J. Exp. Biol. (1971), 54. 587-597 5 8 7 With 1 plate and 9 text-figures
Printed in Great Britain
RHYTHMIC ACTIVITY IN THE.INSECT NERVOUS SYSTEM
I. VENTILATORY COUPLING OF A MANTID SPIRACLE
BY P. L. MILLER Department of Zoology, Oxford
{Received 19 October 1970)
INTRODUCTION
Insect ventilation together with its associated spiracle movements provides a good system for the study of neural oscillatory mechanisms in the central nervous system. In acute highly dissected preparations ventilatory activity continues whereas other forms of rhythmical activity such as flight or stridulation, may be hard to evoke. Rhythmical activity, probably ventilatory in nature, persists in some single abdominal ganglia (Farley, Case & Roeder, 1967); in an isolated locust abdominal ganglion alternating bursts of impulses in motor nerves which supply inspiratory and expiratory muscles can be recorded for 6 h when no external stimulus is applied (Mulloney, personal communication). In some large insects signs of the ventilatory rhythm may be seen in almost every body segment as pumping movements or as synchronized spiracle activity. Spiracle movements synchronized with ventilation and probably helping to produce unidirectional airstreams are known in dragonflies, cockroaches, mantids, crickets, locusts, grasshoppers, bees and beetles (review: Miller, 1966). In locusts, spiracle synchronization is mainly obligatory while in other insects, such as dragonflies and mantids, it appears to be facultative, the spiracles being joined to the ventilating rhythm only intermittently (Miller, 1962). The coupling and uncoupling of an activity to an ongoing rhythm is of some general interest in the study of insect neurophysiology and behaviour; it appears to happen for example in the courtship of cerambycids (Michelsen, 1967), and has been recorded in the insect nervous system (Hoyle, 1964; Bentley, 1969). In this paper the morphology, innervation and normal activity of spiracle 1 of Sphodromantis are described by way of an introduction to the ventilating system of a species which is proving suitable for attempts to understand central control mechanisms.
588 P. L. MILLER
muscle of spiracle i of Schistocerca controls the aperture when the closer relaxes (Miller, i960) and it does not act as a simple antagonist to the closer. This suggested that the two muscles of spiracle 1 of mantids might act in a similar manner. However,'1 it is shown here that they act as synergists receiving identical motor input, but each controls a separate part of the valve and there is limited independence of movement.
MATERIAL
Egg pods and adults of Sphodromantis lineola (Burmeister) were obtained from south-west Nigeria and brought to England in March 1968. Since that time they have been reared in culture. First and second instars are kept together in cages and fed on
Drosophila. Later instars are kept separately in small jars and then in Perspex cages,
21 cm x 21 cm x 10 cm, with a gauze roof and provided with a twig. They are fed two or three times a week on cockroaches of appropriate size. Mature males (the dorsal surface of the abdomen turns dark red when mature) are introduced into females' cages for mating which lasts for 24 h. Provided a male is removed after copulation he can be mated with several females. Females do not normally eat males until copulation is completed. The cages are kept during the day at about 30 °C. Both sexes have been used in experiments.
METHODS
For recording ventilatory and spiracle activity, adult mantids are strapped to a Plasticene block ventral side up. Abdominal movements are recorded with a transducer (RCA 5734) attached to the insect by means of a weak rubber band and thread waxed to the appropriate sclerite. As pointed out by Edwards (1953) mantid ventilatory movements are complex and involve dorso-ventral and longitudinal telescoping movements which give complex wave forms when ventilation is slow.
Spiracle movements were recorded using a small mirror (weight c. o-i mg) fixed to the valve with petroleum jelly. The mirror reflected a focused beam of light on to a phototransistor (Mullard OCP 71) which was positioned close to it and whose output was displayed on an oscilloscope. Angular movements of the valve produced large deflexions whereas movements of the whole spiracle produced negligible readings. The valve movements were not impeded by the mirror. The method provides a convenient means of measuring the occurrence, speed and something of the form of valve move-ments which in all experimove-ments were checked by direct observation through a micro-scope.
The electrical activity of the spiracle muscle was recorded in intact preparations with extracellular platinum-wire electrodes, insulated except for the tip, or with intra-cellular glass micropipettes, filled with o-6 M K2SO4. Before recording, the spiracle plate was fixed firmly with wax to surrounding cuticle to prevent movement. The overlying cuticle was removed and the muscles were then covered with a thin layer of petroleum jelly to prevent desiccation.
Rhythmic activity in the insect nervous system. I
RESULTS
Morphology and action of spiracle 1
Spiracle 1 has a posteriorly directed external valve which is closed by muscular action and opened by cuticular elasticity (Text-fig. 1). In a mature female the valve opens to reveal a narrow atrium, 1050 /im long, from which a dorsal (400 /tm) and a ventral (175 /tm) orifice lead off. The dorsal orifice is joined to a single large trachea which runs anteriorly and supplies prothorax and head. The ventral orifice leads to a tracheal anastomosis from which two large branches pass anteriorly and two posteriorly. A major part of the air supply from spiracle 1 is thus fed anteriorly to the prothorax, the
Ventral orifice Dorsal orifice
Text-fig. 1. External view of spiracle 1 of Sphodromantis from the posterior. Long muscle V Fan muscle
590 P. L. MILLER
enlarged forelegs and the head (tracheae 1-3 in Text-fig. 2), regions which are well removed from the spiracle by the elongation of the prothorax. Further derails of the/ supply will be considered elsewhere.
Closure of the valve cuts off first the ventral orifice from the outside; further closing then seals the dorsal orifice. The valve has a soft sticky cuticular pad which covers the ventral orifice and a flexible mid-region which allows a limited independence of move-ment to the dorsal half.
A fan-shaped muscle arises on the peritreme and inserts on to the valve close to the
hinge. A long muscle arises beside the fan muscle and inserts on to the base of the dorsal orifice trachea which is attached to the valve (Text-fig. 2). The fan muscle is a flattened cone 800 /*m long and 500/tm wide at the base. The long muscle is a flattened cylinder 730 /tm long and 270 /im wide. The fan muscle controls the move-ments of the pad which closes the ventral orifice; the long muscle pulls the remainder
Ventral orifice
Dorsal orifice
Fan muscle
Text-fig. 3. A model of spiracle i of Sphodromantis to illustrate the action of the long and fan muscles. The fan muscle pulls part of the valve down on to the ventral orifice: the long muscle pulls the rest of the valve on to the dorsal orifice.
of the valve down on the dorsal orifice. By destroying each muscle separately it has been shown that a strong contraction of the long muscle can close the whole valve while contraction of the fan muscle closes only the ventral orifice. The action of the muscles is illustrated in the model in Text-fig. 3. Both can accurately be described as closers, but their action is not identical.
Rhythmic activity in the insect nervous system. I 591
stained with Toluidine Blue show that the median nerve contains two axons s-5-6*o /*m in diameter which split at the junction into right and left branches, join the lateral nerve from the mesothoracic ganglion and then leave it to branch and run into both muscles of the spiracle (PI. 1, fig. 1). The two axons expand to 8/im when they run in the lateral nerve; they are about 13 mm long in a mature female. The median nerve also contains 7-8 small axons, 2'$-2'O/im in diameter, and an additional one or two axons
c. 1 /im in diameter. None of these can be traced to the spiracle.
Activity of spiracle 1
Ventilation normally comprises an expiratory stroke followed immediately by an inspiratory stroke and then a pause before the next cycle commences. As the frequency increases the pause is shortened and may disappear. Movements synchronized with ventilation commonly occur in the spiracles.
Text-fig. 4. Mirror records from spiracle i (upper lines: closing upwards) and transducer records of abdominal ventilation (lower lines: expiration upwards) in Sphodromantis. A, spiracle I closes fully during expiration but is wide open for the rest of the cycle. B, following treatment with 5-10% COa spiracle 1 opens only partially. C, brief expiratory closing of
spiracle 1 is followed by momentary full opening before the end of expiration. D, fast ventila-tion in air following mechanical stimulaventila-tion of the abdomen: spiracle 1 opens partially during inspiration and then fully before the subsequent expiration. Scales, 1 s.
Activity of spiracle 1 during the expiratory stroke. During strong ventilation the valve
closes partly or fully at the onset of expiration. It may re-open on the termination (Text-fig. 4 A) or before the end (Text-fig. 4C) of the expiratory stroke. When ventila-tion is shallow a weak closure or no movement may appear in the valve, the spiracle being then uncoupled from the pump.
Activity during the inspiratory stroke. In air the dorsal orifice opens towards the end
592 P. L. MILLER
follows. The ventral orifice remains closed throughout inspiration but opens towards the end of the pause, shortly before the start of the next expiratory stroke (Text-fig. 4D). In 5-10% CO2 the ventral orifice usually remains closed throughout the cycle (Text-fig. 4B)..In higher concentrations of CO2 both orifices open fully during inspiration and the expiratory closing movement may become very brief. Thus in air the dorsal orifice may be open for 80 % of the whole cycle, and the ventral orifice for 20 %, but in low CO2 concentrations the figure for the dorsal orifice is unchanged while the ventral orifice is permanently sealed. Brief opening of the ventral orifice sometimes occurs immediately after expiration but this is an uncommon pattern (Text-fig. 4C). The functional significance of this variety of patterns of synchronized movements in terms of directing airstreams along certain tracheae will be discussed elsewhere. For the present only the neuro-muscular mechanisms which control the movements will be described.
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Text-fig. 5. Simultaneous electromyograms from the muscles of left and right spiracles. A, the synchrony of EPSPs is apparent. Scale, 100 ms. B, short expiratory bursts are shown with lower frequency firing for part of the remainder of the cycle. Electrical activity of this pattern produces the movements shown in Text-fig. 4D.
Text-fig. 6. Intracellular records from fibres in the fan muscle of spiracle 1 in an intact prepara-tion. A, the lower line shows corresponding spikes recorded in the median nerve posterior to G I. B, records from a fibre in which the responses to activity in the two motor axons appear to be of different amplitude. Scale 200 ms and 2 mV.
Electrical recordings from the spiracle nerves and muscles
Recordings taken either from left and right branches of the median nerve, or from the muscles of left and right spiracles, show a 1:1 synchrony of spikes (Text-fig. 5). It is probable therefore that the spiracles are supplied by motor axons in the median nerve which split to supply both sides, as in other insects (Case, 1957).
Rhythmic activity in the insect nervous system. I 593
but left intact centrally, efferent activity in five axons can be recorded which are distinguishable on the basis of spike size. The spikes of largest amplitudes occur in the two motor axons which run to the spiracle muscles. They have been correlated with the occurrence of EPSPs recorded intracellularly in muscle fibres (Text-fig. 6 A). The efferent spikes of smaller amplitude show no pattern correlated with ventilation and do not appear to run to the spiracle. They may occur in motor or neuro-secretory axons. The occurrence of neuro-haemal organs associated with abdominal but not with thoracic median nerves has been described in several insects (Brady & Maddrell, 1967; Smalley, 1970; Finlayson & Osborne, 1970).
After section of the median nerve the spiracles show CO2-sensitive spontaneous activity, probably myogenic in origin like that of spiracle 2 of the locust (Hoyle, 1961). There is no evidence of neurally driven activity after this operation. Moreover stimulation of the first lateral nerve of GII is without effect on the spiracles, so that an additional motor nerve from G i l , which is known to supply spiracle 1 of Schistocerca (Miller, 1965), is absent from Sphodromantis. Intracellular recordings from fibres of each muscle together with electrical stimulation of the median nerve, cut centrally, indicate that every fibre is innervated by two motor axons both of which produce small facilitating EPSPs with no active membrane response. In some fibres the response to each may be an EPSP of 10-15 mV, but in other fibres one axon may produce an
Text-fig. 7. A, simultaneous intracellular records from the long (upper) and fan (lower) muscles of spiracle i, showing the synchrony of EPSPs. In this example large EPSPs in one muscle correspond to small ones in the other. The small apparent hyperpolarizations are artifacts. Scales, 20 ms and 2 mV. B and C, mirror records of spiracle I activity (upper lines: closing upwards) taken during stimulation of the median nerve cut centrally (lower lines). B, short bursts of i ms shocks at ioo Hz produce immediate closing, the spiracle reopening after a short delay. Scale, I s. C, an initial shock frequency of 6 Hz has no visible effect, but when it is increased to 40 Hz the ventral orifice closes. A further increase to 70 Hz (arrow) causes the dorsal orifice to close. Scale, 1 s.
EPSP of 10 mV while the other produces one of only 1-2 mV amplitude (cf. Text-fig. 6B). Unilateral section of the first G i l lateral nerve, intracellular recordings and electrical stimulation of median and lateral nerves all fail to produce evidence for inhibitory innervation of this spiracle, which may therefore be unlike the first spiracle of Blaberus discoidalis in this respect (Miller, 1969). The innervation is comparable to that of spiracle 2 in Schistocerca (Hoyle, 1959).
594 P- L. MILLER
the activity of the other examined alone; (3) intracellular recordings have been taken from a fibre in each muscle simultaneously. Contractions always occur in each together, and there is a 1:1 relationship between EPSPs in the two muscles (Text-fig. 7 A). After removal of GI and Gil, the median nerve has been stimulated with i-o ms electrical pulses to examine the effects of different frequencies on valve closing. The unstimulated valve in a deganglionated preparation usually remains about half closed probably as a result of spontaneous myogenic activity of the spiracle muscles. Single shocks produce very small twitches. Shocks delivered at 30 Hz cause the ventral orifice to close completely, the dorsal orifice remaining open. A frequency of 70 Hz is needed to produce full closing of dorsal and ventral orifices (Text-fig. 7B, C). After stimulation the valve may open fully for several seconds before again closing halfway, possibly as a result of CO2 produced by the preceding contraction. In natural trains of spikes the two axons do not normally fire synchronously, whereas the applied shocks stimulate both axons together. The responses to different frequencies of stimulation cannot therefore be compared directly with naturally occurring frequencies of spikes.
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Text-fig. 8. A, records from the median nerve posterior to G I during high-frequency ventila-tion produced by 10 % COa. The nerves fire strong expiratory bursts but are otherwise silent. B
and C, electromyograms from a spiracle i muscle (upper lines) together with mirror records from the contralateral spiracle 1, in 5 % CO2 (lower lines: closing upwards). Interburst firing keeps
the ventral orifice partly closed while expiratory bursts close the dorsal orifice. All scales, i s. Normal patterns of activity in the motor nerves to spiracle 1
Expiratory closing of the valve is caused by a burst of impulses which usually occur at 50-100 Hz in each axon. Such bursts normally start and end abruptly. In 5 % CO2 interburst activity, in which the ventral orifice remains closed, is caused by lower-frequency firing (c. 10-20 Hz in each axon) (Text-fig. 8 B, C). At such times the tension produced by the fan muscle overcomes the cuticular spring, whereas that produced by the long muscle does not. This is so probably because in the fan muscle more fibres are acting in parallel. High-frequency bursts which cause the long muscle to close the dorsal orifice may also produce further contraction in the fan muscle which can be seen as an inward bending of the cuticle of the valve. In air the ventral orifice opens after inspiration and impulses temporarily cease (cf. Text-figs. 4D, 5B). With higher concentrations of CO2 there may be no interburst activity (Text-fig. 8 A) and the expiratory bursts which produce closure become brief.
Rhythmic activity in the insect nervous system. I 595
in the cockroach (Farley et al. 1967), it appears therefore that G i l l contains the most .anterior ventilatory pacemaker.
Records from the median nerve of an isolated head + prothoracic segment preparation normally show no bursts, but continuous low-frequency firing occurs in both axons similar to interburst firing in intact insects. This activity is comparable to the free running described in a similar preparation from Schistocerca (Miller, 1965). However, after treatment with CO2 or following activity of the forelegs, free running may cease and there is a series of sharp bursts each producing full spiracle closing. In the
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Text-fig. 9. Activity in spiracle i after section of the G I - I I connectives, recorded with a mirror in A—D, and as an electromyogram in E. A and B, continuous record from head and prothoracic segment preparation. Following leg movements the spiracle makes seven abrupt closing movements (upwards) of increasing duration and occurring at longer intervals. C and D, isolated prothorax (continuous record), a gentle stream of CO2 aimed into spiracle i for 20 s
(lower line) produces an immediate partial opening followed by further slow opening during which two bursts occur. With recovery there is a further burst. Opening in response to COa
occurs in two stages because CO2 acts first on the muscle and later at the ganglion to slow the
frequency of the motor output. E, a burst of firing which interrupts free running and produces a sharp closure, following CO2 treatment. It is followed by a postburst depression of free
run-ning. Scales A, B and E, 1 s; C, D, 10 s.
596 P. L. MILLER
depression before free running is resumed (Text-fig. 9E). The depression may be several times longer than the preceding burst and leads to full opening of the valve. It is reminiscent of the pause which often follows an expiratory burst in intact prepara-tions, although there it is usually shorter and only rarely leads to full opening.
In a preparation of the isolated prothoracic segment, free running occurs at a higher frequency than when the head is present, but bursts can still be produced after treatment with CO2 or following leg movements, although commonly only two or three occur (Text-fig. 9C, D). The bursts are not accompanied by other movements, or by signs of activity in other muscles.
DISCUSSION
The valve muscles of spiracle 1 of Sphodromantis are controlled by two excitatory motor axons. Five axons are used in the control of spiracle 1 of Schistocerca (Miller, 1965) and three in that of Blaberus (Miller, 1969). The patterns of activity recorded are similar to those seen in some species of adult dragonfly (Miller, 1962) where at least two axons are involved.
The two muscles of spiracle 1 in Sphodromantis may have evolved from a single closer muscle and they can be regarded as two parts of one muscle. The closer muscle of spiracle 1 of Periplaneta similarly comprises two parts whereas that of Blaberus is a single muscle. The alternative hypothesis that the two muscles correspond to the opener and closer muscles which are found in Schistocerca and other Orthoptera is not supported by their shared innervation or by their common insertion on the peri-treme. Spiracle 1 of Sphodromantis exhibits a similar degree of control as does that of
Schistocerca but with a considerable economy of innervation.
The isolated prothoracic segment of Sphodromantis can produce bursts of impulses in spiracle motor nerves in response to CO2. Such behaviour has not been observed in an equivalent preparation from Schistocerca. Bursts in the isolated segment are similar to those occurring in intact Sphodromantis. This suggests that burst formation may be a normal property of the spiracle motor neurones or interneurones of the prothoracic ganglion and that command interneurones from a ventilatory centre perhaps do no more than trigger the start of a burst. This suggestion is examined further elsewhere. In Schistocerca on the other hand it was suggested (Miller, 1967) that burst formation took place more posteriorly and that bursts were relayed anteriorly by interneurones.
SUMMARY
1. Spiracle 1 of Sphodromantis lineola possesses a moveable valve equipped with two closer muscles which act as synergists, receive identical innervation but act on different regions of the valve.
2. The two muscles are controlled by two motor axons which produce simultaneous small EPSPs in the two muscles of the left and right spiracles.
3. Valve activity can be facultatively coupled to the abdominal ventilator} rhythm. Three patterns of coupling have been recognized.
Journal of Experimental Biology, Vol. 54, No. 3
Plate 1
Rhythmic activity in the insect nervous system. I 597
I am grateful to the Science Research Council for financial support, and to Mrs Unu •^ansrani for technical assistance.
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EXPLANATION OF PLATE
