Neurosecretory control of ovarian development in Schistocerca gregaria

Transcription

1 57 Neurosecretory control of ovarian development in Schistocerca gregaria By K. C. HIGHNAM (From the Department of Zoology, The University, Sheffield 10) With i plate (fig. 5) Summary Cautery of the neurosecretory cells of the pars intercerebralis, or removal of the corpora cardiaca, will prevent the development of the terminal oocytes in the ovaries of the desert locust. Implantation of brains into females whose neurosecretory cells have been cauterized results in some development of the terminal oocytes. Ovariectomy of immature females causes a precocious accumulation of material in the neurosecretory system. Electrical stimulation, drastic wounding, or enforced activity of 14-day-old females reared without males brings about release of material from the neurosecretory system, and also accelerates development of the terminal oocytes. Copulation by these females also results in release of material from the neurosecretory system, and is followed by rapid terminal oocyte development. It is concluded that the neurosecretory system in Schistocerca gregaria exerts a positive control over oocyte development, and that copulation may stimulate release of material from the neurosecretory system. Introduction OVARIAN development in many insects is controlled by a hormone from the corpora allata (Wigglesworth, 1954a). It is now becoming increasingly apparent that this control system is more complex than was at first thought. Thomsen (1952) has shown that the activity of the corpus allatum in Calliphora erythrocephala may be controlled by an 'allatotropic' hormone produced by the median neurosecretory cells in the brain; these cells, in addition, may affect ovarian development directly. Similarly, control of ovarian development in Ipkita limbata by the corpus allatum may be effected through the cerebral neurosecretory cells (Nayar, 1958a). Dupont-Raabe (1951, 1954, 1956) has correlated histological differences in the appearance of the neurosecretory cells in phasmids with the state of development of the ovaries, as have Arvy and Gabe (1952 a, b; 1953 a, b, c, d) in various insects, and Highnam (1961) in Schistocerca gregaria. On the other hand, Engelmann (1957) has shown that the activity of the corpus allatum during ovarian development in Leucophaea maderae is regulated by purely nervous stimuli from the brain and the sub-oesophageal ganglion. Johannson (1958) was unable to find any correlation between histological signs of neurosecretory cell activity and ovarian development in Oncopeltus fasciatus; further, in this species eggs will develop fully in the absence of neurosecretory cells, although the fecundity of the operated females is somewhat reduced. [Quarterly Journal of Microscopical Science, Vol. 103, part 1, pp , March 1962.]

2 58 Highnam Neurosecretory control of ovarian It seems likely that a brain -> corpus allatum > ovarian-development mechanism operates in all these insects, but the participation of the brain in the process may be either humoral or nervous. The experiments described in this paper were performed to determine the role of the neurosecretory system in ovarian development in the desert locust. Material and methods Newly emerged adult females of the desert locust, 5. gregaria, were reared in glass-fronted cages as previously described (Highnam, 1961). Where necessary, heads were fixed in Bouin's fluid under reduced pressure, the cuticle of the head capsule dissected away before impregnation with paraffin wax, and serial sections cut at 10 jit. The sections were stained with paraldehyde-fuchsin (PF) after permanganate oxidation, to show neurosecretory material. Ovaries were removed and examined in saline under a binocular microscope; the length of the terminal oocyte in each ovariole was measured. Operations. Locusts were stored for about an hour in the refrigerator at 3 0 C and then anaesthetized with carbon dioxide gas for 2 min. All operations were performed under a binocular microscope. Cautery of neurosecretory cells. A square of cuticle was cut from the frons, and the cephalic air-sacs overlying the brain removed. An electric cautery needle, mounted on a micromanipulator, was inserted into the head until its point just touched the opaque area in the pars intercerebralis of the brain, which marks the site of the neurosecretory cells. After cautery of the area, the cuticle was replaced and sealed into position with molten paraffin wax. Operated controls were treated in exactly the same way, except that the neurosecretory cells were not cauterized. Mortality in different series of experiments varied between 10% and 25%. Cardiacectomy. The head was bent forwards and downwards until it lay almost parallel to the long axis of the body. A cut was made in the dorsal midline of the tightly stretched cuticle of the neck, and the cervical air-sacs removed to expose the foregut with the overlying aorta. The latter was followed forwards into the head until the bluish-white corpora cardiaca were found, lying just beneath and slightly anterior to the brain. The corpora cardiaca were removed with a pair of fine forceps. The head was then released. The cervical cut was not sealed, being protected by the forwardly projecting prothorax. Operated controls had the cervical air-sacs removed and the aorta torn just behind the corpora cardiaca. Mortality in these operations varied between 60% and 70%. Implantation of brains. Brain implantations were made through a small slit in the side of the second abdominal segment of females which had had their neurosecretory cells cauterized a short time previously. The slit was sealed with paraffin wax. At the end of the experimental period the implanted brains were examined where they lay, to determine whether they had become tracheated. They were then removed, fixed,sectioned, and stained with PF. Ovariectomy. Ovaries were removed through a dorsolateral slit in the second

3 development in Schistocerca gregaria 59 abdominal segment. The slit was afterwards sealed with paraffin wax. Operated controls had a small piece of fat-body removed instead of the ovary. Electrical stimulation. Females were stimulated with 10 V, 5 m sec pulses at a frequency of 40 pulses per min (low frequency) or 40 pulses per sec (high frequency) through the ventral nerve cord, the surface of the brain, or the optic nerves. Platinum electrodes from a square-wave generator were brought up to and maintained in the correct position by a pair of micromanipulators. Stimulation of the optic nerves was achieved by cutting slices of the cuticle from the eyes and moving the electrodes gently to predetermined positions in the head. At the end of the treatment, the heads were dissected to verify that the electrodes had been correctly placed. No quantitative or qualitative differences in the release of neurosecretory material from the corpora cardiaca were apparent after stimulation through the optic nerves, the surface of the brain, or the ventral nerve cords. Operated controls were treated in exactly the same way as the experimental animals, except that no current was passed. Enforced activity. Varying numbers of female locusts were placed in a large conical flask which was rotated by hand in such a way that the insects were repeatedly turned upside down and repeatedly attempted to regain their normal orientation. Control animals were placed in the flask but not rotated. Results Neurosecretory cell cautery The neurosecretory cells in the pars intercerebralis of 3-day-old females were cauterized and the survivors killed 18 days later. No eggs were laid during this time, and the terminal oocytes had a mean length of 1-43 mm when they were measured (table 1). The terminal oocytes of 3-day-old females are 07 mm in length, so the terminal oocytes of the operated females had increased in length by 073 mm in 18 days. The operated controls laid eggs 14 to 17 days after the operation, and their second generation terminal oocytes (confirmed by the presence of red corpora lutea) had a mean length of 4-0 mm when they were examined 18 days after the operation. The original terminal oocytes of the operated controls had therefore increased in length from 07 mm to about 8-o mm, had been laid, and the next generation of oocytes had begun development in the same time that the terminal oocytes of the cauterized females had increased in length by only 073 mm. The slight increase in length of the terminal oocytes in the cauterized females was possibly due to hormone circulating in the blood at the time of the operation, and to the factor leaching out of the corpora cardiaca. The corpora cardiaca and the nervi corporis cardiaci interni (NCCI) contained no PF-stainable material 18 days after cautery of the neurosecretory cells (figs. 1, Q; 2, p). The rapid maturation of the operated controls, compared with normal females, is characteristic of injured desert locusts (Norris, 1954).

4 6o Highnam Neurosecretory control of ovarian Brain implantation into females with cauterized neurosecretory cells The implantation of normal brains into 3-day-old females whose neurosecretory cells had been cauterized resulted in marked oocyte development, compared with the cauterized females alone (table 1). Development was less than in normal females, but this was possibly due to the time taken for the brains to become tracheated and to some upset in the production or release of neurosecretory material by the isolated implanted brain. The neurosecretory cells in such brains were often large, and frequently distorted in shape (fig. 4, j). PF-stainable material was present along the lengths of the NCCI within the brains, and often accumulated at the points of exit of the nerves. This accumulation of neurosecretory material, together with blood-cells, growing tissue, and tracheae recalls Stumm-Zollinger's (1958) description of the formation of 'corpora cardiaca' at the cut ends of the NCCI in Platysamia cecropia. TABLE I Effects of interference with the neurosecretory system upon length of terminal oocyte in S. gregaria Operation cautery of neurosecretory cells operated controls for cautery of neurosecretory cells brain implantation after cautery of neurosecretory cells cardiacectomy operated controls for cardiacectomy Age at operation {days) Length of oocyte at operation* (mm) O-7O;t;OO2 O'7Oj;O'02 O'7O±O-O2 O'7O±O-02 No. of animals Age when dissected (days) O'70±0' Calculated from stock animals, f Second generation oocytes. Time since operation (days) IS IS Length of terminal oocyte (mm) i-09±o'oi 7'33±o ±0-27 f4-oo±o-55 4-oo±i-2 i-8s±o-o+ 6'0O±0-20 Cardiacectomy Very little increase in length of the terminal oocytes followed removal of the corpora cardiaca from 3-day-old females (table 1). This result is in accord with the consequences following cautery of the neurosecretory cells in females of the same age. Ovariectomy A total of 20 females, aged 5 days, were ovariectomized and the neurosecretory systems examined of samples taken 6, 9, and days after the operation. The neurosecretory systems of the samples taken 6 and 9 days

5 development in Schistocerca gregaria 61 after ovariectomy contained large amounts of PF-stainable material (figs, i, L; 2, L; 4, H) compared with the operated controls (figs, i, M; 2, M; 4, D). The neurosecretory systems of the sample taken days after the operation also contained large amounts of material (figs. 1, N; 2, N; 4, B), but so did the neurosecretory systems of the operated controls (figs. 1,0; 2, 0; 4, F). This appearance of the neurosecretory system is characteristic of females of this age (Highnam, 1961). The early appearance of large amounts of material in the neurosecretory systems of the ovariectomized females indicates some reciprocal effect between the system and the ovaries. Electrical stimulation and enforced activity The results of cardiacectomy and cautery of the neurosecretory cells, described above, suggest that the terminal oocytes do not develop in the absence of neurosecretory material. Hodgson and Geldiay (1959) have shown that in Blaberus craniifer, electrical stimulation of the central nervous system, or enforced activity, results in the depletion of neurosecretory material in the corpora cardiaca: the treatments apparently accelerate secretion into the blood. If the same results followed similar treatments in the desert locust, it might be expected that the rate of terminal oocyte development would be increased, owing to the greater availability of the neurosecretory material. Hodgson and Geldiay's methods were therefore repeated with the desert locust to test this hypothesis. Immature females. Ten 5-day-old female locusts were stimulated through the optic nerves with high-frequency electrical shocks for one hour. The heads of 5 individuals were fixed immediately after the treatment; the remaining 5 were replaced in their cage. Ten operated controls were similarly divided. Histological examination of the neurosecretory sytems of the individuals fixed immediately after the treatment showed little difference in the amounts of material in the neurosecretory cells (figs. 3, D), the NCCI (fig. 2, B) or the corpora cardiaca (fig. 1, B), when compared with the operated controls (figs. 3, G; 2, c; 1, c). Normal females of this age possess only small amounts of PFstainable material in the neurosecretory system (Highnam, 1961), so it might be expected that the detection of differences would be difficult. However, 2 weeks after the treatment, the lengths of the terminal oocytes of the stimulated and control females were 4-8 mm and 3-6 mm respectively (table 2); the differences are not statistically significant (p>o-i). It is evident that electrical stimulation of immature females has no appreciable effect in accelerating ovarian development.. Fourteen-day-old females, isolated from males. Fourteen-day-old females, isolated from males at emergence, possess much smaller terminal oocytes than females reared with mature males (Highnam and Lusis, unpublished), and their, neurosecretory systems contain large amounts of PF-stainable material (figs, i, F; 2, F; 3, c). Females in this condition might be expected to be more susceptible to the experimental treatment.

6 62 Highnam Neurosecretory control of ovarian

7 development in Schistocerca gregaria 63 Seven 14-day-old females, isolated from males at emergence, were stimulated with low-frequency electrical shocks through the ventral nerve-cord, brain surface, or optic nerves. The heads were fixed immediately after treatment. Operated controls, not stimulated, were similarly treated. Histological examination showed very large amounts of PF-stainable material along the NCCI (figs. 2, D; 5, E) and within the corpora cardiaca (figs. 1, D; 5, A) compared with the operated controls (figs. 1, E; 2, E). The neurosecretory cells appeared unaffected (figs. 3, E; 3, A). High-frequency stimulation for the same length of time resulted in an almost complete disappearance of PF-stainable material from the NCCI (figs. 2, G; 5, F). A smaller amount of material was present in the corpora cardiaca after high-frequency stimulation (figs. 1, G; 5, B; compare fig. i, E). Material has also left the neurosecretory cells in the pars intercerebralis (fig. 3, F; compare fig. 3, A). These results of low- and high-frequency stimulation are not incompatible. It is probable that both kinds of stimulation accelerate movement of neurosecretory material along the NCCI, through the corpora cardiaca and presumably into the blood. Low-frequency stimulation illustrates the early stages of the process; high-frequency stimulation the final stages. Fourteen-day-old females, isolated from males at emergence, were stimulated with high-frequency shocks for 45 min and fixed 130 min later. The NCCI and corpora cardiaca contained very little PF-stainable material after this treatment (figs, i, H; 2, H). It is clear that electrical stimulation of the central nervous system in S. gregaria results in histological signs of transport and release of material from the neurosecretory system, but that release from the corpora cardiaca occurs more slowly than in B. craniifer (Hodgson and Geldiay, 1959). Changes in the appearance of the neurosecretory cells after stimulation were not apparent in Blaberus. It may be said here that although the differences between the experimental and the operated control animals were clear, the same differences, only in less degree, were apparent between the operated control and unoperated females of the same age. Fourteen-day-old females, isolated from males at emergence, were shaken as described previously for 30 min. Their heads were fixed immediately after the treatment. Similar amounts of PF-stainable material were found in the corpora cardiaca (fig. 1, K) and the NCCI (fig. 2, j) as in the electrically stimulated animals. The controls were similar to normal females of the same FIG. 1. Camera lucida drawings of sections through the anterior parts of the corpora cardiaca of adult female desert locusts to show the amount and distribution of neurosecretory material (black), A, 3-day female; B, 3-day female electrically stimulated; c, operated control for B; D, 14-day female stimulated with low-frequency electrical shocks; E, operated control for D; F, 14-day female, reared without males; G, 14-day female, stimulated with high frequency electrical shocks; H, 14-day female, stimulated with high-frequency electrical shocks and fixed 130 min after the end of the treatment; j, control for K; K, 14-day female shaken for 30 min; L, female ovariectomized for 6 days; M, control for L; N, female ovariectomized for days; O, control for N; p, female taken during copulation; Q, 18 days after cautery of pars intercerebralis neurosecretory cells; R, 24 h after copulation; s, female beginning oviposition; T, female 24 h after oviposition.

8 6 4 Highnam Neurosecretory control of ovarian 400JJ. FIG. 2. Camera lucida drawings of NCCI of adult female desert locusts to show amounts of neurosecretory material (black). A, 3-day female; B, 3-day female electrically stimulated; c, control for B; n, 14-day female electrically stimulated with low-frequency shocks; E, control for D; F, 14-day female without males; G, 14-day females stimulated with high-frequency shocks; H, 14-day female stimulated with high-frequency shocks and fixed 130 min later; j, 14-day female shaken for 30 min; K, control for j; L, ovariectomized 6 days; M, control for L; N, ovariectomized days; o, control for N; p, 18 days after cautery of neurosecretory cells; Q, fixed during copulation; R, 24 h after copulation; s, female beginning oviposition; T, 24 h after oviposition. age (figs. 1, j; 2, K). NO differences were seen in the neurosecretory cells after the treatment (figs. 4, c; 4, E). Electrical stimulation and enforced activity have a marked effect upon the transport and release of PF-stainable material from the neurosecretory systems of 14-day-old females, isolated from the males at emergence. The following experiments were performed to determine whether the material released in this way affected the rate of ovarian development in these females. Ten 14-day-old females, isolated from males at emergence, were stimulated through the optic nerves with high-frequency shocks and their ovaries were examined 7 days later. The terminal oocytes had increased in length to 478 mm (table 2). There was equally rapid development of terminal oocytes in the

9 development in Schistocerca gregaria FIG. 3. Camera lucida drawings of neurosecretory cells from adult female desert locusts. Neurosecretory inclusions black, A, 14-day female, operated control for E; B, 3-day female; C, 14-day female without males; D, 3-day female electrically stimulated; E, 14-day female stimulated with low-frequency shocks; F, 14-day female stimulated with high-frequency shocks; G, control ford; H, female beginning oviposition; j; 24 h after oviposition F H

10 66 Highnam Neurosecretory control of ovarian FIG. 4. Camera lucida drawingsof neurosecretory cells from adult female desert locusts. Neurosecretory inclusions black, A,fixed during copulation; B, ovariectomized days; c, 14-day female shaken for 30 min; D, control for H; E, control for c; F, control for B; G, 24 h after copulation; H, ovariectomized for 6 days; J, implanted brain from female whose neurosecretory cells had been cauterized 18 days previously.

11 development in Schistocerca gregaria 67 operated controls (table 2). The histology of the neurosecretory systems of the operated control animals suggested that wounding the animals may accelerate the release of material from the system. Both experimental and operated control series possessed longer terminal oocytes than uninjured females, isolated from males, of the same age (table 2). TABLE 2 Effects of electrical stimulation and enforced activity upon length of terminal oocyte S. gregaria Treatment Age when treated *Length of oocyte when treated No. animals Age when dissected Time since treatment Length of oocyte when dissected electrical stimulation through optic nerves operated controls for electrical stimulation electrical stimulation through optic nerves operated controls for electrical stimulation enforced activity control handled for weighing control (days) (mm) I-OO±O'O2 I-00±O-O ± '97±i'o6 2-97±i-o6 2'97±i"o6 O'7O±O-O2 initially O'7O±O-O2 initially IS l6 (days) (days) ' total 18 total (mm) 4'8orfcO'97 3-6o±i-o S5±o-S g±o ^ oo±i-4i Calculated from stock animals. Enforced activity, which does not injure the insects, produced the same histological results of depletion of PF-stainable material in the neurosecretory system as electrical stimulation. The experiment was therefore repeated, with enforced activity as the experimental method. Ten 14-day-old females, isolated from males at emergence, were shaken for one hour per day for 3 days. When examined 7 days later, their terminal oocytes were very significantly longer than those of the unshaken controls (table 2). Five other females laid eggs during the week following the treatment, and possessed well-developed second-generation oocytes when examined. It is supposed that these females possessed quite well-developed terminal oocytes, at the beginning of the experiment. In the course of a quite different series of experiments, females, isolated from males, were weighed daily; they were consequently individually handled every day for a period of 18 days. At the end of this time, their terminal oocytes measured about 77 mm in length (table 2). A similar group of females from the same initial stock, which was not so handled, possessed terminal oocytes with a mean length of 5-0 mm (table 2) at the end of 18 days. This group of unhandled females contained 4 individuals with almost fully developed oocytes. They were probably deformed in some way, although this was not ascertained at the time. Excluding these individuals, the mean length of oocyte was only 3-41 mm.

12 68 Highnam Neurosecretory control of ovarian The histological and developmental results of electrical stimulation, drastic wounding, and enforced activity strongly support the conclusion that the neurosecretory material produced by the brain has a positive effect upon the development of the terminal oocytes. Changes in the neurosecretory system during copulation Ten -day-old females, reared without males, were placed in a cage containing mature males. Copulation occurred within 2 days of the sexes being placed together. The heads of 5 females, taken during copulation, were fixed in Bouin's fluid. The remaining 5 females, marked with coloured paint when copulating, were fixed about 24 h after copulation. The neurosecretory systems of females taken during copulation contained very large amounts of PF-stainable material along the NCCI (figs. 2, Q; 5, G) and within the corpora cardiaca (figs. 1, p; 5, c). The neurosecretory cells in the pars intercerebralis were similar to those of females without males (fig. 4, A). The general histological picture was very similar to that of the neurosecretory system of females after low-frequency electrical stimulation (figs.!> D > 5» A > 2 > D > S> E > 3> E )- Twenty-four hours after copulation the corpora cardiaca were almost devoid of PF-stainable material (figs. 1, R; 5, D), as were the NCCI (figs. 2, R; 5, H); the corpora cardiaca and NCCI closely resembled those of immature femaleis (Highnam, 1961; fig. 1, E, F), and of females after high-frequency electrical stimulation (figs. 1, H; 5, B; 2, H; 5, F). The neurosecretory cells in the pars intercerebralis were similar in appearance to those of the immature female (figs. 4, G; 3, B), and to those of females after highfrequency electrical stimulation (fig. 3, F). It would appear that electrical stimulation and enforced activity mimic closely the process of copulation in their effects upon the neurosecretory system. Further, these results give some indication of the way in which the male may accelerate maturation in the female. Changes in the neurosecretory system during oviposition It has already been shown that the neurosecretory system of females possessing fully developed eggs contains large amounts of PF-stainable material (Highnam, 1961). Changes in the amounts and distribution of material in the FIG. 5 (plate). Photomicrographs of sections of corpora cardiaca and NCCI from 14-dayold females, reared without males until the time of treatment. Fixed Bouin; stained paraldehyde-fuchsin after permanganate oxidation. Neurosecretory material black. A, corpus cardiacum after low-frequency electrical stimulation. B, corpus cardiacum after high-frequency electrical stimulation. c, corpus cardiacum during copulation (females were brought into the presence of mature males 2 days previously). D, corpus cardiacum 24 h after copulation. E, NCCI after low-frequency stimulation. F, NCCI after high-frequency stimulation. G, NCCI during copulation. 11, NCCI 24 h after copulation. Notice the similar amounts of neurosecretory material in A and c; B and D; E and a; F and H.

13 B 8 cry 200JJ FIG. 5 K. C. HIGHNAM

14 development in Schistocerca gregaria 69 neurosecretory systems of such females during oviposition were found to be exactly comparable with those which occurred during and after copulation. The large amounts of material in the neurosecretory cells (fig. 3, j), NCCI (fig. 2, s), and corpora cardiaca (fig. 1, s) at the beginning of oviposition had disappeared within 24 h after egg-laying (figs. 1, T; 2, T; 3, H), leaving the neurosecretory system in a condition very similar to that of the immature female (figs. 1, A; 2, A; 3, B). Discussion Cautery of the neurosecretory cells in the pars intercerebralis, or cardiacectomy, greatly retards development of the terminal oocytes. The implantation of brains into females whose neurosecretory cells have been cauterized results in some development of the terminal oocytes. Electrical stimulation, enforced activity, and even wounding, which all bring about release of material from the neurosecretory system, result in increased development of the terminal oocytes in females reared without males. These treatments, which presumably alter the availability of neurosecretory material for developmental purposes, all suggest that the neurosecretory material has a positive effect upon the progress of ovarian development. Ovariectomy, which brings about the precocious appearance of large amounts of neurosecretory material in the corpora cardiaca, the NCCI, and the neurosecretory cells of the pars intercerebralis, suggests some reciprocity between the neurosecretory system and the ovaries. The cerebral control of ovarian development in S. gregaria therefore seems to be humoral, as shown by Thomsen (1952) in C. erythrocephala and Nayar (1958a) in / limbata. Experiments to be described in a further paper suggest that in S. gregaria the corpora allata also play some part in the control of ovarian development. It is considered that the results described in the present paper do not preclude an indirect mechanism of control by the neurosecretory system over ovarian development. In females previously reared without males, copulation results in the same histological signs of transport and release of neurosecretory material as does electrical stimulation and enforced activity. Norris (1954) has shown that the presence of mature males and copulation is necessary for normal egg development and oviposition in S. gregaria. The conclusion is inescapable that copulation can bring about rapid ovarian development in females previously reared without males, by acting through the neurosecretory system. Norris (1954) has also shown that mature males can accelerate the early stages of female maturation through the agency of a pheromone produced by the epidermis of the male (Loher, i960). Immature females reared with mature males are generally very much more active than those reared without males. The present results suggest that both this enhanced activity and the earlier copulation resulting from the presence of mature males would combine to bring about rapid ovarian development by acting through the neurosecretory system. The development of a mechanism in which the rate of ovarian development

15 70 Highnam Neurosecretory control of ovarian is dependent upon the presence of mature males is of obvious advantage in ensuring that the mature eggs are likely to be fertilized, and that the metabolic cost of egg production is not wasted. Enough spermatozoa are obtained from the first copulation to fertilize the first and all subsequent generations of oocytes (Norris, 1954), although a second injection of spermatozoa in some way prevents fertilization by a previous injection (Hunter-Jones, i960). In spite of the fact that all injections of spermatozoa after the first are unnecessary for fertilization, the presence of males and copulation are essential for the normal development and laying of the subsequent oocyte generations (Norris, 1954). This can be explained by the effect of copulation releasing from the neurosecretory system a factor controlling the development of the ovaries. Once the functional mechanism has evolved, it becomes indispensable. The relationship between the release of material from the neurosecretory system and oviposition is not yet clear. Females which are deprived of earth in which to lay may retain their eggs within the oviducts for at least a week (Highnam and Lusis, unpublished) and probably longer, although they will eventually lay eggs on the floor or sides of the cage. Females with eggs in the oviducts always have neurosecretory systems containing large amounts of PF-stainable material (Highnam, 1961). Virgin females, when they lay, will oviposit on the floor and sides of the cage even in the presence of pots of earth. Some relationship evidently exists between presence of males, state of the female neurosecretory system, and oviposition. The present results show gross discharge of material from the neurosecretory system during egg-laying. It is possible that once oviposition has started, some reciprocal mechanism is initiated between the neurosecretory system and the egg-laying process, as described by Nayar (19586) in I. limbata. This problem is being investigated at the moment. Whatever the cause, the neurosecretory system is in the same histological state at the beginning of the developmental cycle of the second generation of terminal oocytes as it was at the beginning of the first cycle. The lack of effect of electrical stimulation on the neurosecretory system of the immature female might be due either to the system being completely inactive at this time and unable to be stimulated into activity by the treatment, or to the system being so active that stimulation can produce no further effect during the time allowed for the experiment. It is considered that the second of these alternatives is the more likely, and evidence in support of this view will be presented in a later paper. Hodgson and Geldiay (1959) have equated the loss of neurosecretory material from the corpora cardiaca of B. craniifer, after electrical stimulation and enforced activity, with the loss of adrenalin-like activity of gland extracts after these treatments. Milburn, Weiant, and Roeder (i960) have shown that extracts of corpora cardiaca can block the general nervous inhibitory centres in the sub-oesophageal ganglion of Periplaneta americana. The increased activity of 5. gregaria females in the presence of mature males, the 'vibration reaction' induced by the near presence of mature males (Loher, i960), and the continued oviposition movements associated with the release of material from

16 development in Schistocerca gregaria 71 the neurosecretory system in 5. gregaria may all be the result of a similar effect upon the nervous system in this species. Blood from an ovipositing female will initiate oviposition movements in another female, as will the implantation of whole corpora cardiaca (Highnam, unpublished). However, Cameron (1953) has shown that extracts of corpora cardiaca of P. americana have an adrenalin-like effect even after the NCCI and II have been cut some time previously, and the glands are presumably devoid of neurosecretory material (Scharrer, 1952). The corpora cardiaca of S. gregaria are each composed of two parts (Highnam, 1916), and evidence is accumulating that the glandular parts secrete an adrenalin-like substance which is distinct from the neurosecretory material secreted from the storage parts. Wigglesworth (19546) has suggested the possibility of the brain hormone being a kind of labile raw material from which hormones manufactured by different parts of the endocrine system may be derived. Hodgson and Geldiay (1959) have compared the release of material from the corpora cardiaca of B. craniifer after electrical stimulation and enforced activity with the effects of stress upon the vertebrate adrenal gland. The results described in the present paper show the experimental treatments inducing neurosecretory release to be comparable with those processes which bring about 'natural' neurosecretory release in a manner which ensures the coordinated development of the terminal oocytes. To press too closely the analogy between 'stress' mechanisms in vertebrates and insects at this stage in our knowledge of insect hormonal processes may obscure more important conclusions concerning the chemical control of developmental processes. I am grateful to the Anti-Locust Research Centre, London, for providing the locusts used in this study; to the Research Fund Committee of the University of Sheffield for a grant for apparatus; and to Mr. W. Mosley, who made the photomicrographs. The research was supported in part by a grant from the Department of Scientific and Industrial Research. References ARVY, L., and GABE, M., 1952a. Ann. Sci. nat. (Zool), 14, Cellule, ss, a. Z. Zellforsch., 38, ft. Arch. Zool. exp. gen., 90, c Biol. Bull. Woods Hole, 106, c?. C.R. Acad. Sci. Paris, 237, 844. CAMERON, M. L., Nature, 172, 349. DUPONT-RAABE, M., Bull. Soc. zool. France, 76, Pubbl. Staz. Zool. Napoli, Suppl. 24, Ann. Sci nat. Zool, 18, 293. ENGELMANN, F., J. ins. Physiol., 1, 257. HIGHNAM, K. C, Quart. J. micr. Sci., 103, 27. HODGSON, E. S., and GELDIAY, S., Biol. Bull. Woods Hole, 117, 275. HUNTER-JONES, P., Nature, 185, 336. LOHER, W., i960. Proc. roy. Soc. B, 153, 380. MILBURN, N., WEIANT, E. A., and ROEDER, K. D., i960. Biol. Bull. Woods Hole, 118, m. NAYAR, K. K., 1958a. Zweites Int. Symp. iiber Neurosekretion, Lund, Berlin (Springer).

17 72 Highnam Neurosecretion in Schistocerca NAYAR, K. K., Proc. Indian Acad. Sci., 47, 233. NOKRIS, M. J., Anti-Locust Bulletin No. 18. SCHARRER, B., Biol. Bull. Woods Hole, 102, 261 STUMM-ZOLLINGER, E., J. exp. Z00L, 134, 315. THOMSEN, E., J. exp. Biol., 29, 137. WIGGLESWORTH, V. B., 1954a. The physiology ofinsect metamorphosis. Cambridge (University Press) Pubbl. Staz. Zool. Napoli, Suppl. 24, 41.