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Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. & F.—II
r. Ins. Physiol., 1963, Vol. 9, pp. 363 to 373. Pergamon Press Ltd.
STUDIES ON THE INITIATION OF GROWTH AND MOULTING IN MIGRATORIA MIGRATORIOIDES R. & F.-II
LOCUSTA THE
Printed in Great Britain
R6LE
OF THE
KENNETH
STOMATOGASTRIC
U. CLARKE and PETER
Department
of Zoology,
University
NERVOUS
SYSTEM
A. LANGLEY of Nottingham
(Received 15 November 1962)
Abstract-A
brief account is given of the anatomy of the stomatogastric nervous system. The role of this system with regard to the postembryonic growth of the locust was investigated by cutting certain of the nerves, removing certain of the ganglia, and observing the effect of these operations on the further growth and moulting of the locust. An account of the technique of each operation is given, and stress is laid on certain asceptic procedures which were found to greatly decrease the mortality rate of the operated animals. All operations were performed on 33-hr-old third instar locusts. Cutting the ventral nerve cord in front of the first abdominal ganglion had little effect on the further growth and moulting of the insects which survived into the adult instar. Cutting the outer oesophageal nerves did not prevent the animals moulting and growing to the fifth instar; however, the growth was slower and the mortality higher than in the operated controls. Removal of the ingluvial ganglion resulted in death within 43 hr of the operation. Cutting the recurrent nerve behind the frontal ganglion did not prevent the animals from moulting and growing to the end of the fourth instar; growth was slower than that of the operated controls. Removal of the frontal ganglion resulted in the immediate cessation of growth, the animal remaining at a constant weight and without moulting until its death. Death occurred at between 260-310 hr which is approximately twice as long as it took the operated control to grow and moult to the next instar. The operated animals were observed to feed frequently, to defecate normally, and upon dissection their guts were found to be full of fresh food. These effects cannot therefore be attributed to a failure to feed. These experiments show that the frontal ganglion has an essential role in the growth of the locust which, however, can survive and show normal activity in its absence.
INTRODUCTION
IN A previous paper it has been shown that the time of activation of the prothoracic gland coincided with the period of ecdysis, and it was suggested that the relaxation and distension of the gut that occurs at this time provided the stimulus for the release of the growth and moulting hormone (CLARKE and LANGLEY, 1963). The present paper substantiates this, and, by surgical interference with the stomatogastric system, establishes the nervous pathways involved. 363
364
KENNETHTJ. CLARKE ANDPETER A. LANGLEY
The visceral nervous system in insects has been described in general terms by SNODGRASS(1935), WIGGLESWORTH(1939), and IMMS (1957). It has been investigated in a wide variety of types by CAZAL(1948) whose description of Metrioptera (Orthoptera, Tettigoniidae) agrees with that given by ALBRECHT (1953) and STAAL (1961) for Locusta. However, the present study has revealed some nerves not described by these workers. To facilitate an understanding of the significance of the operations to be described, a brief account of the anatomy of the stomatogastric system is given here. THE ANATOMY OF THE STOMATOGASTRIC
SYSTEM
The structure of this system has been investigated by dissection of locusts that have been subjected to intra-vitam injections of methylene blue, and by studies of serial sections through the heads of immature and mature locusts. No differences could be found between the larval and adult locusts in the structure of this system. Anteriorly, the stomatogastric system is represented by a frontal ganglion lying in the midline dorsal to the pharynx and a little in front of the brain. It is approximately pear-shaped with its apex pointing posteriorly. From this apex the recurrent nerve passes back in the midline dorsal to the gut to the hypocerebral ganglion which is just behind the brain. From the anterior lateral edges of the frontal ganglion, the frontal connectives pass back on each side of the gut to the tritocerebrum. Close to the brain each frontal connective lies alongside the labral nerve and may join it at a point where it enters the brain. From the lateral borders of the frontal ganglion, just behind the origin of the frontal connectives, a pair of nerves pass laterally to the pharynx, and from each side of the ganglion near the origin of the recurrent nerve a similar pair also pass down to the gut. These are the anterior and posterior pharyngeal nerves respectively. From the hypocerebral ganglion a pair of inner oesophageal nerves pass down to the surface of the crop. Lateral to these are a pair of outer oesophageal nerves which pass posteriorly and ventrally to the ingluvial ganglia. The ingluvial ganglia lie one on each side of the crop just in front of the anterior tips of the mesenteric caeca. From them nerves pass to the posterior crop wall, the gizzard, and inner and outer caecal nerves to the mid-gut (CLARKE and GRENVILLE, unpublished). The corpora cardiaca are situated on the dorsal side of the hypocerebral ganglion. They have been described in detail for Schistocerca gregaria Forsk. by HIGHNAM (1960). They are exactly similar in Locusta migratoria. A pair of nerves, the nervi corporis cardiaci I and II, connect them to the brain; a shorter pair of nerves connect them with the hypocerebral ganglion. A pair of corpora allata are present, one on each side of the crop just behind the brain. A pair of nervi corporis allati join them to the corpora cardiaca. They receive a further nerve originating from the sub-oesophageal ganglion (CAZAL, 1948 ; STAAL, 1961). This nerve in methylene-blue preparations appears to have some fibres passing right through the corpora allata and entering the nervi corporis allati. A short distance from its emergence from the corpus allatum this lateral nerve gives rise to a branch which passes anteriorly and divides into three further
INITIATION OFGROWTH ANDMOIJLTING IN LOCUSTA
MIGRATORIA
MlGRATORIOIDES
365
branches. One,of these passes to the ventral dilator muscles of the pharynx, the second to the muscles of the ventral head apodeme, and the third a small branch which enters the tritocerebrum close to its junction with the circumoesophageal connectives. Near the point of insertion of the lateral nerve with the sub-oesophageal ganglion another branch arises which passes forwards and innervates the anterior dilator muscles of the crop. On the hypothesis that the initiation of a growth and moulting cycle arises from changes in the gut at ecdysis the stomatogastric nerves of especial interest are the frontal ganglion and its branches, the recurrent nerve, the inner and outer oesophageal nerves, and the ingluvial ganglion and its branches. The frontal ganglion has been implicated by FAIVRE (1863) in the control of swallowing movements in Dytticus marginalis L., and this seems to be the generally accepted view of its function. However, ORLOV (1924) has figures of both motor and sensory endings of nerves arising from the frontal ganglion and thus there is a pathway along which a stimulus could pass. The recurrent nerve does not seem to have been assigned a function although neurosecretory material has been described in it by NAYAR (1957). The inner oesophageal nerve has been shown to have a motor function (CLARKE and GRENVILLE, 1960) and has not been further considered here. The outer oesophageal nerves have no clearly defined function in Locusta, although in Calliphora THOMSEN ANDMILLER (1959) and STRANGEWAYSDIXON (1961) have shown they convey neurosecretory ,material to the mid-gut where it is necessary for enzyme formation. No neurosecretory material has been found in these nerves in Locusta (CLARKE and GRENVILLE, unpublished). It was therefore decided to cut the recurrent nerve, the outer oesophageal nerve, and to remove the frontal ganglion and the ingluvial ganglion, immediately after an ecdysis and to determine the effect these operations may have on the further growth and moulting of the locust. MATERIAL
AND METHODS
The insects used were third instar larvae of Locusta migratoria migratorioides R. and F., these being the smallest convenient size for subjection to operative techniques while allowing two further cycles of growth and moulting in which the experimental results could be assessed. The insects were reared under the conditions described previously (CLARKE and LANGLEY, 1963). The growth of each insect was determined by weight at 12-hourly intervals and by determining its instar duration. Each experiment described below was carried out on thirty-six animals all within 12 hr age range. Twelve animals were subjected to the operation (0), twelve to a control operation (CO), and twelve untouched but subjected to the same environmental treatment (C)e A very high percentage (90-100 per cent) of success in recovery from the operation was obtained in all the results reported here. This contrasted with earlier experiments when the recovery rate was low (20-30 per cent). This change was attributed by the authors to the introduction of rigid aseptic procedures and to a
366
KENNETH
U. CLARKEANDPETERA. LANGLEY
definite regime of post-operative treatment suggested by STAAL, (personal communication). Since these do not seem to be current in operative work on insects, they are described below. All glassware was steam pressure sterilized and kept in sterile containers until used. The cages in which the animals were kept, and the instruments used for the operations, were sterilized in ultra-violet light for 30 min immediately before use. Following the operation, it was necessary to keep the animals as dry as possible. Each animal was kept in a glass jar with perforated lid, the jar containing filter paper rolled into a tight cylinder on which the animal could cling and a circle of filter paper on the bottom to absorb any moisture excreted by the animal. Fresh grass was given daily. The grass was cut short and bound into a bundle by a tight wire so that the locust could not get amongst the grass stems and open up its wounds. The jars were kept in a dust-proof cabinet at room temperature (20-22°C) for 48 hr. This kept the animal relatively inactive until its wound had had time to heal. When healed, the locust was returned to the insectary (28°C) and the precautions described above stopped. SURGICAL
TECHNIQUES
Each insect was anaesthetized with di-ethyl ether for approximately 3 min; this rendered the animal unconscious for a sufficient length of time to perform the operation and did not cause death in any instance. Severance of the aentral nerve cord The anaesthetized insect was fastened with its ventral side uppermost on the stage of a binocular dissecting microscope. A transverse incision was made into the intersegmental membrane between the first and second abdominal sternites ; the sternites were parted and the ventral nerve cord exposed. A small section of cord was removed thus isolating the thoracic and abdominal parts of the chain, and minimizing the chance of continuity being established by tissue regeneration. Sealing the wound was found to be unnecessary since the natural overlap of the abdominal sternites ensured adequate protection, and rapid healing occurred. Operations on the stomatogastric system posterior to the brain The anaesthetized insect was placed on the stage of the microscope with its It was held in an extended position with its thorax dorsal surface uppermost. slightly raised and its neck membrane exposed. A small transverse incision was made in the neck membrane. This gave immediate access to the lateral regions of the oesophagus, the crop, and to the ingluvial ganglion. The outer oesophageal nerves were severed by a simple cut about half way along their length. The ingluvial ganglion was freed from its nerves and connective tissue and removed from the body. Again no special sealing of the wound which healed rapidly was necessary.
INITIATION
OF GROWTH
AND
MOULTING
IN LOCLJSTA
MIGRATORlA
MIGRATORIOIDES
367
Operations on the stomatogastra’csystem anterior to the brain The insect was held on the stage of the microscope with the front of the head upwards. A transverse cut was made in the cuticle immediately below the median ocellus and extending from the left to the right carina. Two further cuts were made, one from the end of the lateral cut down the inside of the right carina to the epistomal suture, and a similar cut down the left carina. The flap of cuticle and hypodermis so formed was turned ventrally exposing the anterior air sacs of the
1270.
1150. wt. 1030. mg
. 910.
790.
670,
550
430
70
1
150
.
230
310
hr. FIG. 1. The effect of severing the ventral nerve cord early during the fourth instar on the further growth and development of the locust. +-----0 ventral nerve cord severed; O0 operated control. Time is given as the number of hours that have elapsed since the operation. Arrows mark the fourth and fifth ecdysis respectively. Each curve represents the growth of one individual. For further data see Table 1.
head. These were parted in the midline to expose the upper surface of the pharynx and the anterior surface of the brain. The recurrent nerve was severed by a simple cut between the frontal ganglion and the brain. The frontal ganglion was removed by cutting the recurrent nerve, the frontal connectives, freeing the ganglion from the connective tissue surrounding it, and removing the ganglion completely from the head. Upon completion of the operation the flap was replaced and the wound healed without further treatment.
368
KENNETH U. CLARKE AND PETER A, LANGLEY
RESULTS
(A) Severance of the ventral nerve cord The subsequent growth and moulting of these insects is shown in Fig. 1. All the insects recovered and grew normally throughout the fourth instar. Moulting was slightly delayed in the operated and operated controls probably owing to post-operational shock. Mortality was high at the fourth ecdysis (Table l), owing
hr FIG. 2. The effect of severing the outer oesophageal nerves early in the third instar on the further growth and moulting of the locust. 0 -0 outer oeso0 operated control. The curves are for one phageal nerves severed; Ooperated insect and one operated control. For further data see Table 1. Zero time is at the moment of the second ecdysis, 0 marks the time of the operation, and arrows mark the third ecdysis.
to the difficulties the locusts experienced in shedding their cuticle due to wound adhesions. The two insects that reached the adult instar were dissected and
confirmation obtained that the nerve cord was severed and had not re-united during the intervening growth period. It is concluded from the results of this operation that neither the abdominal ventral nerve cord nor the caudal nervous system was involved in the initiation of the moulting cycle.
C oc 0
Outer oesophageal nerves cut
8 12 10 12 24
C oc
0
8 12 24
Frontal ganglion removed
--
C oc 0
nerve
Recurrent cut
9 15 15
C oc 0
9 15 15 24
12 12 12
Number of locusts used
Ingluvial ganglion removed
---
C oc 0
Ventral nerve cord severed
Operation
9 12 24
II
11 24
15 15
15 15 24
12 12
Survived from operation
---
8 12 20
8 10
8 10 24
9 14 3
9 14 15 24
-
Third instar
>
--
_
6 8
6 8 5
9 12 0 -
9 12 6 18
12 12 12
Fourth instar
_ _
6 8
8 8 0
14
8 8
12 10 6
Fifth instar
6 8 0
8 8 0
8 8 5 17
12 12 6
Fourth ecdysis
Number of insects surviving
None moulted to the fourth instar
7 9
7 9 15
9 14 0
9 14 7 24
-
Third ecdysis
TABLE 1.
-~--_ _ _
-
-
10 10 3
Fifth ecdysis
_ _
-
---
10 10 2 --
-___
Adult
KENNETHU. CLARKE AND PETER A. LANGLEY
370
(B) Severance of the outer oesophageal nerves The subsequent growth and moulting of these insects the survival rates in Table 1. Although growth continued, found in the operated controls and in the normal animals. was delayed, and mortality at the third ecdysis was high. completed their third ecdysis grew and moulted to the fifth
120.
0
100
.
200
is shown in Fig. 2, and it was slower than that Furthermore, moulting Those, however, which instar.
.
’
300
hr
FIG. 3. The effect of severing the recurrent nerve and removing the frontal ganglion early in the third instar on the further growth and moulting of the locust. Orecurrent nerve severed behind the frontal ganglion; 0 0 ----0 frontal ganglion removed; An operated control. Solid lines females, dotted lines males. Each curve represents the growth of one individual, for further data see Table 1. Operations were performed at times between the first and second points on each curve. Arrows mark the time of the third ecdysis.
(C) Removal of the ingluvial ganglion Although all the operated insects recovered from the anaesthesia, all died within 43 hr of the operation (Table 1). (D) Severance of the recurrent nerve The subsequent growth of these insects is shown in Fig. 3. The operated insects grew and moulted to the next instar. Their growth rate was lower than that
INITIATION
OF GROWTH
AND
MOULTING
IN LOCUSTA
MIGRATORIA
MIGRATORIOIDES
371
of the operated controls and ecdysis was delayed by some 40 hr. Only three out of the twenty-four operated animals survived to the fourth instar, where they showed some increase in weight but became moribund and died at 200 hr in the fourth instar. Dissections showed that all three had developed a new cuticle under the old, thus indicating that the growth and moulting cycle of the fourth instar had started. (E) Removal of the frontal
ganglion
The operated insects lost weight during the first 24 hr following the operation, then after a slight increase during the next 48 hr remained practically constant in weight over the next 192 hr, finally dying without moulting (Fig. 3). The operated controls grew normally and moulted to the fourth instar with only a slight delay compared with the unoperated animals. Observations of the insects deprived of their frontal ganglion showed that once the effects of the operation had worn off they fed normally. Dissection at different periods after the operation showed the gut contained normal amounts of food. The failure to grow cannot therefore be due to the failure of these insects to feed: the intake of food, the amount in the gut, and defecation proceeded normally in these animals. DISCWSSION
The surgical techniques described in this paper are fairly straightforward and call for no special comment. Three subsidiary features essential for their success are worth further discussion. Ether was used for anaesthesia and was found to be superior to carbon dioxide in that its effects did not wear off so quickly, and to chloroform in that no deaths resulted from its use. Operations without anaesthesia were always accompanied by a high mortality largely due to the length of time necessary to accomplish the operation when the air sacs are moving and the blood circulating. The use of strictly sterile procedures is not commonplace in work on insects, but our experience is that there is a great reduction in post-operative mortality if care is taken about these matters. Cultures from the cages and solutions prior to sterilization showed the presence of an abundant microflora which these methods served to remove. Care should also be taken to ensure that the wound is not forced open when healing is taking place. This can easily happen if the animal is allowed to force its way amongst grass stems or when other locusts are present. Survival was also aided by keeping it at room temperature (ZO-22°C) until the wound had healed. Excess moisture has a detrimental effect on survival, and filter paper should be present to absorb any moisture present in the grass or excreted by the locust. We are grateful to Dr. G. Staal for his advice on these points at an early stage in this work. The criteria applied to study the effects of the operations described was to follow the growth in weight and the occurrence of subsequent growth and moulting cycles in the operated animals and their operated controls. If, as has been postulated
372
KENNETH U. CLARKE ANDPETERA. LANGLEY
in a previous paper, the initiation of a growth and moulting cycle occurs at an ecdysis, then a cycle will already have been initiated when the operation was performed. Growth and moulting might therefore be expected to follow each operation but that the next cycle will be the one interfered with. As is shown in Table 1, severance of the ventral nerve cord, cutting of the outer oesophageal nerves, and of the recurrent nerve was without effect on the appearance of a second moulting cycle. The removal of the ingluvial ganglion which is responsible for the control of movements of the crop and gizzard, proved rapidly fatal to the locust, probably due to gut paralysis. The compIete cessation of growth that occurred in Locusta when the frontal ganglion was removed has not been reported previously in any insect. The frontal ganglion has been implicated by FAIVRE (1863) in the control of swallowing movements in Dytiscus marginalis L., and this is the function usually attributed to it. For this reason particular care was taken to ensure that the results obtained were not simply due to the failure of the animal to feed. Frequent observation has shown that the animals feed regularly throughout the rest of their lives; and dissection showed the gut to be always full of fresh food. Comparison with starving insects also revealed differences. Starved locusts continually lost weight and did not survive for more than 96 hr. The operated animals stayed at a constant weight and survived for 310 hr. Partially starved animals when given access to food take enormous meals followed by a period of non-feeding (CLARKE,1956). The regular fluctuations in weight so produced are quite different from those found in these operated animals. Other functions for the frontal ganglion have been found. Thus, BOUNHIOL (1938) thought that it was concerned in the metamorphosis of Bombyx mori L. KAMIOKA (1953) showed a decrease in the oxygen consumption of the mid-gut epithelium of Bombyx mori larvae that had been deprived of their frontal ganglion. UNGER (1957) h as suggested that the release of two types of neurohormones in cockroaches might be regulated by nervous impulses from the frontal ganglion and recurrent nerve. Sensory endings in the nerves arising from the frontal ganglion have been reported in Ovyctes larvae (ORLOV, 1924) and in the pharynx and crop of Panorpa (GRELL, 1938), suggesting other than motor functions for this ganglion. A more detailed analysis of the functions of the frontal ganglion is given in the next paper. It is sufficient to say here that its removal results in the complete cessation of growth in the locust and it must therefore form a necessary part of the neuroendocrine system of these insects. REFERENCES ALBRECHTF. 0. (1953) The Anatomy of the Migratory Locust. University of London, Athlone Press. BOUNHIOL J. J. (1938) R81e possible du ganglion frontal dans la metamorphose de Bombyx mori L.
C.R. Acad. Sci., Paris 206, 773-774.
CAZALP. (1948) Les glandes endocrines rCtroc&Cbralis des insectes (Etude morphologique). Bull. Biol., Woods Hole (Suppl.) 32, l-227.
INITIATIONOFGROWTHANDMOULTING IN LOCUSTA
MIGRATORIA
A4IGRATORIOIJX.S
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CLARI~EK. U. (1956) Studies on the relationship between changes in the volume of the tracheal system and growth in Locusta migratoria L. Proc. 10th int. Cong. Ent. 2,205-211. CLARKEK. U. and GRENVILLEH. (1960) Nervous control of movements in the foregut in Schistocerca gregaria Forsk. Nature, Lond. 186, 98-99. CLARKEK. U. and LANGLEYP. A. (1963) Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. & F.-I. The time and nature of the initiating stimulus. J, Ins. Physiol. 9, 287-292. FAIVREE. (1863) C. R. Sot. Biol., Paris 5, 101-104 (quoted in MARCHAL,P. (1911)). GRELL K. G. (1938) Der Darmtraktus von Punorpa communis L. und seine Anhtige bei Larvae und Imago. ZooZ.Jb. (Anat.) 64, l-86. HICHNAM K. C. (1961). The histology of the neurosecretory system of the adult female locust Schistocerca gregaria Forsk. Quart. J. micr. Sci. 102, 27-38. IMMS A. D. (1957) Textbook of Entomology (Revised RICHARDS0. W. and DAVIESR. G.). Methuen, London. KAMIOKA S. (1953) Oxygen consumption of midgut epithelium of the silkworm larvae deprived of frontal ganglion. Oyo Dobutsugahu-Zusshi 17, 155-160. MARCHALP. (1911) Dictionnaire de Physiologie de Charles Richet, p. 273. NAYAR K. K. (1957) Water content and release of neurosecretory products in Iphita Zimbata. Cur. Sci. 26, 25. ORLOV J. (1924) Die innervation des darmes der insekten (Larven vez Lamellicomien). 2. wiss. Zool. 122, 425-502. SNODGRASS R. E. (1935) Principles of Insect Morphology. McGraw-Hill, New York. STAAL G. B. (1961) Studies on the Physiology of Phase Induction in Locusta migratoria migratorioides R. and F. Veenman and Zonen, Wageningen. STRANGEWAYS-DIXONJ. (1961) The relationships between nutrition, hormones and reproduction in the blowfly CaEliphora erythrocephala Meig. II. J. exp. Biol. 38,637-646. THOMSENE. and MOLLER I. (1959) N eurosecretion and intestinal proteinase activity in an insect, Calliphora evythrocephala Meig. Nature, Lond. 183, 1401-1402. UNGER H. (1957) Untersuchungen zur neurohormonalen steurung der herztHtigkeit bei Schaben. Biol. Zbl. 76, 204-255. WIGGLESWORTH V. B. (1939) The Principles of Insect Physiology. Methuen, London.
STUDIES ON THE INITIATION OF GROWTH AND MOULTING IN MIGRATORIA MIGRATORIOIDES R. & F.-II
LOCUSTA THE
Printed in Great Britain
R6LE
OF THE
KENNETH
STOMATOGASTRIC
U. CLARKE and PETER
Department
of Zoology,
University
NERVOUS
SYSTEM
A. LANGLEY of Nottingham
(Received 15 November 1962)
Abstract-A
brief account is given of the anatomy of the stomatogastric nervous system. The role of this system with regard to the postembryonic growth of the locust was investigated by cutting certain of the nerves, removing certain of the ganglia, and observing the effect of these operations on the further growth and moulting of the locust. An account of the technique of each operation is given, and stress is laid on certain asceptic procedures which were found to greatly decrease the mortality rate of the operated animals. All operations were performed on 33-hr-old third instar locusts. Cutting the ventral nerve cord in front of the first abdominal ganglion had little effect on the further growth and moulting of the insects which survived into the adult instar. Cutting the outer oesophageal nerves did not prevent the animals moulting and growing to the fifth instar; however, the growth was slower and the mortality higher than in the operated controls. Removal of the ingluvial ganglion resulted in death within 43 hr of the operation. Cutting the recurrent nerve behind the frontal ganglion did not prevent the animals from moulting and growing to the end of the fourth instar; growth was slower than that of the operated controls. Removal of the frontal ganglion resulted in the immediate cessation of growth, the animal remaining at a constant weight and without moulting until its death. Death occurred at between 260-310 hr which is approximately twice as long as it took the operated control to grow and moult to the next instar. The operated animals were observed to feed frequently, to defecate normally, and upon dissection their guts were found to be full of fresh food. These effects cannot therefore be attributed to a failure to feed. These experiments show that the frontal ganglion has an essential role in the growth of the locust which, however, can survive and show normal activity in its absence.
INTRODUCTION
IN A previous paper it has been shown that the time of activation of the prothoracic gland coincided with the period of ecdysis, and it was suggested that the relaxation and distension of the gut that occurs at this time provided the stimulus for the release of the growth and moulting hormone (CLARKE and LANGLEY, 1963). The present paper substantiates this, and, by surgical interference with the stomatogastric system, establishes the nervous pathways involved. 363
364
KENNETHTJ. CLARKE ANDPETER A. LANGLEY
The visceral nervous system in insects has been described in general terms by SNODGRASS(1935), WIGGLESWORTH(1939), and IMMS (1957). It has been investigated in a wide variety of types by CAZAL(1948) whose description of Metrioptera (Orthoptera, Tettigoniidae) agrees with that given by ALBRECHT (1953) and STAAL (1961) for Locusta. However, the present study has revealed some nerves not described by these workers. To facilitate an understanding of the significance of the operations to be described, a brief account of the anatomy of the stomatogastric system is given here. THE ANATOMY OF THE STOMATOGASTRIC
SYSTEM
The structure of this system has been investigated by dissection of locusts that have been subjected to intra-vitam injections of methylene blue, and by studies of serial sections through the heads of immature and mature locusts. No differences could be found between the larval and adult locusts in the structure of this system. Anteriorly, the stomatogastric system is represented by a frontal ganglion lying in the midline dorsal to the pharynx and a little in front of the brain. It is approximately pear-shaped with its apex pointing posteriorly. From this apex the recurrent nerve passes back in the midline dorsal to the gut to the hypocerebral ganglion which is just behind the brain. From the anterior lateral edges of the frontal ganglion, the frontal connectives pass back on each side of the gut to the tritocerebrum. Close to the brain each frontal connective lies alongside the labral nerve and may join it at a point where it enters the brain. From the lateral borders of the frontal ganglion, just behind the origin of the frontal connectives, a pair of nerves pass laterally to the pharynx, and from each side of the ganglion near the origin of the recurrent nerve a similar pair also pass down to the gut. These are the anterior and posterior pharyngeal nerves respectively. From the hypocerebral ganglion a pair of inner oesophageal nerves pass down to the surface of the crop. Lateral to these are a pair of outer oesophageal nerves which pass posteriorly and ventrally to the ingluvial ganglia. The ingluvial ganglia lie one on each side of the crop just in front of the anterior tips of the mesenteric caeca. From them nerves pass to the posterior crop wall, the gizzard, and inner and outer caecal nerves to the mid-gut (CLARKE and GRENVILLE, unpublished). The corpora cardiaca are situated on the dorsal side of the hypocerebral ganglion. They have been described in detail for Schistocerca gregaria Forsk. by HIGHNAM (1960). They are exactly similar in Locusta migratoria. A pair of nerves, the nervi corporis cardiaci I and II, connect them to the brain; a shorter pair of nerves connect them with the hypocerebral ganglion. A pair of corpora allata are present, one on each side of the crop just behind the brain. A pair of nervi corporis allati join them to the corpora cardiaca. They receive a further nerve originating from the sub-oesophageal ganglion (CAZAL, 1948 ; STAAL, 1961). This nerve in methylene-blue preparations appears to have some fibres passing right through the corpora allata and entering the nervi corporis allati. A short distance from its emergence from the corpus allatum this lateral nerve gives rise to a branch which passes anteriorly and divides into three further
INITIATION OFGROWTH ANDMOIJLTING IN LOCUSTA
MIGRATORIA
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branches. One,of these passes to the ventral dilator muscles of the pharynx, the second to the muscles of the ventral head apodeme, and the third a small branch which enters the tritocerebrum close to its junction with the circumoesophageal connectives. Near the point of insertion of the lateral nerve with the sub-oesophageal ganglion another branch arises which passes forwards and innervates the anterior dilator muscles of the crop. On the hypothesis that the initiation of a growth and moulting cycle arises from changes in the gut at ecdysis the stomatogastric nerves of especial interest are the frontal ganglion and its branches, the recurrent nerve, the inner and outer oesophageal nerves, and the ingluvial ganglion and its branches. The frontal ganglion has been implicated by FAIVRE (1863) in the control of swallowing movements in Dytticus marginalis L., and this seems to be the generally accepted view of its function. However, ORLOV (1924) has figures of both motor and sensory endings of nerves arising from the frontal ganglion and thus there is a pathway along which a stimulus could pass. The recurrent nerve does not seem to have been assigned a function although neurosecretory material has been described in it by NAYAR (1957). The inner oesophageal nerve has been shown to have a motor function (CLARKE and GRENVILLE, 1960) and has not been further considered here. The outer oesophageal nerves have no clearly defined function in Locusta, although in Calliphora THOMSEN ANDMILLER (1959) and STRANGEWAYSDIXON (1961) have shown they convey neurosecretory ,material to the mid-gut where it is necessary for enzyme formation. No neurosecretory material has been found in these nerves in Locusta (CLARKE and GRENVILLE, unpublished). It was therefore decided to cut the recurrent nerve, the outer oesophageal nerve, and to remove the frontal ganglion and the ingluvial ganglion, immediately after an ecdysis and to determine the effect these operations may have on the further growth and moulting of the locust. MATERIAL
AND METHODS
The insects used were third instar larvae of Locusta migratoria migratorioides R. and F., these being the smallest convenient size for subjection to operative techniques while allowing two further cycles of growth and moulting in which the experimental results could be assessed. The insects were reared under the conditions described previously (CLARKE and LANGLEY, 1963). The growth of each insect was determined by weight at 12-hourly intervals and by determining its instar duration. Each experiment described below was carried out on thirty-six animals all within 12 hr age range. Twelve animals were subjected to the operation (0), twelve to a control operation (CO), and twelve untouched but subjected to the same environmental treatment (C)e A very high percentage (90-100 per cent) of success in recovery from the operation was obtained in all the results reported here. This contrasted with earlier experiments when the recovery rate was low (20-30 per cent). This change was attributed by the authors to the introduction of rigid aseptic procedures and to a
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definite regime of post-operative treatment suggested by STAAL, (personal communication). Since these do not seem to be current in operative work on insects, they are described below. All glassware was steam pressure sterilized and kept in sterile containers until used. The cages in which the animals were kept, and the instruments used for the operations, were sterilized in ultra-violet light for 30 min immediately before use. Following the operation, it was necessary to keep the animals as dry as possible. Each animal was kept in a glass jar with perforated lid, the jar containing filter paper rolled into a tight cylinder on which the animal could cling and a circle of filter paper on the bottom to absorb any moisture excreted by the animal. Fresh grass was given daily. The grass was cut short and bound into a bundle by a tight wire so that the locust could not get amongst the grass stems and open up its wounds. The jars were kept in a dust-proof cabinet at room temperature (20-22°C) for 48 hr. This kept the animal relatively inactive until its wound had had time to heal. When healed, the locust was returned to the insectary (28°C) and the precautions described above stopped. SURGICAL
TECHNIQUES
Each insect was anaesthetized with di-ethyl ether for approximately 3 min; this rendered the animal unconscious for a sufficient length of time to perform the operation and did not cause death in any instance. Severance of the aentral nerve cord The anaesthetized insect was fastened with its ventral side uppermost on the stage of a binocular dissecting microscope. A transverse incision was made into the intersegmental membrane between the first and second abdominal sternites ; the sternites were parted and the ventral nerve cord exposed. A small section of cord was removed thus isolating the thoracic and abdominal parts of the chain, and minimizing the chance of continuity being established by tissue regeneration. Sealing the wound was found to be unnecessary since the natural overlap of the abdominal sternites ensured adequate protection, and rapid healing occurred. Operations on the stomatogastric system posterior to the brain The anaesthetized insect was placed on the stage of the microscope with its It was held in an extended position with its thorax dorsal surface uppermost. slightly raised and its neck membrane exposed. A small transverse incision was made in the neck membrane. This gave immediate access to the lateral regions of the oesophagus, the crop, and to the ingluvial ganglion. The outer oesophageal nerves were severed by a simple cut about half way along their length. The ingluvial ganglion was freed from its nerves and connective tissue and removed from the body. Again no special sealing of the wound which healed rapidly was necessary.
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Operations on the stomatogastra’csystem anterior to the brain The insect was held on the stage of the microscope with the front of the head upwards. A transverse cut was made in the cuticle immediately below the median ocellus and extending from the left to the right carina. Two further cuts were made, one from the end of the lateral cut down the inside of the right carina to the epistomal suture, and a similar cut down the left carina. The flap of cuticle and hypodermis so formed was turned ventrally exposing the anterior air sacs of the
1270.
1150. wt. 1030. mg
. 910.
790.
670,
550
430
70
1
150
.
230
310
hr. FIG. 1. The effect of severing the ventral nerve cord early during the fourth instar on the further growth and development of the locust. +-----0 ventral nerve cord severed; O0 operated control. Time is given as the number of hours that have elapsed since the operation. Arrows mark the fourth and fifth ecdysis respectively. Each curve represents the growth of one individual. For further data see Table 1.
head. These were parted in the midline to expose the upper surface of the pharynx and the anterior surface of the brain. The recurrent nerve was severed by a simple cut between the frontal ganglion and the brain. The frontal ganglion was removed by cutting the recurrent nerve, the frontal connectives, freeing the ganglion from the connective tissue surrounding it, and removing the ganglion completely from the head. Upon completion of the operation the flap was replaced and the wound healed without further treatment.
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KENNETH U. CLARKE AND PETER A, LANGLEY
RESULTS
(A) Severance of the ventral nerve cord The subsequent growth and moulting of these insects is shown in Fig. 1. All the insects recovered and grew normally throughout the fourth instar. Moulting was slightly delayed in the operated and operated controls probably owing to post-operational shock. Mortality was high at the fourth ecdysis (Table l), owing
hr FIG. 2. The effect of severing the outer oesophageal nerves early in the third instar on the further growth and moulting of the locust. 0 -0 outer oeso0 operated control. The curves are for one phageal nerves severed; Ooperated insect and one operated control. For further data see Table 1. Zero time is at the moment of the second ecdysis, 0 marks the time of the operation, and arrows mark the third ecdysis.
to the difficulties the locusts experienced in shedding their cuticle due to wound adhesions. The two insects that reached the adult instar were dissected and
confirmation obtained that the nerve cord was severed and had not re-united during the intervening growth period. It is concluded from the results of this operation that neither the abdominal ventral nerve cord nor the caudal nervous system was involved in the initiation of the moulting cycle.
C oc 0
Outer oesophageal nerves cut
8 12 10 12 24
C oc
0
8 12 24
Frontal ganglion removed
--
C oc 0
nerve
Recurrent cut
9 15 15
C oc 0
9 15 15 24
12 12 12
Number of locusts used
Ingluvial ganglion removed
---
C oc 0
Ventral nerve cord severed
Operation
9 12 24
II
11 24
15 15
15 15 24
12 12
Survived from operation
---
8 12 20
8 10
8 10 24
9 14 3
9 14 15 24
-
Third instar
>
--
_
6 8
6 8 5
9 12 0 -
9 12 6 18
12 12 12
Fourth instar
_ _
6 8
8 8 0
14
8 8
12 10 6
Fifth instar
6 8 0
8 8 0
8 8 5 17
12 12 6
Fourth ecdysis
Number of insects surviving
None moulted to the fourth instar
7 9
7 9 15
9 14 0
9 14 7 24
-
Third ecdysis
TABLE 1.
-~--_ _ _
-
-
10 10 3
Fifth ecdysis
_ _
-
---
10 10 2 --
-___
Adult
KENNETHU. CLARKE AND PETER A. LANGLEY
370
(B) Severance of the outer oesophageal nerves The subsequent growth and moulting of these insects the survival rates in Table 1. Although growth continued, found in the operated controls and in the normal animals. was delayed, and mortality at the third ecdysis was high. completed their third ecdysis grew and moulted to the fifth
120.
0
100
.
200
is shown in Fig. 2, and it was slower than that Furthermore, moulting Those, however, which instar.
.
’
300
hr
FIG. 3. The effect of severing the recurrent nerve and removing the frontal ganglion early in the third instar on the further growth and moulting of the locust. Orecurrent nerve severed behind the frontal ganglion; 0 0 ----0 frontal ganglion removed; An operated control. Solid lines females, dotted lines males. Each curve represents the growth of one individual, for further data see Table 1. Operations were performed at times between the first and second points on each curve. Arrows mark the time of the third ecdysis.
(C) Removal of the ingluvial ganglion Although all the operated insects recovered from the anaesthesia, all died within 43 hr of the operation (Table 1). (D) Severance of the recurrent nerve The subsequent growth of these insects is shown in Fig. 3. The operated insects grew and moulted to the next instar. Their growth rate was lower than that
INITIATION
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of the operated controls and ecdysis was delayed by some 40 hr. Only three out of the twenty-four operated animals survived to the fourth instar, where they showed some increase in weight but became moribund and died at 200 hr in the fourth instar. Dissections showed that all three had developed a new cuticle under the old, thus indicating that the growth and moulting cycle of the fourth instar had started. (E) Removal of the frontal
ganglion
The operated insects lost weight during the first 24 hr following the operation, then after a slight increase during the next 48 hr remained practically constant in weight over the next 192 hr, finally dying without moulting (Fig. 3). The operated controls grew normally and moulted to the fourth instar with only a slight delay compared with the unoperated animals. Observations of the insects deprived of their frontal ganglion showed that once the effects of the operation had worn off they fed normally. Dissection at different periods after the operation showed the gut contained normal amounts of food. The failure to grow cannot therefore be due to the failure of these insects to feed: the intake of food, the amount in the gut, and defecation proceeded normally in these animals. DISCWSSION
The surgical techniques described in this paper are fairly straightforward and call for no special comment. Three subsidiary features essential for their success are worth further discussion. Ether was used for anaesthesia and was found to be superior to carbon dioxide in that its effects did not wear off so quickly, and to chloroform in that no deaths resulted from its use. Operations without anaesthesia were always accompanied by a high mortality largely due to the length of time necessary to accomplish the operation when the air sacs are moving and the blood circulating. The use of strictly sterile procedures is not commonplace in work on insects, but our experience is that there is a great reduction in post-operative mortality if care is taken about these matters. Cultures from the cages and solutions prior to sterilization showed the presence of an abundant microflora which these methods served to remove. Care should also be taken to ensure that the wound is not forced open when healing is taking place. This can easily happen if the animal is allowed to force its way amongst grass stems or when other locusts are present. Survival was also aided by keeping it at room temperature (ZO-22°C) until the wound had healed. Excess moisture has a detrimental effect on survival, and filter paper should be present to absorb any moisture present in the grass or excreted by the locust. We are grateful to Dr. G. Staal for his advice on these points at an early stage in this work. The criteria applied to study the effects of the operations described was to follow the growth in weight and the occurrence of subsequent growth and moulting cycles in the operated animals and their operated controls. If, as has been postulated
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KENNETH U. CLARKE ANDPETERA. LANGLEY
in a previous paper, the initiation of a growth and moulting cycle occurs at an ecdysis, then a cycle will already have been initiated when the operation was performed. Growth and moulting might therefore be expected to follow each operation but that the next cycle will be the one interfered with. As is shown in Table 1, severance of the ventral nerve cord, cutting of the outer oesophageal nerves, and of the recurrent nerve was without effect on the appearance of a second moulting cycle. The removal of the ingluvial ganglion which is responsible for the control of movements of the crop and gizzard, proved rapidly fatal to the locust, probably due to gut paralysis. The compIete cessation of growth that occurred in Locusta when the frontal ganglion was removed has not been reported previously in any insect. The frontal ganglion has been implicated by FAIVRE (1863) in the control of swallowing movements in Dytiscus marginalis L., and this is the function usually attributed to it. For this reason particular care was taken to ensure that the results obtained were not simply due to the failure of the animal to feed. Frequent observation has shown that the animals feed regularly throughout the rest of their lives; and dissection showed the gut to be always full of fresh food. Comparison with starving insects also revealed differences. Starved locusts continually lost weight and did not survive for more than 96 hr. The operated animals stayed at a constant weight and survived for 310 hr. Partially starved animals when given access to food take enormous meals followed by a period of non-feeding (CLARKE,1956). The regular fluctuations in weight so produced are quite different from those found in these operated animals. Other functions for the frontal ganglion have been found. Thus, BOUNHIOL (1938) thought that it was concerned in the metamorphosis of Bombyx mori L. KAMIOKA (1953) showed a decrease in the oxygen consumption of the mid-gut epithelium of Bombyx mori larvae that had been deprived of their frontal ganglion. UNGER (1957) h as suggested that the release of two types of neurohormones in cockroaches might be regulated by nervous impulses from the frontal ganglion and recurrent nerve. Sensory endings in the nerves arising from the frontal ganglion have been reported in Ovyctes larvae (ORLOV, 1924) and in the pharynx and crop of Panorpa (GRELL, 1938), suggesting other than motor functions for this ganglion. A more detailed analysis of the functions of the frontal ganglion is given in the next paper. It is sufficient to say here that its removal results in the complete cessation of growth in the locust and it must therefore form a necessary part of the neuroendocrine system of these insects. REFERENCES ALBRECHTF. 0. (1953) The Anatomy of the Migratory Locust. University of London, Athlone Press. BOUNHIOL J. J. (1938) R81e possible du ganglion frontal dans la metamorphose de Bombyx mori L.
C.R. Acad. Sci., Paris 206, 773-774.
CAZALP. (1948) Les glandes endocrines rCtroc&Cbralis des insectes (Etude morphologique). Bull. Biol., Woods Hole (Suppl.) 32, l-227.
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CLARI~EK. U. (1956) Studies on the relationship between changes in the volume of the tracheal system and growth in Locusta migratoria L. Proc. 10th int. Cong. Ent. 2,205-211. CLARKEK. U. and GRENVILLEH. (1960) Nervous control of movements in the foregut in Schistocerca gregaria Forsk. Nature, Lond. 186, 98-99. CLARKEK. U. and LANGLEYP. A. (1963) Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. & F.-I. The time and nature of the initiating stimulus. J, Ins. Physiol. 9, 287-292. FAIVREE. (1863) C. R. Sot. Biol., Paris 5, 101-104 (quoted in MARCHAL,P. (1911)). GRELL K. G. (1938) Der Darmtraktus von Punorpa communis L. und seine Anhtige bei Larvae und Imago. ZooZ.Jb. (Anat.) 64, l-86. HICHNAM K. C. (1961). The histology of the neurosecretory system of the adult female locust Schistocerca gregaria Forsk. Quart. J. micr. Sci. 102, 27-38. IMMS A. D. (1957) Textbook of Entomology (Revised RICHARDS0. W. and DAVIESR. G.). Methuen, London. KAMIOKA S. (1953) Oxygen consumption of midgut epithelium of the silkworm larvae deprived of frontal ganglion. Oyo Dobutsugahu-Zusshi 17, 155-160. MARCHALP. (1911) Dictionnaire de Physiologie de Charles Richet, p. 273. NAYAR K. K. (1957) Water content and release of neurosecretory products in Iphita Zimbata. Cur. Sci. 26, 25. ORLOV J. (1924) Die innervation des darmes der insekten (Larven vez Lamellicomien). 2. wiss. Zool. 122, 425-502. SNODGRASS R. E. (1935) Principles of Insect Morphology. McGraw-Hill, New York. STAAL G. B. (1961) Studies on the Physiology of Phase Induction in Locusta migratoria migratorioides R. and F. Veenman and Zonen, Wageningen. STRANGEWAYS-DIXONJ. (1961) The relationships between nutrition, hormones and reproduction in the blowfly CaEliphora erythrocephala Meig. II. J. exp. Biol. 38,637-646. THOMSENE. and MOLLER I. (1959) N eurosecretion and intestinal proteinase activity in an insect, Calliphora evythrocephala Meig. Nature, Lond. 183, 1401-1402. UNGER H. (1957) Untersuchungen zur neurohormonalen steurung der herztHtigkeit bei Schaben. Biol. Zbl. 76, 204-255. WIGGLESWORTH V. B. (1939) The Principles of Insect Physiology. Methuen, London.