity, and sound envelope of a four-syllable chirp in an intact G. campestris male. This typical recording was taken from a long continuous song bout. The four repeated movements can be easily distinguished. Between chirps, a characteristic resting position (about two-thirds open) is assumed. The first syllable is started with a further opening motion from this resting position. The wings remain in this maximal open position for about 5–10 ms. Then the first closing of the wings produces the sound of the first syllable. Four opening and closing phases can be seen. Sound is always and only produced during the closing phase. The closing speed is only about one-third of the opening speed. The time course and peak speed of the opening phase vary between syllables. In contrast to this, the time course of the closing phase is remarkably constant. At the beginning of the closing phase, wing speed rises in a ramp-like fashion, and later remains at an almost constant value, which we will call the ‘closing speed base value’. While the maximum opening position of the wings is the same for each syllable, the endpoint of the closing motion proceeds further inwards for each subsequent syllable in the chirp. This means that, starting on the same point of the file, more and more lateral teeth are used in the later syllables of the chirp. After each closing, the wings are opened again to the same maximum opening position. Thus, the systematic increase in syllable duration is caused by the wings moving further inwards in each syllable. At the end of the fourth syllable, the wings are closed furthest. Then they are moved back to the interchirp resting position. To obtain a good data base of reference recordings, wing motion and sound envelope were recorded from all experimental animals in the intact state. They showed a very close correspondence in the features described above.

After lesions of the cubital nerve proximal to the CCS, the wing movement recordings were resumed. Examples taken from stable calling songs are presented in Fig. 3B—F (same animal as Fig. 3A). In Fig. 3B, the wings do not close (arrow) after the opening for the second syllable. Accordingly, no sound is produced before the third syllable, when the wing is opened somewhat further than the normal opening position. Fig. 3C shows a motion stop after the second syllable. After the failure of the third syllable, there is a further opening starting at the (successful) fourth syllable. The base value of the closing speed (about 0·7° ms⁻¹; Koch, 1980) is only kept for a short time. After this, the closing speed becomes very fast and rises to three times the opening speed. At the same time, syllable duration is markedly reduced. In these cases the sound envelope often has sharp peaks and other deformations.

Examples of other missing syllables are shown in Fig. 3D—F. In Fig. 3F, all four syllables are missing. The inward movement always stops at the same position, which is approximately the interchirp resting position. Although very many combinations of missing and deformed syllables have been found, deformation and/or loss of the third and fourth syllable seem to occur more often.

Further examples of the variability in the movement patterns are shown in Fig. 4. The upper part shows the intact song, the lower part the songs after the lesions. One can clearly distinguish the alteration in the lesion song. Sometimes, sound is even produced during the opening phase (Fig. 4A). In lesion songs, the closing speed base value is often not maintained. Whenever higher closing speeds occur together with effective sound emission, the closing speed can be assigned to a multiple of the basal value (Fig. 5A,B). Several of these multiples can occur in a single syllable; the speed record then shows ‘steps’ (e.g. Fig. 5A, last syllable; Fig. 5B, penultimate syllable). As mentioned above, these increased speeds result in a strongly reduced syllable duration (sometimes less than 5 ms).

The movement recording results can be summarized as follows.

• (1) The plectrum slides over the file without sound production at a high speed (‘slipping’). The slipping speed may be as high as three times the opening speed. Slipping can occur during the whole closing phase, which leads to a missing syllable (Fig. 5C). If slipping occurs during a part of the syllable (often near the end of the closing phase), syllable duration is reduced accordingly.

• (2) The plectrum stops its motion: it ‘gets stuck’ on the file. Sticking can occur in any part of the closing phase and may persist for very short times or for the whole syllable cycle. Some sticking may also occur at the beginning of intact syllables, but it is always followed by ‘normal’ sound and normal speed. Often, there is a small sound emission (one-quarter of normal intensity) at the beginning of a syllable just before sticking takes place (e.g. Fig. 3E,F).

• (3) Anomalously high closing speed with sound emission (‘overspeed’). This often occurs after some initial sticking and leads to syllable shortening (e.g. Fig. 3B,C).

• (4) In intact males, one never observes sound production during the opening phase. Even in lesioned males it occurs rarely, except where the male changes permanently to a left-over-right singing position (Elliott & Koch, 1983; Schäffner, 1985).

• (5) The interchirp resting position and the maximum opening position show a stronger variance in lesioned songs.

These findings also explain why syllable shortening was found in one-sided lesion experiments, whereas missing syllables were only prominent in two-sided lesions.

A plausible explanation for the movement anomalies could be changes in the centrally programmed neuromotor activation pattern of the main opener and closer muscles (described by Huber, 1965; Ewing & Hoyle, 1965; Kutsch, 1969; Innenmoser, 1974; Weber, 1974; Elepfandt, 1980). We therefore made EMG recordings of the M99 (opener) and M90 (closer) muscles in males with lesions of the cubital nerve proximal to the CCS.

The potentials of the opener and closer muscles (M99 and M90) are unaffected by the lesions of the cubital nerve (Fig. 6). The time pattern of the EMG potentials corresponds very closely to the patterns reported for the intact male. The closer muscle M90 is activated 5–7 ms before the appearance of the sound. This delay is caused by the time necessary to activate the muscles and the wing mechanics. As in the intact male, several motor units may be activated, especially near the end of the chirp. The opener muscle M99 is activated in alternation with M90, marked by arrows in Fig. 6A. As a result of cross-talk, the trace of the opener muscle also contains the closer muscle signal.

The muscle potentials show a striking stability, even where the sound intensity envelope indicates missing or mutilated syllables. This is illustrated in Fig. 6B-D,F (asterisks). Although the M90 trace shows an undisturbed four-syllable pattern, the sound envelope signal in Fig. 6B shows only three syllables: the second syllable is missing. Fig. 6C shows a strong deviation from the usual 30 Hz syllable pattern. The sound is generated between the second and third syllables, and the third syllable is missing, although M90 had been activated correctly. Presumably, the wings got stuck at the beginning of the second syllable and only closed when the peak of muscular power was reached, just before the opening stroke for the third syllable. Such events were recorded in 3–4 % of the chirps and must be especially unattractive to the female (see Thorson, Weber & Huber, 1982; Schäffner & Koch, 1986). In addition, it can be shown that the three-syllable chirps occurring in lesion songs even within long continuous singing bouts are in fact four-syllable chirps with a missing first or last syllable (e.g. Fig. 6F).

In order to give a more quantitative evaluation of these findings, we used joint-interval (J–I) histograms of the EMG and the sound envelope signals of the same 1-min sections of stable calling song. The J–I histogram of the closer muscle signal (Fig. 7A) shows remarkable stability. The data points scatter by only ±6 ms.

This is in good agreement with the data found in intact G. campestris (Weber, 1974). In the J–I histogram of the sound envelope signal, however, a much larger scatter can be seen, corresponding to substantial disturbances in the acoustic output of the lesion song. Although some increase in the scatter can be assigned to the normal irregularities in the onset of sound, a number of data points (filled arrows) located at twice the standard interval indicate the occurrence of missing syllables (7 out of 600 data points). A further set of data points (open arrows) is closer to the average yet clearly distinguishable from the main ‘cloud’. These points represent strong syllable displacements (see Fig. 6C) or syllable shortenings.

The irregularities found in the sound output and in the movement recordings in males with lesions of the CCS cannot be found, therefore, at the level of muscle potentials in the main stridulatory-muscles. The centrally generated pattern persists as if the male were intact. It is not changed during even the most severe disturbances of the sound output.

In wing movement recordings from males with lesioned cubital nerves, one prominent feature is the enhanced closing speed, which is often associated with no sound production. In examples where sound is produced at high closing speeds, the speed values are always multiples of the basal closing speed. These findings accord with the ‘clockwork’ hypothesis (Elliott & Koch, 1985; U. T. Koch, C. J. H. Elliott, K.-H. Schäffner & H.-U. Kleindienst, in preparation), which proposes that the resonance properties of the harp control the closing speed of the wings. In the intact male, the plectrum jumps from one file tooth to the next, and this takes the time of one sound cycle. If the plectrum jumps over n teeth per sound cycle – that means the animal uses every nth tooth – the closing speed must be n times the base value. Intermediate speeds, such as 1–5 times the base value, should not occur, as indeed they do not (see Fig. 5). Syllables with closing velocities of twice the base velocity were also observed in males with a reduced syllable duration (see Elliott & Koch, 1983). In contrast, when the wings close without sound production (slipping), the harp is not engaged in speed control. Accordingly, a continuous spectrum of closing speeds up to very high values is observed (Figs 3B–E, 5C) (see U. T. Koch, C. J. H. Elliott, K.-H. Schäffner & H.-U. Kleindienst, in preparation).

EMG recordings present the surprising result that the neuromuscular pattern generates perfect opening and closing strokes while severe defects are seen in the sound signal. This must mean that the central pattern generator for singing, presumed to be located in the thoracic ganglia (Bentley, 1969; Kutsch & Otto, 1972), is not affected in its basic rhythmicity by the removal of influence from the CCS. These statements are based on recordings from the two most important opener and closer muscles, but they do not exclude a CCS influence on some of the less prominent muscles (see Schäffner, 1985).

For the hypotheses formulated below we should bear in mind the following.

(i)Male crickets have special campaniform sensilla, which are located and oriented on the wing in such a way that they are able to measure thrust forces along the file. The extracellular recordings from the cubital nerve show song-correlated nerve potentials mostly when the file is at maximum thrust load near the end of the syllable.

(ii)Lesions of the cubital nerve that remove the influence of the CCS cause a high frequency of missing syllables, significantly reduce syllable intervals and increase their variance, and lead, in part, to drastic reductions in syllable duration. Songs modified in this way have a significantly reduced attractiveness in female phonotaxis (Schäffner & Koch, 1987).

(iii)The alterations in the lesion songs can be traced back to changes in the wing movement, to an increased wing closing speed with sound production (overspeed) resulting in shorter syllables, to wing closing without sound production (slipping) causing syllables to be missed or markedly shortened, and to stops in wing motion (sticking) causing missing or shortened syllables. These alterations can affect only parts of a syllable, and/or several of them can affect the same syllable.

(iv)In spite of these well-documented changes in the wing movement, the motoneuronal excitation pattern to the major wing opening and closing muscles in lesioned males remains unaffected by the CCS lesion.

The following hypotheses should help to explain the alterations seen in lesion songs.

(1)Since the development of the muscular power for the closing movement is unaffected, the changes described in iii must be caused by alterations in the forces that are orthogonal to the file and to the wing surface. These will be labelled ‘engaging forces’. Engaging forces that are too high would thus result in ‘sticking’, while forces that are too low would either cause ‘slipping’ or ‘overspeed’. The concept of slipping caused by insufficient engaging force is evident. The sticking due to excess engaging forces can only occur if the form of the file teeth and plectrum are such that effective locking is possible, as in a ratchet. Scanning micrographs show that the teeth and plectrum profiles are in fact adequate to produce locking (Schäffner, 1985; G. Breutel, personal communication).

(2)Using postural muscles, which may be tonically active, or asymmetries in the excitation of the major muscles (see Kutsch, 1969), the cricket could adjust the engaging pressure such that sliding as well as sticking are avoided, and optimum operation of the clockwork mechanism is achieved.

This postulated control system needs two sensing elements, one measuring wing position or velocity, such as the wing hinge stretch receptor and chordotonal organ, and the other measuring thrust along the file (closing force), such as the CCS. The aim of this control loop would be to apply as high closing forces as possible (avoiding sliding), while keeping the wings in motion (avoiding sticking). The result of this action would be a maximum sound power output p, which depends on closing velocity v and closing force f in the following way: p = f × v. Here, v need not be measured with precision as long as it is not zero, since the clockwork mechanism regulates v automatically. In such a system, the removal of the force transducer would cause the control loop to increase the engaging forces, since ‘sliding’ is signalled by ‘no closing force’ information. The primary result would then be sticking, which would be sensed by the movement detector and cause the system to reduce engaging pressure, resulting in ‘slipping’.

The hypotheses outlined above should predict disturbances if the movementsensing elements (stretch receptor and chordotonal organ) were removed from the system. Moss (1971) reported no changes in the song pattern when he removed the stretch receptor. This is not necessarily a contradictory result, since Moss left the chordotonal organ intact, which may have led to a situation similar to the one-sided lesions of the CCS : one of the two sensory systems keeps up the system’s performance to a large extent. In addition, Moss (1971) looked for changes of the song pattern only at the level of the EMG signals which we have shown to be unaffected even during severe disturbances of the sound output.

A closer inspection of the proposed feedback system raises several questions.

(1)Reaction time. Since the syllable duration is only 15 ms and the sensory information from the CCS and the velocity sensors is spread over the syllable duration, it seems unlikely that the feedback loop could correct wing engaging forces within the same syllable. Rather, we assume that the sensory information is integrated and stored for the adjustment of engaging forces in the next cycle, in a way similar to the position-control system of the postcubital hair fields (Elliott, 1983).

(2)Generation of engaging forces. Preliminary experiments with an isolated wing–thorax preparation (U. T. Koch, unpublished results) show that a torque around the longitudinal axis of the tergal plate can generate the engaging forces. Such a torque could be generated by the slight asymmetries in the timing between left and right closer muscles, as observed by Kutsch & Huber (1970). As in locust steering reactions (Zarnack & Mòhl, 1977), information from the sensory systems could be used to change the muscle timing and thus change the engaging forces. The results of Kutsch (1969) seem to contradict this scheme. He showed that the cricket was still capable of singing when all wing muscles on one side had been cut or denervated, demonstrating the very strong coupling between both sides of the thorax.

A further interesting candidate for the generation of engaging forces is M85. In the locust, M85 has been shown to produce wing twisting leading to pronation (Pfau, 1983), which may be analogous to the wing twist required to generate engaging forces. In addition, a reflex connection between campaniform wing sensilla and M85 was found (Heukamp, 1983; Wendler & Heukamp, 1983). Light microscopic observations in the cricket (Schäffner, 1985) showed that cubital nerve projections end in an area of the mesothoracic ganglion which is occupied by ramifications of M85. This supports the idea that a reflex connection between M85 and the CCS may exist. However, because of the lack of knowledge about the precise functional morphology of the cricket wing hinge, the ideas about the role of M85 or other muscles in the generation of engaging forces must remain speculative.

The influences of wing sensory systems described here stabilize the sound output of the singing cricket in such a way that sound pulses of homogeneous quality are reliably produced, thus transforming the very stable basic motor spike pattern into an equally stable sound pattern. The results of the phonotaxis experiments (Schäffner & Koch, 1987) underline the biological relevance of this control system. Male crickets without information from the cubital campaniform sensilla produce faulty calling songs and have a much reduced success in luring females for mating.

We gratefully acknowledge the support and constant interest of Professor Franz Huber. Much help was offered by our fellow scientists in Huber Abteilung. We are especially grateful to Chris Elliott, Theo Weber, Hans-Ulrich Kleindiest and Günter Kàmper for their technical support and helpful discussions.