Pupal diapause in Mimas tiliae can be terminated by keeping the pupa for at least 4 weeks at 3° C. The adult emerges about 15 days after transfer to 25° C.

Histological examination shows that the neurosecretory cells in the brain are inactive in the diapausing pupa, but they elaborate intracellular material during the first 3 weeks at 3° C. The material is passed to the corpora cardiaca. The neurosecretory cells are again inactive by the end of the low-temperature period.

The brain/cardiaca system shows little sign of secretory activity during the subsequent period at 25° C. The corpora cardiaca undergo phagocytosis and reorganization during this time. This suggests that conditions for further development are established by the end of the low-temperature period. This hypothesis is supported by the fact that development of the non-endocrine organs begins immediately the pupa is transferred to 25° C after 4 weeks at 3° C.

Extirpation and implantation experiments involving the brain, with and without its associated corpora cardiaca, support the histological results, indicating that the brain is necessary for diapause development at 3° C and that the corpora cardiaca are involved in the release of the brain factor.

There is evidence that diapause in insects is under the control of hormonal factors. Rhodnius prolixus nymphs live in a quiescent state (diapause) after decapitation, and it has been shown that the operation removes the source of developmental hormones (Wigglesworth, 1934). This author suggests that the diapause obtaining normally in many insect species results from the absence of developmental hormones. Further, Williams (1946) has shown that the termination of diapause in Platysamia cecropia is caused by factors secreted by the brain-factors produced mostly at low temperatures, but absent in diapausing pupae.

In Bombyx mori diapause in the egg is initiated by the sub-oesophageal ganglion of the parent (Fukuda, 1951 a, b;Hasegawa, 1951). Possibly pupal diapause also is caused by the secretion of a diapause factor (Hinton, 1953). It would appear then that in some cases diapause may result from the absence of a hormone; in other cases from the presence of one. Correspondingly, termination of diapause may result from the production of a hormone or the disappearance of one. Alternatively, the two processes of induction and termination of diapause may be due to the functioning of two different hormones.

The present investigation was begun in an attempt to resolve some aspects of this problem. It soon became evident that the process of diapause ‘break’ in Mimas tiliae differed in detail from that described by Williams (1946) in the Platysamia cecropia pupa. The description of thi!; difference is the main concern of this paper. The histological observations are of particular interest since Williams has published no comparable data on the endocrine system in the pupa of P. cecropia during diapause and diapause ‘break’.

Diapausing and experimentally produced post-diapause pupae of the Lime Hawk Moth, M. tiliae, were used.

Samples of the pupae for histological study were fixed in Bouin, DuboscqBrazil, Susa, or Zenker. After fixation, the thick cuticle was dissected away from the tissues beneath, which were subsequently embedded in paraffin wax and sectioned at 6μ. A few pupae were embedded whole in ester wax after the cuticle had been softened with diaphanol. The serial sections were stained with Weigert’s haematoxylin and eosin, with Masson’s or Mallory’s polychrome staining techniques, or with the chrome-haematoxylin phloxine technique of Gomori to show neurosecretory material. The haematoxylin and eosin method after Bouin fixation gave excellent results for the purpose of this study.

To give support to the results obtained from the histological study, a series of experiments involving removal or implantation of the brain and corpora cardiaca was performed. The methods followed will be described in the appropriate section (p. 82).

Low temperature and the termination of diapause

Under natural conditions, the Lime Hawk Moth pupates during September and enters a pupal diapause lasting about 8 months, adult development beginning again about the middle of the following May. In the laboratory diapause can be extended to 12 months or longer by keeping the pupa in a saturated atmosphere at a temperature of 25° C. Although the period of diapause can be considerably lengthened by keeping the pupa at this temperature, it does not continue indefinitely. The adults always eventually emerge.

The diapause can be terminated precociously by subjecting the pupa to a low temperature (3° C) for at least 4 weeks, and then transferring it to a temperature of 25° C; the adult emerges about a fortnight later (table 1). A number of pupae (10% in table 1) emerge after less than 4 weeks at 3° C, but the great majority require the full period. The process by which diapause is brought to an end is called ‘diapause development’ (Andrewartha, 1952). In the pupa of M. tiliae the rate of diapause development is evidently very low at 25° C, but is greatly increased at 3° C.

TABLE 1.

Adult emergence from pupae subjected to a temperature of 3° C

Adult emergence from pupae subjected to a temperature of 3° C
Adult emergence from pupae subjected to a temperature of 3° C

The figures in the last column represent days on which the first and last emergences in the samples occurred. In each sample, the largest number of emergences occurred during the four teenth and fifteenth days.

The histology of the neurosecretory cells and corpora cardiaca during diapause

The endocrine system has four components-neurosecretory cells (located in the cerebral ganglia), corpora cardiaca, corpora allata, and prothoracic glands. The neurosecretory cells are arranged in four groups, an inner and an outer group in each half of the brain. The inner groups lie close together on each side of the midline, on the dorsal side of the anterior region of the pars inter-cerebralis. Together they comprise 20 cells. Each outer group contains 5 cells and is situated dorsally about half-way along each cerebral lobe. The corpora cardiaca and allata are both paired (fig. 1), lying lateral to the aorta, from which they are completely separate (‘type lateralise’ of Caza!, 1948). Each corpus cardiacum is a small pyriform body, connected with the brain by the usual pair of nervi corporis cardiaci. The roughly spherical corpora allata lie immediately behind the corpora cardiaca so that no nervi allati are visible. The prothoracic glands consist of two bilaterally placed masses of tissue in intimate association with the branches of the prothoracic tracheae. In general appearance they resemble a tightly clumped string of beads. Fig. 1 shows the appearance of part of the tissue lying posterior to the brain.

FIG. 1.

Drawing of a dissection of the anterior part of the diapausing M. tiliae pupa to show the general arrangement of the endocrine system. Only the anterior lobes of the prothoracic glands are shown.

FIG. 1.

Drawing of a dissection of the anterior part of the diapausing M. tiliae pupa to show the general arrangement of the endocrine system. Only the anterior lobes of the prothoracic glands are shown.

The neurosecretory cells

The cells are generally pear-shaped in outline with a prominent axon leaving the cell at the apex of the narrow end. They are readily identified by their staining reactions, the cytoplasm becoming deep pink or red with eosin, red with acid fuchsin, and reddish blue with chrome haematoxylin. There are, however, two distinct types of cell, different in size and with some difference in staining reaction. The first type is small (mean diameter about 24μ) with a markedlyacidophil cytoplasm and an extranuclear meshwork which stains strongly with haematoxylin. The second type is larger (figs. 2, A; 3, A), with a homogeneous acidophil cytoplasm containing a few acidophil globules or clear vacuoles. The extranuclear meshwork is less obvious than in the smaller cells. There are more of the smaller type of cell, but both types are present in the several groups of neurosecretory tissue. No sign of any secretory material can be found along the paths of the nervi cardiaci at this time. The neurosecretory cells (fig. 3, B) in the brains of a small number of diapausing individuals show signs of the sort of secretory activity which will presently be described. It is thought that these are the individuals which break diapause prematurely when subjected to a low temperature for periods of less than 4 weeks (p. 74 and table 1).

FIG. 2.

Camera lucida drawings of sections of brain through the pars inter-cerebralis region to show the neurosecretory cells. A, vertical section, diapausing pupa. The neurosecretory ce11s contain no inclusions and the extranuclear meshwork is very obvious (stages a and b of the secretory cycle). B, horizontal section, after I week at 3° C. One cell contains inclusions and a few peripheral vacuoles (end of stage c). c, vertical section, after :z. weeks at 3° C. Three cells in different stages of the secretory cycle are shown.

FIG. 2.

Camera lucida drawings of sections of brain through the pars inter-cerebralis region to show the neurosecretory cells. A, vertical section, diapausing pupa. The neurosecretory ce11s contain no inclusions and the extranuclear meshwork is very obvious (stages a and b of the secretory cycle). B, horizontal section, after I week at 3° C. One cell contains inclusions and a few peripheral vacuoles (end of stage c). c, vertical section, after :z. weeks at 3° C. Three cells in different stages of the secretory cycle are shown.

FIG. 3.

(plate). Photomicrographs of neurosecretory cells in the pars intercerebralis, A, diapausing pupa. Two cells with obvious extranuclear meshworks are shown. No inclu-sions (stage b). B, diapausing pupa. Cells with inclusions (present in a very small proportion of the diapausing pupae). C, after r week at 3° C. Cell with small inclusions shown, together with a and b cells. D, after 3 weeks at 3° C. a, c, and d cells shown. E, after 4 weeks at 3° C. Only a cells present. F, neurosecretory material along the axons of the nervus corporis cardiacum. A, B, and F, Masson’s trichrome. S, D, and E, Weigert’s haematoxylin and eosin.

FIG. 3.

(plate). Photomicrographs of neurosecretory cells in the pars intercerebralis, A, diapausing pupa. Two cells with obvious extranuclear meshworks are shown. No inclu-sions (stage b). B, diapausing pupa. Cells with inclusions (present in a very small proportion of the diapausing pupae). C, after r week at 3° C. Cell with small inclusions shown, together with a and b cells. D, after 3 weeks at 3° C. a, c, and d cells shown. E, after 4 weeks at 3° C. Only a cells present. F, neurosecretory material along the axons of the nervus corporis cardiacum. A, B, and F, Masson’s trichrome. S, D, and E, Weigert’s haematoxylin and eosin.

The corpora cardiaca

Each corpus cardiacum (fig. 6, A, p. 81) is surrounded by a thin membrane which stains blue or green with Mallory’s or Masson’s technique. The membrane is continuous with the thicker membrane over the corpus allatum. A large part of each gland is made up of the nerve-fibres from the nervi corporis cardiaci (fig. 6, A). Numbers of nerve-cells are also present, especially where the nervi cardiaci enter the gland. A few connective tissuecells beneath the bounding membrane and cells of the tracheal epithelium can be identified.

FIG. 4.

Camera lucida drawings of neurosecretory cells from the pars intercerebralis region to show the 4 stages of the secretory cycle. Semi-diagrammatic.

FIG. 4.

Camera lucida drawings of neurosecretory cells from the pars intercerebralis region to show the 4 stages of the secretory cycle. Semi-diagrammatic.

FIG. 5.

Camera lucida drawings of brain sections through the pars intercerebralis region. A, vertical section, after 3 weeks at 3° C. Neurosecretory cells in three stages of the secretory cycle similar to fig. 2, C. B, vertical section, after 4 weeks at 3° C. One cell only shows a few inclusions, Most cells are in stage a of the cycle (similar to fig, 2, A),

FIG. 5.

Camera lucida drawings of brain sections through the pars intercerebralis region. A, vertical section, after 3 weeks at 3° C. Neurosecretory cells in three stages of the secretory cycle similar to fig. 2, C. B, vertical section, after 4 weeks at 3° C. One cell only shows a few inclusions, Most cells are in stage a of the cycle (similar to fig, 2, A),

FIG. 6.

Camera lucida drawings of sections of corpus cardiacum. A, in the diapausing pupa. The gland is large, but there are no inclusions. B, after I week at 3° C. Note the presence of acidophil inclusions, especially in the region of the nervus corporis cardiacus. There is a fine basiphil extranuclear meshwork in the cytoplasm of the glandular cells. c, after 3 weeks at 3° C. The gland is still large and the acidophil incl sions arc numerous. A vacuolated haemo cyte is evident near the centre of the gland. D, after 4 weeks at 3° C. The gland is small com pared with the previous stage. There are no inclusions. The glandular cells are small and relatively inconspicuous.

FIG. 6.

Camera lucida drawings of sections of corpus cardiacum. A, in the diapausing pupa. The gland is large, but there are no inclusions. B, after I week at 3° C. Note the presence of acidophil inclusions, especially in the region of the nervus corporis cardiacus. There is a fine basiphil extranuclear meshwork in the cytoplasm of the glandular cells. c, after 3 weeks at 3° C. The gland is still large and the acidophil incl sions arc numerous. A vacuolated haemo cyte is evident near the centre of the gland. D, after 4 weeks at 3° C. The gland is small com pared with the previous stage. There are no inclusions. The glandular cells are small and relatively inconspicuous.

In the posterior part of each corpus cardiacum are four or five large cells (fig. 6, A), very different in appearance from the other components of the gland.

The cells have a mean diameter of about 35μ with one or two long prolongations ending blindly in the tissue of the gland. Around the nucleus and spreading through the cytoplasm of each cell are traces of a meshwork similar to that described from the neurosecretory cells in the brain. These large cells are thought to be the glandular elements of the corpora cardiaca. Since the corpora cardiaca develop as evaginations from the foregut in the neighbourhood of the hypocerebral ganglion (Roonwal, 1937), they are considered to be modified autonomic nerve-ganglia. On this basis, the large cells in the corpora cardiaca are possibly modified nerve-cells, and therefore analogous to the neurosecretory cells in the central nervous system, which they resemble.

The histology of the neurosecretory cells and corpora cardiaca during diapause development at 3° C

The neurosecretory cells

By the end of the first week at 3° C, the staining reactions of the neurosecretory cells are very intense, enabling the cells to be easily identified. Close examination shows that the same two types of cell are present that were identifiable in the brain of the diapausing pupa (figs. 2, B; 3, C) but these now constitute a smaller proportion of the total number of cells (table 2). The greater number of the cells contain masses of inclusions, while others are vacuolated (fig. 2, B). These cells are not of new origin, since the total number of cells remains unchanged. These different neurosecretory cells are thought to represent stages in an asynchronous cycle of elaboration and release of the intracellular material. The cycle can be differentiated into 4 stages, according to the size of the cells (table 2) and their contents (fig. 4). Though it is not possible to correlate the changes in each cell with a definite time sequence, examination of the material suggests the cycle to be as follows.

TABLE 2.

Size of neurosecretory cells at different stages in the secretory cycle, and the proportion of different stages present during diapause and diapause development at 30 °C

Size of neurosecretory cells at different stages in the secretory cycle, and the proportion of different stages present during diapause and diapause development at 30 °C
Size of neurosecretory cells at different stages in the secretory cycle, and the proportion of different stages present during diapause and diapause development at 30 °C

The ‘resting’ stage (stage a) in which each cell is small (figs. 2, B; 4, A) with a mean diameter of about 24μ (table 2). The nucleus is small and globular. The cytoplasm takes up stain rather poorly, when compared with later stages. Around the nucleus and extending through the cytoplasm is a meshwork of interconnected basiphil strands. This stage corresponds almost exactly with the smaller kind of neurosecretory cell described from the diapausing pupa.

The second stage (stage b) only differs from the preceding one in that it is slightly larger (table 2), and the cytoplasm stains more deeply. At the points of junction of the extranuclear meshwork are small globules of intensely basiphil material (fig. 4, B). This stage is similar to the second type of ‘diapausing cell.

The next stage (stage c) includes several intergrading phases which show a progressive accumulation of intracellular material. The basiphil globules which are present in stage bat the junctions of the meshwork strands are considerably larger, and smaller globules are apparent along the lengths of the strands. Acidophil globules almost completely fill the cell in many cases (figs. 2, B; 4, C), and in these the meshwork has disappeared, or is perhaps hidden by the accumulated intracellular material. At the end of this stage, the cell is much larger (table 2). Its nucleus, too, is larger than in either of the preceding stages, and may be distorted from a spherical shape, possibly as a result of the pressure of the elaborated material in the cell (fig. 4, C).

No intracellular material is present in the fourth stage (stage d), and the cytoplasm is vacuolated, especially around the cell periphery (fig. 4, o). Some cells with few vacuoles have the proximal part of the axon swollen and packed with intracellular material, which can also be seen along the lengths of the nervi corporis cardiaci (fig. 3, F), whose origins lie in the neurosecretory cell groups. There is little doubt that this stage is derived from the preceding one (stage c) by the passage of elaborated material from the cell-body along the axons. The cell has decreased from the size shown in stage c (table 2).

Stage c and d cells are present (figs. 2, C; 3, C, D; 5, A) in about the same proportion after 2 and 3 weeks at 3° C (table 2). It is probable, therefore, that there is no accumulation of material within the groups of neurosecretory cells as a whole. It is possible that the rate at which material is produced may change during the first 3 weeks at 3° C, but no evidence for such a change can be obtained by the present method of analysis, which gives only the proportions of cells at different stages present at the time of fixing. But no matter what the rate of production is, the material is passed from the cell-bodies after formation, as can be seen by its presence along the nervi cardiaci.

By the end of the fourth week at 3° C, stage c and d cells are no longer visible in quantity (table 2). The majority of the cells are in stage a, with a few in b. The general appearance of the groups of cells is very similar to that in the diapausing pupa (figs. 3, E; 5, B; compare figs. 2, A; 3, A).

The appearance of the neurosecretory cells in the brain during diapause and after 4 weeks at 3° C, compared with their appearance after 1, 2, and 3 weeks at this temperature, indicates that intracellular material either is not being produced in the diapausing pupa and in the pupa after 4 weeks at 3° C, or is being produced at such a slow rate, or by so few cells, that stage c cells are not obvious. Further, the transference of the diapausing pupa to the low temperature in some way initiates or speeds up the production and release of the material by the neurosecretory cells (so that stage c and d cells are visible in quantity) during the first 3 weeks (table 2). Production of the material finishes abruptly during the fourth week at the low temperature.

Scharrer (1941) and Dupont-Raabe (1951) described comparable stages in the elaboration of intracellular material in the neurosecretory cells in the brains of cockroaches and phasmids respectively. Both authors assume that the smaller cells without inclusions or vacuoles represent a stage near the beginning of the secretory cycle, before the accumulation of material has begun.

The corpora cardiaca

By the end of the first week at 3° C, the corpora cardiaca appear rather larger than the glands in the diapausing pupa (fig. 6, B). There is little difference between the glandular cells of the corpora cardiaca at this time compared with those in the diapausing pupa. But a characteristic feature of the glands now is the presence of large numbers of intensely acidophil globules, sometimes in vacuoles, in the body of each gland. The globules are of various sizes, the largest being almost 5 μ in diameter. They are especially abundant near the points of entry of the nervi corporis cardiaci (fig. 6, B). Similar material can also be seen in association with the fibres of these nerves. It would seem that the material elaborated by the neurosecretory cells in the brain is being transferred along the nervi cardiaci to the corpora cardiaca. Such transfer hasbeen shown tooccur in other insectspecies (Scharrer, 1946, 1952a, b;Thomsen, 1954). Nayar (1954) describes cytoplasmic inclusions (spheroids) in the corpora cardiaca of adult Locusta migratoria, but it is not clear whether the spheroids are neurosecretory material from the brain stored in the corpora cardiaca, or whether they are a secretion product of the glands themselves.

The spheroids differ considerably in size and appearance from the masses of large acidophil inclusions which are present in the corpora cardiaca of nymphs of Periplaneta americana shortly before moulting (unpublished observation), which more nearly resemble the globules in the corpora cardiaca of the pupa of M. tiliae described here. The material in the corpora cardiaca in the pupa of M. tiliae is present until the end of the third week at 3° C (fig. 6, c), but by the end of the fourth week the corpora cardiaca are very much smaller, the glandular cells are small and stain feebly, and there is no sign of any acidophil material in the glands (fig. 6, D). This agrees closely with the absence of the neurosecretory material in the brain at this time (p. 80). It is to be inferred from these observations that the material elaborated by the neurosecretory cells in the brain is passed to the corpora cardiaca, but does not remain long in the glands. It is difficult to demonstrate how the material leaves the glands. The membrane around the corpora cardiaca is incomplete in places, and it is possible that the m terial contained in the glands is passed directly out into the blood. In a few series (fig. 6, C), however, vacuolated haemocytes have been found inside the corpora cardiaca, and it is possible that the material within the glands is actively removed by these blood-cells (see Wigglesworth, r955a).

Beginning of post-diapause development in non-endocrine organs of the body

The hypodermis, thoracic muscles, and anterior imaginal ring (of the ali mentary canal) were examined in detail throughout the diapause and diapause development periods. During these times the histological appearance of the tissues remains unchanged. The low temperature produces no visible effect upon them. But almost immediately the pupa is transferred to a temperature of 25° C (after at least 4 weeks at 3° C) intense mitotic activity begins in all three tissues, followed by differentiation and development until the adult form is attained. This immediate development of the non-endocrine organs of the body is due to the establishment of conditions favouring development by the end of the prior low-temperature treatment. Further evidence for this is given by the fact that at the subsequent high-temperature period, no secretory activity of any part of the endocrine system is visible (except a short burst of activity by the neurosecretory cells in the brain at about the sixth day after transfer to the high temperature, an activity that can have nothing to do with the initiation of development in the general body-tissues). Indeed, during the period at 25° C before adult emergence, the corpora cardiaca and allata are invaded by phagocytic blood-cells, and undergo a process of reorganization very similar to that occurring in the non-endocrine organs. The prothoracic glands disappear entirely, after being invaded by blood-cells, by about the twelfth day after the transfer to the high temperature.

Removal and implantation of the brain and the corpora cardiaca

Several series of experiments were carried out on the extirpation and re implantation of the various parts of the endocrine system in order to follow more closely its effect in terminating diapause.

The operations were carried out on pupae anaesthetized with ether. The pupae were placed in a block of wax which had been hollowed out at an angle to the vertical to form a pit of such form and size that each specimen was held securely, with only the anterior part of the body projecting above the wax. On one side of this deep pit was a shallow depression which was kept filled with physiological saline.

A roughly rectangular area of cuticle was cut out from the dorsal side of the head, and this was placed in the saline in the shallow depression. The appropriate organs were then removed. Any loss of blood which had occurred was made good by the addition of physiological saline. A few crystals of phenyl thiourea were placed in the body of the pupa to prevent the browning and eventual death of the operated animals, which results from the enhanced activity of the tyrosine-tyrosinase system after wounding (Williams, 1947). The cuticle was then replaced, the surface of the pupa dried, and the cut edges sealed with molten paraffin wax. Finally, the animals were placed on cotton-wool in an atmosphere saturated with water vapour.

Extirpation of the brain from the diapausing pupa

Two series of 10 diapausing pupae were operated upon for the removal of the brain. After removal of the cuticle from the dorsal part of the head, the brain with the corpora allata and cardiaca was immediately obvious. The nervi corporis cardiaci were severed, as also were the circum-oesophageal connectives. The nervous tissue making up the brain and sub-oesophageal ganglion is very condensed in the pupa of M. tiliae and it is consequently difficult to identify the circum-oesophageal connectives as such. In all cases the cuts were made at about the middle of the lateral nervous tissue. All other nerve connexions with the anterior parts of the body were cut and the brain lifted out. The cuticle was replaced and sealed into position, and the pupa placed on cotton-wool in a saturated atmosphere and stored in a refrigerator at 3° C for 4 weeks. The sample was then transferred to 2 5° C.

None of the pupae died during the low-temperature treatment, but 12 of the total of 20 died during the first week at 25° C. The remaining 8 survived for more than 2 weeks.

Ten control animals were subjected to the same operation except that the brains were not removed. Three of these control pupae died in about 2 weeks at 25° C.

Two of the brainless pupae were dissected at the end of the second week at 25° C. They were found to be almost identical in internal appearance (green blood, compact fat-body, no thoracic musculature, &c.) with normal diapausing pupae and showed no sign of adult development. Unoperated pupae, subjected to the same temperature treatments as these brainless pupae, would have been almost ready for hatching by this time (table r). The remaining brainless pupae survived for a further 2 weeks at 25° C without any sign of adult development. Histological examination of the tissues confirmed this result.

The 7 surviving operated control pupae also had not hatched after 15 days at 25° C (the normal time for hatching after previously chilling for 4 weeks). But dissection revealed that adult development had proceeded normally, since the blood was creamy (being filled with haemocytes and fat-body cells), and adult hypodermis (with scales) and thoracic musculature were well developed. Diapause had been broken in these pupae, although the normal hatching process had not proceeded to completion.

It may be concluded from this experiment that the brain is necessary for diapause development in the tiliae pupa, and that only when the brain is present can the low temperature cause diapause ‘break’. These results agree with those of Williams (1946) on the cecropia pupae.

Implantion of chilled brains alone into diapausing pupae

Ten diapausing pupae, previously maintained at 25° C in a saturated atmosphere, were each implanted with a brain taken from a pupa which had been subjected to a temperature of 3° C for not less than 4 weeks. Care was taken during the removal of the ‘chilled’ brains to exclude all traces of the corpora cardiaca and allata. Implantation was carried out by anaesthetizing the experimental pupae and making a small slit in the arthrodial membrane between two abdominal terga. The ’chilled’ brain was pushed through the slit into the body, a few crystals of phenylthiourea added, and the slit sealed with molten paraffin wax. The insects were then returned to the saturated atmosphere at 25° C. Nine of the 10 animals successfully survived the operation.

Fifteen days after the implantation none of the surviving pupae had hatched. Three were dissected and it was found that the chilled brain implant had ‘taken’ satisfactorily, tracheae having grown into the implant from the host. Otherwise the animals were identical with untreated diapausing individuals (green blood, compact fat-body, no thoracic musculature, &c.). No hatching occurred in the following two months. At the end of this period the survivors were dissected, but none showed any sign of adult development.

This result was quite unexpected, in view of the results obtained by Williams (1946) from similar experiments with the pupa of Platysamia cecropia. In this species Williams found that implanted chilled brains would terminate diapause in unchilled diapausing hosts. There would appear to be three possible explanations of the result of the present experiment.

  1. The implanted chilled brains had been unaffected by the low temperature to which they had been previously subjected. This would seem to be unlikely, in view of the results from the brain extirpation experiment (p. 83).

  2. The factor had been produced by the implanted brain during the low temperature, but could not be released into the blood of the diapausing host. Scharrer (1946, 1952a, b) has shown that section of the nervi corporis cardiaci in Leucophaea maderae acts like a ligature in preventing the exit of neurosecretory material. The nervi cardiaci were cut when chilled brains were removed from the M. tiliae pupae. It is possible, therefore, that failure of the brain factor to pass the cut ends of the nerves accounts for the results of the present experiment. This effect of section of the nervi cardiaci cannot be of universal occurrence, however, since unchilled Platysamia cecropia pupae will terminate diapause when implanted with chilled brains (Williams, 1946). This indicates that the factor is being released from the brain, although the nervi cardiaci have presumably been cut.

  3. The factor had been produced by the brain during the low-temperature period, but was no longer present at the time of the operation. This is the most likely possibility for the histological results already described (p. 80) indicate that the material produced by the neurosecretory cells in the brain is passed along the nervi cardiaci to the corpora cardiaca before the end of the low-temperature period. The observation suggested the following experiment.

Implantation of chilled brains, corpora cardiaca, and corpora allata into diapausing pupae

In this experiment it would have been desirable to implant only the brain and corpora cardiaca. Unfortunately this proved impossible owing to the intimate association between the corpora cardiaca and allata and the consequent difficulty of separating them completely. It is unlikely, how ever, that the inclusion of the corpora allata has influenced the result, since the small size of the glands and the absence of inclusions and vacuoles, &c., at the time of the operation indicates that they are not actively secreting (unpublished observation).

The brain with the attached corpora cardiaca and allata were removed from the pupae previously chilled for 24-28 days and implanted into the abdomens of pupae diapausing at 25° C. Fifteen implants were made into a similar number of diapausing pupae. Both host and donor pupae were then transferred to a saturated atmosphere at 25° C. The subsequent development of the host pupae is described below; that of the donor pupae in the next section.

Eleven host pupae survived for 4 weeks or longer at the high temperature, but none hatched out. When dissected, however, it was found that in 7 of the 1 I diapause had been broken, whereas in the other 4 there was no sign of adult development. In the first group the blood was creamy, the thoracic leg and wing-muscles were developing, and the hypodermis possessed scales. In the second group the blood was still clear and green and the fat-body compact. It is thus clear that whilst the implantation of chilled brains alone into diapausing pupae will not break diapause, the implantation of chilled brains plus corpora cardiaca and allata may do so. The failure of this latter treatment to break diapause in four of the experimental pupae suggests that the factor responsible for this event was not available in these particular cases. The most probable explanation for this is that the factor had already been released into the blood of the donor and was. thus not available to the host pupa. If so, the donor pupae might be expected to break diapause despite the loss of the brain-gland complex; evidence presented in the next section indicates that this did in fact occur. Thus 24-28 days appears to be the critical time for carrying out transplantation after exposure to 3° C.

Extirpation of the brain, corpora cardiaca, and corpora allata from chilled pupae

The animals from which the chilled brains and glands were removed in the previous experiment were subsequently kept at 25° C. Twelve of the fifteen pupae survived for more than 15 days after the operation. No adults emerged from any of the pupae, but dissection of the animals on death revealed interesting differences between two groups of pupae. Nine of the animals, al though dehydrated, possessed a compact fat-body identical with that of the diapausing pupa. The other three were dark-brown or black internally, and showed no sign of a well-organized fat-body. The cuticle was thin in the latter group and there were signs of developing thoracic musculature. Adult development had begun in 3 of the operated animals, whereas it had not in the other 9. This is the complement of the results of the previous experiment (p. 85). It indicates that the factor initiating development leaves the brain gland complex soon after the 24th day at 3° C.

The marked difference between the appearance of the neurosecretory cells during the diapause and low-temperature periods is a very interesting feature of the cells in the pupa of M. tiliae, since such differences have rarely been described before. In Rhodnius prolixusWigglesworth (1940) states ‘ ⃛ there are no differences in the [neurosecretory] cells during fasting and at the height of secretion of the moulting hormone’; i.e. the appearance of the neurosecretory cells does not differ between their resting and active phases which have been experimentally determined. Similarly, Scharrer (1941) was unable to find any rhythmic activity of the groups of neurosecretory cells as a whole in the cockroach species which she examined. More recently, however, Dupont Raabe (1951) has demonstrated in the phasmid Cuniculina annamensis that the elaboration of intracellular material by the neurosecretory cells in the brain is particularly intense a certain time after each larval moult, and also during egg-laying in the adult. Jones (1956) has shown that the neurosecretory cells in the embryo Locustana pardalina reach a period of maximal activity on the fourth day after the quiescent egg has been wetted, and Fraser (1957), working with Lucilia caesar, has shown differences in activity during diapause and after in several types of cerebral neurosecretory cell. The conclusions drawn from the examination of the neurosecretory cells in tiliae are in accord with these recent studies.

The present histological observations indicate that the production of material by the neurosecretory cells in the brain is not proceeding, or is proceeding very slowly, during the diapause period. Chilling the pupa stimulates the production of intracellular material, and this is then passed to the corpora cardiaca, where it is stored for some little time. The period of diapause development in the pupa of M. tiliae is thus correlated with the production of material by the neurosecretory cells in the)?rain. The intracellular material from these neurosecretory cells is related in some way to the hormone (or hormones) produced by the brain which influences growth and development throughout the life of the insect (Wigglesworth, 1955b). It seems that conventional techniques stain a biologically inert ’carrier-sub stance’ (Clarke, 1956) and not the active brain factor itself; but it is reason able to conclude that when the carrier-substance is produced, so is the associated hormone.

The conclusions arrived at from the examination of the neurosecretory cells and the corpora cardiaca during diapause development, together with the results of implantation and removal of these organs, suggest that the conditions governing the further development of the pupa are established by the end of the low-temperature period. This hypothesis is supported by the fact that the non-endocrine organs of the body (e.g. hypodermis, thoracic muscles, anterior imaginal ring) begin development almost immediately the pupa is transferred to 25° C after at least 4 weeks at 3° C.

In Platysamia cecropia (Williams, 1946, 1947, 1948, 1949) the diapausing pupa has to be kept at the low temperature for a minimum period of 6 weeks, and diapause is then terminated after a further 2 weeks at 25° C. Williams postulated that during the 2 weeks at 25° C the actual release of the brain factor and stimulation of the prothoracic glands takes place. M. tiliae differs from Platysamia cecropia in this respect, since the present results show that no‘latent period’ at 25° C is necessary in this animal before adult development begins. Recently Williams (1956) has shown that by greatly extending the low-temperature period, the time at 25° C before diapause is broken can be reduced to as little as one day. The difference between M. tiliae and Platysamia cecropia therefore seems to be that the brain factor is released slowly at low temperatures in P. cecropia and rapidly in M. tiliae. A consequence of the fact that the brain factor is released slowly at the low temperature in Platysamia cecropia is that the brain of a pupa chilled for 6 weeks will initiate development when implanted into an unchilled diapausing pupa (Williams, 1946). The present histological evidence suggests that no hormone is present, or is produced, in the brain of the M. tiliae pupa at the end of the low-temperature period and that therefore the brains of pupae chilled for 4 weeks cannot initiate development when implanted into diapausing pupae. The experimental implantation of chilled brains (p. 84) supports the histological evidence.

It is assumed that the effect of the brain factor in initiating development is mediated through the prothoracic glands. This has not yet been tested experimentally, but the experiments of Williams (1947) together with histological observations on the activity of the prothoracic glands (to be described in a later paper) support the assumption.

I am grateful to Professor L. E. S. Eastham for his keen interest and encouragement at all times, to Dr. F. Segrove, who made many valuable suggestions while the manuscript was in preparation, and to Mr. J. Hancock, who made the photomicrographs.

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