Regenerative growth in insect nerve axons

Ins. Physiol., 1962, Vol. 8, pp. 79 to 92. Pergamon Press Ltd.

Printed in Great Britain

REGENERATIVE GROWTH IN INSECT NERVE AXONS D. Department

M.

GUTHRIE

of Zoology, (Recekd

University

19 Septembw

of Leicester

1961)

Abstract-The podial nerves of adult and larval specimens of Periplunetu americana were crushed or sectioned, and observations made on the speed and nature of regeneration of the larger axons. Diagnosis of repair was by examination of gait, dissection, stimulation mainly of the tibia1 depressor muscle through the ‘fast’ nerve fibre system, and examination of stained sections. Adult roaches repaired much less well than larvae. In larvae a larger proportion of operated insects repaired when the nerve was crushed rather than sectioned. Adults that had only recently metamorphosed did not exhibit greater regenerative properties than older insects. Growth and degeneration changes were described. INTRODUCTION

THE insect nervous system has been the object

of much study, but little of this interest has been directed towards its regenerative properties despite the remarkable growth phenomena manifested by the epidermis of insects. FRIEDRICH (1930) and SUSTER (1933) have demonstrated the effect of denervation on limb growth in Carausius, whereas NEEDHAM(1945) studied similar phenomena in the crustacean Asellus. One of the most important guides to this field of study in invertebrates is furnished by the work of SERENI and YOUNG (1932) on the regeneration of peripheral nerve in cephalopods. The formation in normal and regenerative growth of sensory neurones in Rhodnius has been described by WIGGLESWORTH (1953), whereas more recently BODENSTEIN(1957) has demonstrated that severed peripheral nerves in Periplaneta may grow again very rapidly in both larvae and adults. The main aims of the work described here are: (i) to investigate regeneration and return to function of n.5 of PRINGLE (1939)-the large podial nerve-in larvae as compared with adults; (ii) to compare the growth of crushed nerves with sectioned nerves; and (iii) to identify stages in regeneration. METHODS Operative procedures. All experiments were carried out on the fifth podial nerve of PRINGLE. This nerve can be seen clearly through the transparent cuticle at the base of the coxa when the insect is examined from the ventral aspect. With the insect under light ether anaesthesia the nerve can be cut or crushed with flamed scissors or forceps. Simple aseptic precautions appear to have a marked effect in reducing mortality. Histological methods. A simple staining method was required that was rapid in application and invariably successful enough to allow the state of the nerve to be judged. Well-oxidized Hansen’s Trioxyhaematin was used for these reasons, rather 79

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than a silver technique, and gave a dark brown colour to the fibres that defined them clearly. Animals were cut open, and fixed in warm Duboscq-Brasil, embedded in a hard wax and cut at 4-8 p. Physiological methods. In its most complete form the test for recovery of function was as follows: (i) The insect was made to move at different speeds and examined carefully forsigns of flexion in the operated limb. In a few instances the insects were made to run over smoked paper and the scores made by the limbs of either side compared. (ii) The operated nerve was exposed by dissection and its form noted. The nerve was then isolated by being lifted away from surrounding tissues on a cork block, or cut through and tied up to a support by means of a hair, these measures being designed to prevent direct spread of current to the muscles. Olive oil was occasionally employed to isolate a part of the nerve. The nerve was then stimulated by means of hooked platinum-platinic chloride electrodes connected to a neon-tetrode rectangular pulse generator. Stimulation was mainly at 20 c/s and 60 c/s, with the potentiometer voltage at l-3 V. (iii) The insects were mounted so that the tibia1 flexor muscle in the femur could be made to pull on a tubular glass lever writing on a smoked drum. Bearing in mind criticisms made by HOYLE (1957), propagated impulses were monitored by means of an a.c.-coupled amplifier system connected to an oscilloscope. * It was also possible to control spread of current from the stimulator by this means. The full procedure was not carried out on all the insects used in the work as indicated in the results. THE NORMAL MORPHOLOGY OF THE MAIN PODIAL NERVE (n.5) OF THE MESOTHORAX Most experiments were carried out on the left mesothoracic podial nerve, n.5 of PRINGLE (1939), and since there does not appear to be much information on the structure of the whole nerve some attempt has been made to supply this. The distribution of the branches of this nerve to the muscles and the sense organs is shown in Fig. 1, as worked out by means of sections, dissections, and whole mounts, the latter stained with methylene blue prepared by Unna’s method (PANTIN, 1946). From Fig. 1 it can be seen that n.5 supplies most of the muscles and sense organs of the limb. Nerve 3b of PRINGLE, however, innervates part of the trochanteral levator, the femoral reductor, and the tibia1 levator, and in the more distal part of the limb gives off a few sensory branches. In sections it is seen to contain only a few large fibres. Thus almost all the sensory fibres of the limb appear to be gathered into n.5 (Fig. 1). An assumption is made throughout this paper that the large fibres in the nerve (that is, more than 5 p in diameter) are for the most part motor fibres, while the fibres smaller than this are predominantly sensory. This assumption is based on the following observations: (i) Axons stained whole in methylene blue and seen to end * Grant for apparatus from D.S.I.R.

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in muscle were 5-20 t.~in diameter where they issued from the main nerve trunk, in most preparations examined. In similar parts of the limb single axons from trichoid sensillae were usually less than 1 ,u across. (ii) Large fibres followed out in transverse sections can be seen to terminate in the muscles. (iii) The proximal stump of a degenerated nerve contains many large fibres, the distal stump mainly fine fibres.

Smm 1. The nerves of the mesothoracic leg as seen from the ventral side. N.3b, 5, and 6-podial nerves according to PRINGLE’S (1939) enumeration, . a, plane of transverse sections ; cu, connective tissue union between nerves 3b and 5. FIG.

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In the intact nerve there are over eighty fibres with diameters over 15 p at the point where it enters the limb, but these taper rapidly to smaller diameters. The numbers of fibres less than 15 p in diameter could only be estimated, since there appear to be groups of very fine fibres aggregated together that are very difficult to distinguish apart. There appeared to be at least 350 fibres in this category at this point in the nerve. Because of the difficulty in distinguishing the smaller fibres with the light microscope, most attention has been given to the appearance and disappearance of large fibres, that is, more than 5 p in diameter, in the distal stump. For the purpose of estimating the extent of recovery of n.5, sections were taken through the nerve at or near level ‘a’ indicated in Fig. 1. The fibres in the normal nerve could be grouped into three diameter categories: (1) greater than 25 p, (2) between 25 and 10 cc, and (3) 10-5 p. The formula for this part of the nerve usually approached the following example (E24fS7): 3/25/80+ (more than eighty small fibres could be resolved). At this point in the nerve many of the largest fibres have been given off to the coxal muscles. The appearance of the normal nerve is shown in Fig. 4. The sheath cells that according to HESS (1958) envelope several fibres can be seen. A point of importance with regard to nerve 3b is that in some insects it appears to fuse with nerve 5 for a short distance at the point where both nerves pass through the trochanter, the fibres then separate again to form a distinct nerve to the tibia1 levator. GENERAL

DESCRIPTION

OF THE COURSE REGENERATION

OF DEGENERATION

AND

The material was found to be extremely variable with regard to the speed at which growth changes took place. The times given, therefore, are those characteristic of insects with a fairly high growth-rate, that is, most larvae and some adults when maintained at about 27°C. A small number of insects developed poorly after operation due to parasitic infections and excessive growth of scar tissue. Regeneration of severed nerve offers a greater task to the tissues involved, and the results are more varied than with crushed nerve experiments, but there is usually no doubt of the extent of the damage, so that this type of experiment may be used as a standard. After the operation a clot is formed over the opening in the integument and the cut ends of the nerve draw away from one another. Within 48 hr the epidermis has secreted a darkened strip of cuticle closing the wound area externally and, later, internal cuticular bodies may be formed. The behaviour of the epidermal wound tissue is important as it may affect the course of nerve regeneration. The cuticular bodies are often formed round parasite cysts. After section the end of the proximal stump may withdraw some way towards the ganglion and become coiled on itself; the distal stump, on the other hand, may shrink a short distance fom the wound area, but it tends to be held in position by branches to the coxal muscles. During the first post-operative week the proximal stump retracts and then extends again. Some of the smaller fibres become narrower

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and the axon membranes thicker. This narrowing of the larger fibres can also be observed in the distal stump and may be due to the lowering of internal pressure inside the axon accompanying loss of axoplasm. By the end of the first week the extending proximal stump is near the scar, and its further progress will be affected by the form and size of the epidermal cell mass lying over the scar. In some insects the nerve appears to be deflected by this obstruction.

-rf

FIG. 2. Diagrammatic representation of n.5 to show the course of new proximal stump fibres following section of the nerve. bca, blood cell aggregate; ds, distal stump; ps, proximal stump; pz, proliferation zone of muscle; rz, reduction zone of musk; st, scar tissue.

During the second and third weeks the large fibres in the proximal stump sprout and divide at their outer ends to produce numbers of fine axon processes. These processes were not seen to be distinctly club-shaped as in cephalopods (SERENI and YOUNG, 1932), but tapered to rounded ends. They are surrounded

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..:. :

‘..‘...- ,...1.:

. .. . . . .._...

_,.....

I.”

:.. _, :.: ..:.

1OA

FIG. 3. The appearance of degenerated nerve axons in distal stumps as they are seen in transverse sections. The shrunken and irregular axon membranes can be seen within the sheaths.

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and separated by the cells of BODENSTEIN’S ‘blood-cell aggregate’ (BODENSTEIN, 1957). Most sprouts in proximal stump outgrowths of this kind appear to have grown towards the distal stump head, but many others can be observed to have grown away from this region, often following the course of trachea and other structures. The large fibres in the distal stump that have been disconnected from This proceeds very slowly, in that few their cell bodies undergo degeneration. changes occur before the insect is 2 weeks old post-operatively and degenerated large fibres were found in the distal stump up to 9 weeks. Final disappearance of these large fibres with their many sheaths (HESS, 1958, figured these clearly) does not appear to be promoted by amoebocyte cells as in cephalopods (SERENI and YOUNG, 1932). The axon sheath slowly crumples inwards, becomes much contorted and disintegrates (Fig. 3). The sheath cell lamellae become thinner, and in the older distal stumps, they fragment into isolated globules of darkly staining material. It was difficult to decide from sectioned material whether the bridge of fib&s uniting the two stumps was formed at first by proximal stump rather than distal stump fibres, although proximal fibres were the more obvious in sections through this region. Fibres of large size as already defined begin to make their appearance in the distal stump at about 5 weeks. They are, at first, narrow and thick-walled, with dense axoplasm. Even the largest fibres never attained the diameters of those on the intact side. Seventy-eight insects recovered at the end of various experiments were sectioned through both legs at the same level, and the numbers of fibres in the three sizegroupings defined in the description of the normal nerve ascertained by means of a calibrated grid. The formulae for the operated nerves varied widely, but in only eight instances were first category fibres found on the operated side even when physiological tests had indicated a functional nerve; there were usually at least three first category fibres in the intact nerve at the same level. In five instances only a single fibre was present. The number of secondary fibres in the operated nerve was extremely variable (two to twenty) but there were usually at least half as many as on the unoperated side. The area of the whole nerve section was also measured in most insects and the thickness of the operated nerve was about 80 per cent of the unoperated nerve at the same level. Anomalous structures Muscle proliferation. The cutting of n.5 was sometimes followed by remarkable changes in parts of the trochanteral abductor muscle lying adjacent to the main nerve trunk in the region at which the main nerve branches to this muscle arise. In the most extreme examples, a large mass of muscle tissue consisting of narrow muscle columns, similar to those occurring in the early stages of muscle growth, was developed (POISSON, 1924). SCHARFCER (1945) has described cancerous growths produced in the roach by nerve section.

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Skeletal terata. In some operated limbs a remarkable structure was to be found lying in the region of the coxa through which transverse sections were taken. This was a hollow cuticular structure with many internal and external processes lying in the position of the trochanteral depressor apodeme, to which the larger muscles of the coxa are attached. This teratological structure was often very large, displacing and obscuring most other structures. It was not a limb bud rudiment. The form of this structure resembled in some way that of the scar, and suggested that it might have been formed by the hyperactivity of epidermal cells triggered off by the making of the incision. The diagnosis of successful repair

One of the aims of many of the experiments was to determine the percentage successful regenerates in any given group. In practice two groups were defined: (i) those in which a considerable measure of growth and recovery of function could be discerned, and (ii) those in which these characteristics are slightly or not at all developed. The main tests employed, histological, physiological, and observational (outlined in the section on Methods), had somehow to be added up for comparative purposes. To be able to employ several tests together helped to reduce the effect of the diagnostic inaccuracies peculiar to each. A negative result in the dissection or the electrical test did not always mean much, and even sectioned material was frequently equivocal. Therefore a somewhat arbitrary scoring system was resorted to, whereby each insect was scored out of 35-50 points according to the number of tests it had been subjected to. To be regarded as a successfully repaired insect, half or a larger fraction of the total points applicable had to be awarded, and this required a high score in at least two out of four tests. Two examples from the same experiment show that: E24b3. General condition poor. Leg slightly damaged ; gait and usage of leg conspicuously poor-4 out of 15 points. Few points available for the dissection as these two examples were pinched nerves, but in this insect the nerve was in good condition and showed little sign of a constriction-5 out of 5 points. Electrical tests showed only a very slight response of the tibia1 depressor to high-frequency stimulation-3 points out of 10 points. Examination of sections demonstrated an unusually thick nerve on the intact side-30,000 pL2in section, with a normal grouping of fibre sizes-Z/25/80. The operated nerve was much narrower9000 p2, but the detailed structure was not very clear. Some larger fibres were present-O/2/10. The muscles on the operated side were well developed. Sections difficult to assess-6 points out of 15. Total: 18 points out of a possible 4.5. Only slight signs of regeneration and recovery. E24b4. General condition good. Limb in perfect condition. Gait and usage of limb subnormal, but fairly advanced-7 out of 15 points. Nerve trunk at point of crushing still showed slight signs of constriction when dissected-4 points out of 5. Electrical stimulation of the nerve produced strong contractions of the tibia1 muscles at low and high frequencies-10 out of 10 points. Sections showed the p2, and the operated nerve somewhat intact nerve to be of normal diameter-16,200

["l{;. 4. Transverse section of the normal n.5. l:l{~. 5. Transverse section of a largely repaired n.5. lqc;. {) Transverse section of distal s t u m p of n.5 3 xxeeks after cuttinv sla~)x~in~ def{eneration well advanced. FIG. 7. "l'l'ansxcrse section (~f n.5 durinv regeneration. Note the darkly stainin~ sheulh cell glol~ules.

l"l(;, S. Transverse section t h r o u g h the proxilnal slt_lnlp of n.5 and the z~me ~f muscle' proliferation. Igibres from the s t u m p (upper left) can bc seen enterinv the proliferation zone in th,: lower part of the photograph.

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reduced at 10,200 ps, The fibres were rather large in both nerves, the fibre formulae SIight reduction of some of the muscles was being 4/45/50+ and l/13/20+. apparent on the operated side-9 out of 15 points. Total: 30 out of 45 points. Definite signs of regeneration and recovery. All but a few of the insects were given as full an examination as the examples quoted. EXPERIMENTAL

RESULTS

A. Preliminary experiments (i) Control of cutting techniques. N.5 was cut in twenty adult standard method. Examination after 3 days showed that in one survivors the nerve was only partially severed, in the remainder completely divided. This result indicated that while care had to cutting, the level of error due to faulty cutting would probably not 5.5 per cent.

roaches by the of the eighteen the nerve was be exercised in be greater than

of

(ii) The mortality rate normal adults. It was important to be able to compare normal expectation of life with mortahty following nerve section. A batch of twenty-eight insects were kept under normal conditions, and it was found that the average rate of dying was one per 8 days over a period of 64 days, giving an expectation of life of 231 days for an adult 1 week old when it entered the experiment. The number dying during the course of an experiment, due to causes unconnected with nerve operation, could be calculated as follows: Number of days of experiment Average expectation

of life (224 days)

x Size of batch = Number died.

The chief assumption made here is that the youngest insect in a batch of twenty-eight insects chosen from a large breeding box containing larvae and adults would not be much older than a week. B. Regeneration of n.5 after section in the adults Group 1; Twenty-two insects. These insects were kept in the high-temperature room (25-28°C) after operation and fed dogfood and water. After periods ranging from 3 to 8 days, the insects were dissected, fixed, and sectioned. The proximal stump was found to be separated from the distal stump by an interval of about 1 mm in most instances. The head of the proximal stump was swollen but smooth, and the outer sheath of the nerve appeared to have closed over the cut end. The distal stump head was also enlarged, but had contracted towards the point at which the nerve gives off many branches. The end of the distal stump was irregular. Transverse sections of the distal stump below the enlarged head showed that very little change had taken place and many large fibres were present. As GAMBLE and GOLDBY (1957) found in Lacerta, degenerative processes may occur very slowly in peripheral nerve. The distance separating the two stumps must be a very critical determinant of successful reunion as pointed out by YOUNG (1948). In a few insects, the proximal

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stump was found to be much coiled and turned away from the rest of the nerve; it is doubtful whether reunion occurred in such instances. Group 2 ; Thirteen insects. These insects were kept under the same conditions as those placed in group 1 but were killed after a post-operative life of from 2 to 6 weeks. The electrical test was only applied to two of these insects, otherwise the tests outlined above were employed with all. Out of thirteen insects, four showed junction of the stump ends and in sections taken from them, small dense regeneration fibres of a distinct type were visible. Many of the other insects showed a close approximation of the two stumps. Degeneration was well advanced in some of these insects, the larger fibres breaking up and disappearing, leaving behind an incomplete network of sheath cell lamellae and axon membranes. The two insects tested electrically were relatively little advanced. In this group as in the others, the correlation between age and growth stage was not obvious. The four most advanced insects were between 15 and 35 days old. Group 3 ; Thirty-seven insects. All but two of these insects were over 9 weeks old post-operatively, and were examined for type of gait, state of nerve as shown by dissection, tested electrically, and the nerve made into sections. The two others were 45 days old post-operatively, and both showed signs of regeneration; they were not tested electrically. Out of ten insects that were killed after 9 weeks, four showed definite signs of nerve regeneration. Out of ten insects that were killed after 13 weeks, two showed definite regenerative changes. Out of fifteen insects killed after 20 weeks, seven showed definite signs of recovery. Thus in thirteen insects out of thirty-five, examination by the methods outlined In the remaining above indicated a considerable measure of regeneration. twenty-two insects, there was either no evidence or slight evidence of regeneration beyond the most elementary stage. It was clear that union of the stumps might occur without any further changes ensuing. Indeed, in as many as twenty-three insects out of the thirty-five a union of some kind had been formed, but in ten of these no further changes followed this junction. Placing the insects of the three groups together, it can be seen that only seventeen out of fifty killed after over 2 weeks of post-operative life showed definite evidence of regeneration, whereas BODENSTEIN (1957) had found that most of his adults repaired rapidly. It was thought that the conditions of rearing or the line of roaches used might be responsible for the small percentage of regenerates (34 per cent). It was hoped that experiments with larvae would define the role of extrinsic factors in nerve regeneration. C. The regeneration Group

instars),

of n.5 after section in the larvae

1. Thirty insects, larvae towards the middle of larval life (fifth to eighth were maintained for between 14 and 18 days in the hot room.

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Six insects died. The remaining twenty-four were examined superficially only; that is, the form and movement of the limb were examined. Twelve out of twenty-four showed considerable evidence of functional recovery. In the remaining twelve, the operated limb was imperfectly flexed as compared with that of the other side. Group 2. Forty insects, larvae of the same age as those in group 1, were maintained under standard conditions for between 40 and 51 days. Four insects died. Thirty-two of the remaining thirty-six insects showed great evidence of recovery as evinced by a study of gait, and stained sections of the operated limb. In its most advanced form, the operated nerve was usually thinner than the unoperated nerve of the other side. There was a good deal of variation in the number of larger fibres present. The insects of group 1 were almost certainly of the age when regrowth of nerve was just well established. Those of group 2 gave a better indication of the growth capacity of the larva, approximately 80 per cent having attained a considerable measure of repair and functional recovery. D. Regeneration of n.5 in the adult after crushing with forceps Regeneration could be expected to be more rapid and more complete when the outer sheath of the nerve remained intact than when the nerve was severed and the two ends allowed to draw away from one another (YOUNG, 1948). Flat-faced jewellers’ forceps were used, the nerve being forcibly manipulated two or three times until a continuous clear zone separated the slightly opaque column of fibres within the main nerve sheath or perilemma. Seventy insects were operated on by this method, sixteen dying towards the end of the longest period of 96 days. One insect surviving was found to have entered the insect boxes from outside, so that fifty-three genuine survivors of these experiments were obtained. Testing of these insects was varied, but thirty-seven out of fifty-three were examined in at least three of the four ways described in Methods. The remainder (sixteen) were examined externally and dissected or sectioned. Group I. Sixteen insects less than 40 days old, Twelve of these insects were post-operatively 21 days old: of these, six showed clear evidence of regeneration. The other four insects were killed 15, 20, 25, and 35 days after operation respectively. The first and last showed good evidence of recovery, the other two only slight signs of nerve regrowth. Group II. Fourteen insects, killed 56-68 days after operation. Nine of them showed considerable positive signs of nerve regrowth and return to function. Group III. Twenty-two insects, post-operative age 84-96 days. The number exhibiting successful regrowth of nerve was eighteen, or 77 per cent. From these results, it can be seen that for the three age groups, the percentages of successful regeneration were : 50,64, and 77 per cent. The percentage regenerated out of the total of fifty-three insects was just over 67 per cent (thirty-three).

90

E.

D. M. GUTHRIE The regeneration of 12.5 in young adults

BODENSTEIN(1957) described the formation of a new continuous nerve in most adult roaches operated on by section. This was not observed in the present work, in which a high failure rate was encountered amongst adults regrowing nerves after section; 49 per cent stumps did not unite, and 66 per cent failed to achieve any considerable measure of repair. These figures may be compared with the results of larval nerve regrowth. In these, some 89 per cent good recoveries were made in older groups of larvae. It seemed a possibility that some of the adults selected at random from the boxes might be too old to retain much capacity for regrowth of nerve. Twenty-one adults were selected which were known to have metamorphosed from the last larval instar not more than 4 weeks previously. N.5 was sectioned, and the insects were killed 49 days later. Three animals died or escaped. Of the remaining eighteen insects, seven showed considerable signs of recovery, or under 39 per cent. Since this percentage success shows little improvement on that already obtained for adults (34 per cent), no other similar experiments were tried. F. The effect of larval blood on the growth of n.5 after section in the adult It seemed possible that bathing the nerve in blood richer in blood cells, metabolites, and growth-promoting hormones than that normally surrounding it might have the effect of accelerating the growth of the nerve. The experiment consisted of joining a middle-instar larva through the prothorax to an adult with n.5 sectioned on one side. Unfortunately, considerable post-operative mortality occurred, largely due to the very lively nature of the larvae. Cutting away the tarsal claws of the larvae sometimes appeared to aid survival of the parabiotic pairs. Of thirty-two pairs set up successfully, the majority died within the first week and only five pairs survived beyond 40 days. Four pairs survived for 46 days, and one pair for 54 days. On dissection, all these adults showed a well-healed nerve which gave evidence of ability to conduct impulses. The oldest adult showed a particularly perfect nerve. G. The effect of denervation on the leg muscles It was a common observation that muscles deprived of nerves for any length of time underwent regressive changes, that is to say, the muscles of the operated coxa appeared reduced as compared with those of the other side when dissected and examined as transverse sections. Reduction of the coxal musculature was most marked when little progress in the repair of the nerve had been made over a long post-operative period. As with the regenerating nerves, it was useful to divide the coxal musculature types into two groups; those in which reduction was advanced and those of normal or near-normal appearance. The first group was defined as consisting of those coxal muscles with a cross-sectional area plainly less than half as great as that of the normal coxal muscles in the unoperated limb in the same transverse section. The second group, comprising the rest, included a number showing various degrees of slight reduction, but temporary denervation, and

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dehydration due to damage, were invoked to explain their occurrence. Of the 102 adult specimens examined for muscular atrophy, 61 per cent fell in the latter group of relatively well-developed coxal muscles, and only 28 per cent showed no reduction at all. The percentage of the whole batch described as showing clear signs of regenerative growth of n.5 was 52 per cent, a yomparable proportion to those with near normal muscles. Fewer larvae were examined for muscular atrophy but observations suggested that this condition was rarer than it was in adults; thus, amongst twenty-eight denervated larvae, only three showed slight reduction of the coxal muscles. The youngest insect post-operatively to show muscular atrophy was an adult 15 days subsequent to denervation, but most insects showing clear signs of muscular reduction were 5-6 weeks post-operatively. II. Retardation of limb growth in the larva following nerve section It was found that in most larvae that had had n.5 crushed, and then survived for periods between 20 and 60 days, the operated limb when compared with the undamaged mesothoracic leg was often found to be slightly shorter. These were insects that had not automized the operated limb as far as was known. Although a few measurements were made, in many insects it was merely noted that the unoperated limb was clearly longer than the operated one. In forty-two insects that had moulted, eighteen examples of short operated limbs were found. That the difference could be considerable is shown by the following groups of measurements in millimetres of the two limbs of four 50-day larvae (unoperated limb-first figure) : 23122, 24/23+, 26123, 26&/21. In the other four members of this group the mesothoracic legs were of equal length.

DISCUSSION

It appears from the results of the work described that the union of the stumps in the severed nerve takes place rapidly in most larvae, and is usually followed by a return towards normal of the nerve, accompanied by a considerable measure of functional recovery. In the adult, however, a similar return to normal structure and function occurs in less than 50 per cent of insects operated by section, and though a greater percentage of successes occur when the nerve is crushed rather than completely severed, this still falls short of the recovery figures for larvae. In this respect, these observations differ from those recorded by BODENSTEIN (1957), whose adult roaches regenerated as competently as the larvae. The cockroaches used for these experiments sometimes contained haemocoelic parasites, but animals that died during the course of experiments did not contain noticeably larger numbers of parasites ; one adult insect was noted in which an unusually rapid repair of the nerve had occurred together with a heavy infestation of protozoa1 cysts. Considerable regrowth of nerve after only 15 days had occurred in one larva. It may be concluded that the parasitism did not appear to affect nerve regeneration greatly in these experiments.

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Examination of most of these results fails to indicate the increased number of repaired insects with time, which would be expected if growth potentialities were the same in most insects of an experimental group. These potentialities must be very variable. REFERENCES BODENSTEIN D. (1957) The regeneration of nerves in insects. J. exp. Zool. 136, 89-117. FRIEDRICH H. (1930) Zur Kenntnis der Regeneration der Extremitaten bei Curausius momsus. 2. z&s. Zool. 137, 578-605. GAMBLEH. J. and GOLDBYF. (1957) The effect of temperature on the degeneration of nerve fibres. Nature, Lond. 179, 527. HESS A. (1958) The fine structure and morphological organization of the peripheral nerve fibres and nerve trunks of the cockroach Periplaneta americana. Quart. J. mim. Sci. 99, 333-340. HOYLE G. (1957) The nervous control of insect muscle. In Recent Advances in Invwtebrate Physiology (Ed. by B. T. SCHEER). University of Oregon Press. NEEDHAMA. E. (1945) Peripheral nerve regeneration in Crustacea. J. exp. Biol. 21,144--146. PANTIN C. F. A. (1946) Notes on Microscopical Technique for Zoologists; Cambridge University Press. POISSONR. (1924) Etudes sur les Hemipteres aquatiques. Bull. biol. 58, 51-306. PRINGLEJ. W. S. (1939) The motor mechanism of the insect leg. J. exp. Biol. 16, 220-231. SCHARRERB. (1945) Experimental nerve tumours after nerve section in an insect. Proc. Sot. exp. Biol., N. Y. 60, 184-188. SERENIE. and YOUNGJ. 2. (1932) Nervous regeneration and degeneration in Cephalopods. Pubbl. Staz. zool. Napoli 12, 173-206. SUSTERP. M. (1933) Beinregenerationnach Ganglionextirpation bei Sphodromantis bioculata Burm. Zool. yb. (Physiol.) 53, 49-66. WIGGLESWORTH V. B. (1953) The origin of sensory neurones in an insect Rhodnius prolixus. Quart. J. micr. Sci. 94, 93-112. YOUNG J. 2. (1948) The growth and differentiation of nerve fibres. Symp. Sot. exp. Biol. 2, 57-73.