J. Exp. Biol. (1964), 41, 331-343 33I With 12 text-figures Printed in Great Britain

FACTORS ALTERING SPIRACLE CONTROL IN ADULT : WATER BALANCE

BY P. L. MILLER* Department of Zoology, Makerere College, Kampala,

{Received 5 October 1963)

INTRODUCTION The valves of all the spiracles of adult dragonfiies are controlled by single closer muscles. These muscles respond to carbon dioxide directly (Miller, 1962) probably through the same mechanism as that which occurs in spiracle 2 of the locust (Hoyle, i960). The threshold of the response to carbon dioxide is determined in part at least by the frequency of motor impulses in the nerve to the closer muscle. Hypoxia, flight and abdominal ventilation have been shown to alter this frequency in spiracle 2 of dragonfiies in different ways (Miller, 1962), and the water balance is here shown to be another factor which may affect it and hence exert an influence over spiracle control. In dragonfiies which are partially desiccated spiracle control is tight and the threshold of the carbon dioxide response is high, while in well-hydrated the threshold is low. Mellanby (1934) and Ramsay (1935) have clearly demonstrated the importance of the spiracle valves in reducing water loss from an . Changes in spiracular activity following alteration of the water content, however, have only recently been recognized (Bursell, 1957), although the likelihood of such changes has been envisaged for many years (see Wigglesworth, 1953, p. 433). Some initial observations were made in the field in Uganda on about 60 specimens of (Burmeister) and Trithemis aimulata (Palisot de Beauvois) (). They indicated that spiracle control varies very much in different individuals caught on the same occasion and examined immediately after capture. For example spiracle 2 might close instantly after a burst of struggling or it might continue to make movements synchronized with abdominal ventilation for a further 2-3 min. Such variation may depend on many factors including the age, condition, sex and temperature of the and on the interval since the last meal, but the experi- ments reported here indicate that water balance may also be a contributory factor.

MATERIAL Most of the experimental work was carried out in Uganda on ferox Rambur (). Methods for obtaining teneral and mature adults have already been described (Miller, 1962). Other used are mentioned in the appropriate sections. • Present address: Department of Zoology, Oxford, England. 332 P. L. MILLER

METHODS The means of registering spiracle valve movements and of recording nerve impulses have already been described (Miller, 1962). In all experiments the central recording site was chosen and paraffin oil was not employed. The experiments were carried out at 23-250 C. and at 60-70% R.H. The frequency of nerve impulses was determined from counts made on films of the oscilloscope trace. The values given represent the sum of the frequencies in the two motor axons to the spiracle, which were seen to fire at more or less the same frequency under all experimental conditions. They are derived from the mean of 10-20 counts each of 200 msec, duration. The physiological saline used contained 153-1 mM./l. sodium, 2-6 mM./l. potassium, i-8 mM./l. calcium and 24 mM./l. glucose, pH 6-8-7-0. Other techniques are described under the appropriate experiments.

RESULTS Experiment 1. The effect of changed water balance on the control of spiracle 2 This experiment was carried out in order to determine whether dragonfiies which have been made to drink copiously exercise a different degree of control over their spiracles compared with those which have been partially desiccated. In addition to , several small libellulid species were used: Brachythemis leucosticta (Burmeister), B. lacustris Kirby, Trithemis amtulata (Palisot de Beauvois) and Orthetrum iulia Kirby in Uganda, and Sympetrum striolatum Charp. in England. Mature insects were chosen 1-2 days after their capture, by which time most of their gut contents had been voided. The tests were each made on batches of 4-7 insects and in all over 100 have been examined. Dragonfiies were made to drink by allowing them to chew at the end of a pipette from which distilled water was expressed. By adding 0-5% methylene blue to the water and subsequently dissecting a few specimens the occurrence of drinking was verified by the presence of the dye in the gut. The amount of water imbibed was determined by weighing the insect on a torsion balance after blotting off superficial drops. As much as a 25 % increase in weight was brought about in this way. Dehydration was achieved by keeping insects in dry air over phosphorus pentoxide. The rate of desiccation was accelerated in some individuals by lifting a flap of cuticle from the pterothorax which was later waxed back in position. As an alternative method some dragonfiies were placed in a stream of 1 o % carbon dioxide in which their spiracles remained open. The progress of desiccation was followed by periodic weighings on a torsion balance, and in some insects up to 10% of the original body weight was lost after several hours' treatment. Dragonflies which defaecated during the experiment or which were unduly active were rejected. Many batches of insects were alternately desiccated and hydrated several times. They survived this treatment well and changes in weight brought about by these means were maintained for several hours. At intervals during treatment the valve activity and hence the control exercised by spiracle 2 was checked in 2-5 % oxygen, 5 % carbon dioxide in air, and after a 10 sec. flight. This flight was performed with the insect glued to a small glass rod in front of a Spiracle control in adult dragonflies: water balance 333 wind tunnel: it was initiated by pinching the abdomen and terminated by allowing the legs to grasp a small ball of paper. The rod was weighed with the insect and its weight was subsequently subtracted. In Fig. 1 the percentage change in weight of I. ferox is plotted against the amount of opening of spiracle 2 in 3 % oxygen. After a gain in weight of 10%, brought about by drinking, spiracle 2 opens fully in this gas mixture, whereas after a weight loss of 3-6 %, accompanying desiccation, it remains more or less closed in the same gas.

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O 00 c. 20- c o —r~ —1— —I— -8 -6 -4 0 +2 + 4 + 6 ~7iT Change In weight (%) Fig. 1. Ictmogompkus ferox. Changes in weight brought about by desiccation or hydration plotted against the percentage opening of spiracle 2 in 3 % oxygen in nitrogen.

In Fig. 2 the loss in weight with desiccation of initially hydrated Sympetrum striolatum is plotted against the interval between the end of a 10 sec. flight and the full closing of spiracle 2. After a weight loss of 6-13% the spiracle closes with no delay after a short flight.- Prompt closing also follows considerably longer flights in desiccated insects. Fig. 3 shows the increase in length of the interval between the end of a 10 sec. flight and the full closing of spiracle 2 as the dragonflies are progressively hydrated, following earlier desiccation. Fig. 4 shows the rise in the threshold of the response to 2 % carbon dioxide following prolonged treatment with 10% carbon dioxide. After these last tests saline was injected into the thorax and there was an immediate lowering of the threshold to a value similar to that in hydrated insects. This suggests that prolonged treatment with 10% carbon dioxide does not itself bring about fatigue of the response but has its effect through the water balance. The results show that variations in the amount of spiracle control exercised correspond to the state of hydration of the insect. The experiments discussed below were designed to throw light on the mechanism involved.

Experiment 2. Measurements of the frequency of nerve impulses in the spiracle nerve following desiccation or hydration At least two mechanisms could account for the results reported in Expt. 1: the water balance might affect the spiracular response to carbon dioxide directly, or the action 334 P. L. MILLER

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Fig. 2. Sympetrum striolatum. The percentage loss in weight of four dragonflies, initially well hydrated, plotted against the interval between the end of a io sec. flight and full closing of spiracle 2.

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60 Time (sec.) Fig. 3. Four Brackythemis lacustris (C © • O) and three B. leucosticta ( x A A). The gain in weight, after drinking, plotted against the interval between the end of a 10 sec. flight and the full closing of spiracle 3. Spiracle control in adult dragonflies: water balance 335 might take place through the central nervous system. If the latter holds, then changes in spiracular sensitivity might be reflected in changes in the frequency of motor impulses to the closen Since the threshold to hypoxia as well as to carbon dioxide varies with water balance, and since hypoxia has no direct action on the spiracle muscle (Miller, 1962), it seemed likely that the central nervous system was involved. The following experiments show this to be so, and evidence discussed later suggests that in fact both central and peripheral mechanisms play a part in regulating spiracle move- ments with respect to the water balance.

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§ 60 120 180 Time In 10% CC^ (mln.) Fig. 4. Sympetrum striolatum. The percentage opening of spiracle 2 in 2 % carbon dioxide plotted against the time spent in a continuous stream of 10 % carbon dioxide. The experiment started with twelve dragonflies; after 90 min., one was dead; after 180 min. a further four were dead. Table 1. Correlation of the frequency of motor impulses in the nerve to spiracle 2 with the behaviour of the valve in 2 % oxygen after dehydration or desiccation Weight change Species Regime (mg.) Impulses/sec. Valve activity Macromia reginac Dry -7o (-11%) 108 0-10 % open Wet + 40 ( + 6%) 95 95-100% open Ictinogomphui ferox Dry -60 (-7%) 85 o-5 % open Wet + 75 ( + 8%) 43 100% open Dry -90 (-9%) 54 0-25 % open Wet + 35 ( + 4%) 11 100% open Ictinogomphus ferox and a large corduliid dragonfly, Macromia reginae Le Roi, were subjected to dry and wet regimes as described in Expt. 1. The response of spiracle 2 was tested in 2% oxygen; the spiracle nerve was then dissected out as quickly as possible and a recording was taken. The results indicate that an increased frequency of motor impulses accompanies desiccation while a decrease follows hydration; some examples are shown in Table 1. This technique; however, is not wholly satisfactory since time is not allowed for the insect to recover from the operation, and this may account for the great variation in the results. In consequence an attempt was made to reproduce the conditions of hydration and desiccation by perfusion of the operated dragonfly with saline solutions of different concentrations. 22 Exp. Biol. 41, 2 336 P. L. MILLER

Experiment 3. The effect of varying the strength of the perfusing salines on the frequency of impulses to spiracle 2 Salines of the following concentrations were made up: 0-5,0-75, i-o, 1-5, 2-0, 2-5 and 3-0 x normal. The appropriate solution was then perfused through the inverted insect by running it into the base of a prothoracic leg from a small pipette and allowing it to leave from an incision at the posterior end of the abdomen. In some experiments the solution was added through an opening in the fronts but the site of perfusion did not affect the results. Between 1 and 2 ml. saline was added before recordings were made, and more was pipetted in between readings. Although nearly all the haemolymph was replaced by saline, insects survived well for many hours. As an indicator of 'good condition' the ability to perform flight movements on pinching the abdomen was used. After more than about 2 hr. perfusion with 2*0 x normal saline, the condition began to deteriorate, but this was fully reversible on return to saline of normal strength. In 3-0 x normal the dragonfly was in poor condition after 15-30 min., but again the effect was reversible. 120-1

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Time (sec) Fig. 5. Ictinogotnpkus ferox. Histogram of the frequency of motor impulses in the two axons to spiracle 2 in successive 200 msec, periods, with the insect perfused with double strength, normal strength and half strength saline. The cyclical rise and fall in frequency results from the activity of abdominal ventilation centres and both crests and troughs are affected by the perfusing saline. Recordings from the spiracle nerve were taken at intervals during perfusion until no further change was detected. The response was often slow to appear and might not be complete in less than 30 min. Faster responses were obtained with stronger solutions. In 0-5 x normal saline the discharge was sometimes completely extinguished after long periods of perfusion. The insect showed no ill effects and spiracle closure could still be evoked by stroking the valve hairs. This showed that the neuromuscular mechanism had not been blocked by the dilute solution, although spontaneous firing of the motor neurones had ceased. On return to normal saline firing was soon resumed. This has been repeated several times on the same preparation. Spiracle control in aduh dragonflies: water balance 337 In Fig. 5 the frequencies in 0-5, i-o and 2-0 x normal saline are shown from the same dragonfly as consecutive readings every 200 msec. It can be seen that the high- frequency bursts, coinciding with expiration (Miller, 1962), as well as the troughs of lower frequency are affected by the perfusing saline. In Figs. 6 and 7 the effect of switching between 0-5 and 2-0 x normal salines is shown in .teneral and mature insects. In these preparations the connectives between the thorax and abdomen were cut so that there was no interference from the abdominal ventilation centres and steady readings were obtained. After perfusion with a different saline, teneral insects normally achieve a new value more rapidly than mature specimens, and the changes in tenerals are usually greater than in matures.

Saline xO-5 x20 xO-5 x2-0 x20 xOS x2O

40 60 40 60 Time (mln.) Time (mln.) Fig. 6. Ictinogomphus ferox. A, Teneral individual 7 days after emergence. The frequency of motor impulses to. spiracle 2 is plotted against time and the effect of switching between half and double strength saline is shown. B, Teneral insect 1 day after emergence, as in A. In Fig. 8 the results from many tests are summarized. In 0-5 x normal saline the mean frequency from 34 tests is 31/sec. (s.E. + 2-10); in normal saline it is 43-5/sec. (S.E. ± 2-4) from 33 tests; and in 2-0xnormal it is 56-5/sec. (s.B. ± 1-95) from 31 tests. Experiment 4. The effect of varying the osmotic pressure of the solution independently of the ionic concentration For this experiment 0-5 x normal saline was made isosmotic with i-o and 2-0 x normal by the addition of 31 and 92 g./l. glucose respectively. In Fig. 9 the results of perfusing /, ferox with 2-0 x normal saline followed by 0-5 x normal and 0-5 x normal + glucose are shown. The low frequency achieved in 0-5 x normal saline 338 P. L. MILLER is maintained when this is changed to a solution containing excess glucose, but is re- placed by a high frequency when 2-0 x normal saline is again perfused through the insect. In Fig. 10 comparable results are shown in which the order of perfusions is changed; this experiment was performed on Phyllogomphus orientate, a gomphid of about the same size as /. ferox. A similar result has been obtained from six other I. ferox. It seems likely, therefore, that the concentration and perhaps the ratios of specific ions in the haemolymph have an important regulatory function in controlling the frequency of motor impulses to the spiracles, while the osmotic pressure plays no part.

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40 60 80 100 0-5 10 2-0 Time (mln.) Saline strength (log scale) Fig. 7 Fig. 8 Fig. 7. Ictinogompkus ferox. Mature insects treated with salines of various strengths as in Fig. 6. Fig. 8. Ictinogomphus ferox. The frequency of motor impulses in both axons to spiracle 2 in teneral and mature insects plotted against the strength of the perfusing saline. Vertical lines show maximum and minimum values; horizontal bars denote standard errors.

Experiment 5. Localization of the site of the response to water balance The mesothoracic ganglion of /. ferox is partially fused to the metathoracic ganglion. For the following experiments, therefore, Acanthagyna villosa (Gruenberg) (Aeshnidae) was used since in this species the ganglia are well separated. After cutting all the lateral nerves and the posterior and anterior connectives, the mesothoracic ganglion still responds to'changes in the concentration of the perfusing saline (Fig. 11). This preparation is successful only when the ventral tracheal trunks supplying the ganglia are undamaged and the abdomen continues to ventilate them. Thus complete de- afferentation does not abolish the response and it may therefore depend on intra- Spiracle control in adult dragonflies: water balance 339 ganglionic receptors or alternatively it may take place through a direct action on interneurones or on motor cells. The possibility of additional receptors outside the central nervous system is not excluded but their presence seems unlikely.

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20 AO Time (mln.) Fig> 9 Ictinogomphiu ferox. The frequency of motor impulses to spiracle 2 in a teneral insect plotted against time, with double strength, half strength, half strength + glucose (isosmotic with double strength) (o'5 G), and double strength saline successively perfusing through the thorax and abdomen.

The removal of parts of the central nervous system may have certain long-lasting effects on the discharge in the nerve to spiracle 2. These are summarized in Fig. 12, in which results from I. ferox and A. villosa are shown. The values given are all from insects perfused with small amounts of normal saline. When the cord is intact the nerve to spiracle 2 of A. villosa passes impulses at a frequency two or three times higher than that in /. ferox. After decapitation this falls to less than half the original value, the frequency now becoming similar to that in the nerve to spiracle 1, as already reported (Miller, 1962). When the supra-oesophageal ganglion alone is cauterized there is no change and the sub-oesophageal ganglion seems to be responsible for the maintenance of a high frequency in the spiracle 2 nerve in this species. The possible significance of this in dragonflies with a crepuscular flight period will be discussed elsewhere. Decapitation oil. ferox produces no change in the frequency of impulses to spiracle 2 (Fig. 12). Removal of the prothoracic ganglion in either species may cause a temporary drop, but there is then usually a return to the value held before the operation. After cutting the central nervous system posterior to the mesothoracic ganglion, the 34° P. L. MILLER discharge is unaffected except in so far as the removal of abdominal ventilatory centres may abolish synchronizing bursts (Miller, 1962). To summarize, in /. ferox removal of parts of the central nervous system anterior to the mesothoracic ganglion has no appreciable effect on the discharge, whereas in A. villosa removal of the sub-oesophageal ganglion depresses the frequency to less than half the value in the intact insect.

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Time (mln.) Fig. 10. Phyllogompkus orientalis. Details as in Fig. 9, with the order of perfusing solutions changed

DISCUSSION Lengthy discussion of the results seems premature at present when the experimental work is incomplete. Experiments are now in progress on British species to determine which components of the physiological saline are important in setting the frequency of the endogenous motor output. It is also hoped to compare by analyses of the haemolymph of hydrated and desiccated insects the natural changes in ionic concentra- tions with those used in perfusing solutions. The latency of the reaction is probably accounted for by diffusion through the outer layers of the ganglion. The time course described here is of the same order as Treherne (19626) found when he measured the rate of spike height reduction in the cord of the cockroach bathed in a solution containing 70 mM. K+/1. Although the neural lamella does not present a barrier to the diffusing ions a Donnan equilibrium is maintained across the sheath (Treherne, 1962 a). Hence, while the composition of the fluid in contact with the nerve cells may be very different from that in the haemo- lymph, changes in the latter will probably alter the position of the equilibrium and so affect the environment of the neurones. Spiracle control in adult dragonflies: water balance 341 Changes in the concentration of potassium and calcium ions are known to affect spontaneous activity in the central nervous system of the cockroach, calcium lack lowering the level and potassium excess (more than 20 mM./l.) raising it, this latter being antagonized by calcium (Roeder, 1948). Raised potassium level might act directly on the spiracle motor cells slightly depolarizing them and thereby increasing their rate of firing. That changes in the ionic composition of the haemolymph occur naturally in some insects is clear from the work of Hoyle (1954, 1956; see also Shaw & Stobbart, 1963). If potassium concentrations are found to be significant for regulating the frequency in the spiracle nerves, then the high potassium level which Hoyle (1956) has found in moulting locusts may not only reduce movement in the insect by de- polarizing muscles, but also reduce water loss by increasing spiracle control at a time when feeding temporarily ceases.

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40 50 Tl/ne (min.) Fig, 11. Acanthagyna villosa. The frequency of motor impulses in the transverse nerve from the mesothoracic ganglion with all lateral nerves and the posterior and anterior connectives cut. The insect is perfused successively with the following salines: i-o, i's, 2-o, i-oxnormal strength.

However, while it is likely that changes in the frequency of motor impulses to the spiracle determine the threshold of the spiracular response to carbon dioxide, the mechanism of the response to hypoxia is less clear. This depends on a ganglionic rather than a peripheral mechanism and will be the subject of discussion in a later paper. The mechanism in the dragonfly is clearly different from that in the slug, where a reduction of the osmotic pressure following dilution of the bathing medium causes an 342 P. L. MILLER increase in the spontaneous activity of the central nervous system. As in the dragonfly, this result also can be interpreted functionally (Hughes & Kerkut, 1956; Kerkut & Taylor, 1956).

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1 1 1 1 1 1 1 20 40 60 80 100 120 20 40 60 80 Time (mln.) Time (min.) Fig, 12. Ictinogomphu ferox and Acanthagyna villosa. A, Frequency of motor impulses to spiracle 2 before and after decapitation. B, The same before and after section of the nerve cord between the prothoracic and mesothoracic ganglia. •, Acanthagyna villosa; A, A. villosa with supra-oesophageal ganglion cauterized; O, Ictinogompkus ferox; A, /. ferox with supra- oesophageal ganglion cauterized.

Water balance may affect the spiracle closer muscle directly in addition to its action through the nervous system. After denervation of the spiracle close to its muscle the valve sometimes shows autonomous activity by closing and responding to low carbon dioxide tensions in a way familiar in other insects (Beckel& Schneiderman, 1957; Hoyle, 1961). When removed from the insect and floated on saline it opens, but when floated on saline of 1-5 or 2-0 x normal strength it closes and still shows sensitivity to carbon dioxide. These results are comparable with those of Hoyle (1961) from spiracle 2 of the locust, where he showed that the haemolymph potassium produced contracture of the denervated spiracle muscle when its concentration was more than 30 mM./l. Although this mechanism has not been demonstrated to act in the intact preparation, indirect evidence suggests it is operative there too, and it was at first taken to be the only one involved in the spiracular response to water balance (Miller, 1961). However, it now seems likely that there are two separate mechanisms: the first depends on the response of the ganglion to the concentration of one or more unidentified ions and the second on the action of high potassium levels on the closer muscle. Spiracle control in adult dragonflies: water balance 343

SUMMARY 1. Dragonflies caught in the wild display a marked variation in the degree of control exercised over their spiracles. 2. In the laboratory desiccation produces tighter control and hydration looser control of spiracle 2: that is, in a partially desiccated insect the thresholds of the spiracular responses to carbon dioxide and to oxygen lack are raised. 3. In desiccated insects the frequency of motor impulses to the spiracles is higher than in hydrated individuals. These effects can be reproduced by perfusion with physiological salines of various strengths. 4. The reaction does not depend on the osmotic pressure of the solution but on the concentration of one or more of its constituents. 5. The isolated mesothoracic ganglion is able to mediate this reaction.

I am most grateful to Mr Jonathan Kingdon for assistance in the capture of dragon- flies. My thanks are due to the Tropical Medicine Research Board for the award of a grant for the purchase of apparatus.

REFERENCES BECKBL, W. E. & SCHNEIDERMAN, H. A. (1957). Insect spiracle as an independent effector. Science, 136, 352-53- BURSELL, E. (1957). Spiracular control of water loss in the tsetse fly. Proc. R. Ent. Soc. Lond. A, 33, 21-9. HOYLE, G. (1954). Changes in the blood potassium concentration of the migratory locust (Locusta rmgratoria imgratorioides R. and F.) during food deprivation and the effect on neuromuscular activity. J. Exp. Biol. 31, 260-70. HOYLE, G. (1956). Sodium and potassium changes ocurring in the haemolymph of insects at the time of moulting and their physiological consequences. Nature, Lond., 178, 1236—7. HOYLE, G. (i960). The action of carbon dioxide gas on an insect spiracular muscle. J. Ins. Pkytiol. 4, HOYLE, G. (1961). Functional contracture in a spiracular muscle, jf. Int. Pkytiol. 7, 305-14. HUGHES, G. M. & KERKUT, G. A. (1956). Electrical activity in a slug ganglion in relation to the con- centration of Locke solution. J. Exp. Biol. 33, 282-94. KBRKUT, G. A. & TAYLOR, B. J. R. (1956). The sensitivity of the pedal ganglion of the slug to osmotic pressure changes. J. Exp. Biol. 33, 493-501. MELLANBY, K. (1934). The site of water loss from insects. Proc. Roy. Soc. B, 116, 139-49. MILLER P. L. (1961). Spiracle control in dragonflies. Nature, Lond., 191, 623. MILLER, P. L. (1062). Spiracle control in adult dragonflies (). J. Exp. Biol. 39, 513-35. RAMSAY, J. A. (1935). The evaporation of water from the cockroach. J. Exp. Biol. 12, 373-83. ROEDER, K. D. (1948). The effect of potassium and calcium on the nervous system of the cockroach, Periplaneta americana. J. Cell. Comp. Physiol. 31, 327-38. SHAW, J. & STOBBART, R. H. (1963). Osmotic and ionic regulation in insects. In Advances in Insect Physiology, pp. 315-99. Ed. J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth. Academic Press. TREHERNE, J. E. (1962a). Distribution of water and inorganic ions in the central nervous system of an insect (Periplaneta americana). Nature, Lond., 193, 750-2. TREHERKE, J. E. (19626). Some effects of the ionic composition of the extracellular fluid on the electrical activity of the cockroach abdominal nerve cord. J. Exp. Biol. 39, 631—41. WiGGiBSWORTH, V. B. (1953). The Principles of Insect Physiology, 5th ed. London: Methuen.