Effects of bursicon on cuticular properties in Locusta migratoria migratorioides

J. Insect Phyriol., 1971, Vol. 17,pp. 625 to 636.

Pergamon Press. Printed in Great Britain

EFFECTS OF BURSICON ON CUTICULAR PROPERTIES IN LOCUSTA MIGRATORIA MIGRATORIOIDES J. F. V. VINCENT* Department of Zoology, The University, Sheffield SlO 2TN (Received 3 December 1969 ; revised 5 September 1970) Abstract-Bursicon, the cuticular hardening and darkening hormone, is shown to be necessary as a hormone distinct from ecdysone if the insect is to ‘choose’ the time at which it ecdyses. Ligaturing experiments on ecdysing fourth and fifth instar Locusta migratort’a mi’atorioides larvae show that bursicon is released from at least the terminal abdominal ganglion, but not the corpus cardiacum, and that it affects darkening, the deposition of endocuticle, water loss, and tracheal emptying. It is suggested that bursicon influences cuticular dehydration, and so may also be involved in changes in cuticle plasticization. INTRODUCTION BURSICONhas been reported to occur in several species in the orders Orthoptera, Hemiptera, Coleoptera, and Lepidoptera, where its occurrence in the blood appears to be confined to stages which tan after ecdysis (FRAENKELand HSIAO, 1965; COTTRELL, 1962a-d). This paper describes certain effects of bursicon on the cuticle of a locust. The mode of action and the functional significance of the hormone are discussed. MATERIALS

AND METHODS

Insects used Locusta migratoria mi’atorioides

were bred in a 12 hr photoperiod

at 28°C

and were fed on lettuce and bran.

Mechanical disturbance of larvae Fifth instar L. m. migratorioides larvae about to make the final ecdysis were placed in 1 lb. honey jars with a piece of card to sit on and the mouths of the jars covered with gauze. The jars were put onto a Griffin flask shaker. The shaker was set at 8 to 10 cycles/set at which frequency there is sufficient jerk at the end of each cycle for the larvae to be unable to hold onto the sides of the jar or the card. The jars had a vertical movement of 5 mm. The larvae were judged to be ready to emerge within the next day or so when the dorsal carina of the prothorax could not be felt when the prothorax was squeezed dorsi-ventrally between the thumb and forefinger. * Present address: Department of Zoology, Earley Gate, Reading RG6 2AL. 625

626

J. F. V. VINCENT

Ligaturing experiments

Ligatures were placed behind the head or on the abdomen between the fourth and fifth abdominal ganglia. The ligatures were made with cotton in an overhand loop pulled tight. Weight changes were followed on an Oertling ‘Release-Omatic’ balance weighing to O-1 mg. Histological examinations were made on sections taken from the widest point on the hind femur fixed in Bouin, cut at 7 to 10 p, and stained in Mallory’s trichrome. Terminal abdominal ganglianectomy

Fifth instar L. m. migratorioides larvae were anaesthetized with CO, then laid inverted in a metal cradle and held in position with a rubber band. The arthrodial membrane between stemites seven and eight was torn and forceps inserted into the abdomen to lift out the nerve cord posterior to the incision. The terminal abdominal ganglion was then removed and the remainder of the nerve cord replaced within the insect. The wound was then sealed with ‘Nu-Skin’. RESULTS

Mechanical disturbance of the larvae before ecdysis

It is possible that the time of ecdysis, and thus of the release of bursicon, is dependant on previous events such as the release of ecdysone. Casual observation suggests that mechanical disturbance of the locust when it is ready to ecdyse delays the ecdysis. To quantify this effect, fifth instar larvae were mechanically disturbed on a Griffin flask shaker for 2 to 3 days. In the experiments two groups of six insects were confined in 1 lb. jars and the number of adults emerging recorded. The results are shown in Table 1. In all the insects cuticular darkening occurred after ecdysis. Since therefore bursicon seems to be intimately associated with ecdysis, the events of the larval-adult ecdysis were studied more closely so that any disturbance of the process such as ligaturing could be placed on a definite time scale. Time scale of ecdysis of L. m. migratorioides

About 10 to 15 min before ecdysis is due to start the locust hangs inverted from a suitable place making pumping movements with its abdomen. The insect from this stage through to the immediate pre-ecdysis stage shows extreme irritability when disturbed. Having settled itself with a good hold for its hind and middle legs, it curves its forelegs back (if hanging from a vertical surface) or holds them loosely away from the surface (if it is hanging upside down from a horizontal surface). It then starts longitudinal contractions of the abdomen, approximating the segments and relaxing them again, from the posterior forwards. These movements are only slight at first, and indeed are merely larger and more definite versions of movements which are also made between the pumping movements of the first stage. After about a minute these contractions become more marked and the cuticle is moved back slightly between each set of contractions. This point is

EFFECTSOF BUBSICONON LOCUSTA

taken as the start at this stage and insect then starts first split appears

627

MIGRATORIA

of ecdysis sense strictu. The wing pads also spread out and down the cuticle in the midline of the pterothorax is stretched. The to arch the entire thorax which further stretches the cuticle. The in the midline of the pterothoracic cuticle and extends over the

TABLE ~-DELAYING

ECDYSIS

IN Lowsta BY MECHANICAL DISTURBANCE Experimentals

Controls Time (hr)

Emerged

Dead

Emerged

Dead

Experiment 1 0 5 24 29 43 48

0 1 3 4 5 5

0 0 0 0 0 0

0 0 1 1 1 2

0 0

Experiment 2 0 5 19 23 43 66

0 0 2 3 4 5

0 0 0 0 0 1

0 0 1 1 2 3

0 0 0 0 0 0

0

1 1 1

Significance of the results: test the null hypothesis that disturbance of the insects does not affect their willingness to ecdyse. Then the expected number ecdysing in each group should be the maximum, i.e. all six in each group. Calculate xa = 4.14; 0.05 >P>O*O2 for N (d.f.) = 1.

prothorax and eventually over the head. The cuticle is then shed by a combination of longitudinal abdominal contractions and alternate pulls on the mid- and hindlegs of each side. The prothoracic cuticle is free from the pronotum some 4 to 5 min after the start of ecdysis. It is a little before this stage that the insect becomes oblivious to external stimuli, paying no attention to whatever is done to it. About 15 min after the start of ecdysis the insect is hanging by the tip of the abdomen from the old cuticle. It hangs like this for about 10 min after which it drops, or curls round on itself, finally to hang, head uppermost from a vertical surface or beneath a horizontal surface, by its fore- and mid-legs. The 10 min rest period seems to be associated with the preliminary hardening of the legs, since these are too soft for the insect to be able to walk properly if it is disturbed before it is ready. The insect then expands its wings. By 1 hr after the start of the moult the wings are nearly completely expanded with just the last few millimetres of the forewing tip curled over. At 14 hr the wings are just ready to be folded over the abdomen and the cuticle is noticeably darker than in the emerging insect.

628

J. F. V. VINCENT

The removal by hand of the larval cuticle from the pharate adult Larvae within an hour or two of their ecdysis (as judged by their behaviour) had the larval cuticle removed by hand. The subsequent effects of this operation mimic those of the abdominal ligature. While undergoing the artificial removal of the cuticle, the insect struggles until the cuticle has been removed from most of the head and pterothorax. It then calms down and starts the longitudinal abdominal contractions and the thoracic movements of ecdysis, which it continues even after the entire larval cuticle has been removed. Removing the cuticle from the abdomen first does not induce contractions: it appears that some sort of proprioception from the thoracic or head region is involved.

The effects of ligatures on emerging locusts Since bursicon is probably a neurosecretory hormone and is found in the largest amounts in the brain and corpus cardiacurn (cc) (VINCENT, 1971), the most likely place for its release is the cc. Accordingly, emerging adults were ligatured behind the head within a few minutes of the start of ecdysis. In such ligatured insects the head remains small and shrivelled, but the rest of the body in both larvae and adults tans normally. (Such a head ligature also stops the insect from continuing its ecdysis behaviour after the 10 min rest stage is reached.) This suggests that the ligature has prevented the head from hardening and darkening by preventing the movement of a factor from behind the ligature. This result is surprising in view of the amount of bursicon found in the cc (VINCENT, 1971) and shows that the hormone is not released from this site at the ecdysis. Following MILLS’ et al. (1965) suggestion that bursicon is released from the terminal abdominal ganglion in Periplaneta americana, a series of experiments was performed on Locwta in which this ganglion was ligatured off at various times during the fifth instar to adult acdysis. Previous casual observation had shown that if the old larval cuticle is removed by hand from a pharate adult which is within an hour or two of ecdysis, the resulting adult locust dries and shrivels markedly and does not harden its cuticle. It appeared that bursicon might be affecting water loss indirectly-lack of it causing less tanning of the cuticle. Accordingly the insects were weighed at intervals to determine the rate of water loss. But the water loss which occurs from such ligatured locusts, which apparently harden and darken normally, could be due to a thinner endocuticle. So the femora were examined histologically after 100 hr. Because the ligatured insects could not defaecate all the insects were starved. They were kept individually in 1 lb. jars with gauze tops and had a piece of card to sit on. When being weighed the insects were handled as little as possible to avoid damaging the wax layer of the cuticle: they could usually be induced to jump straight into the tube in which they were weighed. The various groups are tabulated below: control (untreated) ; C LC ligatured directly after total emergence (i.e. about 20 to 25 min after the start of ecdysis);

EFFECTS OF BURSICON

ON LOCUSTA MIGRATORlA

629

cuticle removed artificially from the pharate adult, but the insect not then ligatured ; AL cuticle removed artificially from the pharate adult and the insect then ligatured on the abdomen; ligatured at the first signs of ecdysis ; LE EL ligatured about 10 min after the start of ecdysis; LCV ligatured after the wings have expanded (i.e. at about 14 to 2 hr after the start of ecdysis); terminal abdominal ganglionectomized. This group is composed of only two insects, hence no standard error is shown for them but they are included in the analysis of variance. Unfortunately removal of the last abdominal ganglion leads to lack of rectal control and the insects are liable to lose water at a rather high and irregular rate, probably mainly in undehydrated faeces. This leads to a high mortality rate, and means that the loss in weight is meaningless for the present experiments. Fig. 1 shows the loss in weight as percentage body weight of the insects at the end of the 100 hr experimental period ; LCl is an additional LCV group. Analysis of variance shows a high significance at the O-1 per cent level (F,,32,erptl. = 14.74; AC

40-

30:

20-

+-? IO-

ac o-

al

le

el

Fl Ic

c

Icv

ICI

FIG. 1. Percentage weight loss of starved ligatured adult locusts over a period of 100 hr after the start of ecdysis. Means and standard errors from five readings each.

As stated above, the rate of water loss might be affected by the thickness of the cuticle. This parameter was measured in the femur rather than anywhere else because: (1) it is easy to localize an area on the femur where measurements may be taken-in this instance the widest part of the femur was chosen; (2) the femur is the first major locomotory organ to become effective after ecdysis. It presumably

630

J. F. V. VINCENT

therefore will have its cuticle deposited at the highest rate and any differences in cuticle thickness will be readily apparent. The insects were killed after 100 hr and the hind femora fixed in Bouin for histological processing and examination. The endocuticle and exocuticle were measured at three different sites (Fig. 2) and the thickness of the endocuticle expressed as a percentage of the total thickness of the cuticle (Figs. 3-5). The F-values for these three distributions are shown in Table 2.

FIG. 2. Diagrammatic transverse section of a locust femur. The areas used for measurements of the thickness of the cuticle are shaded and numbered.

Comparing the cuticles of the experimental and control insects, the endocuticle of the control insects is often more yellow than that of the experimental insects, which is almost entirely blue. Near its outer surface the exocuticle is often less fuchsinophil, being more of an amber or yellow in the control insects. In the larva of Locusta, the black colour of the cuticle is due to melanin (UVAROV, 1966). When the abdomen of a fourth instar larva is ligatured at the start of ecdysis, the part of the abdomen behind the ligature of the emerged fifth instar insect remains undarkened. More interestingly, when the ligature is placed between the thorax and the abdomen at this time the darkening of the body is much reduced and generally patchy. The wings often remain white or grey. This is a similar appearance to that of a fifth instar larva which has had the cuticle removed by hand just before its fourth-fifth instar ecdysis.

EFFECTS

ac

OF BUBSICON

al

le

ON

el

LOCUSTA

59x

631

hllGRATORfA

IC

c

Icv

ICI

0

FIG. 3. Percentage thickness of the endocuticle in 100 hr old starved ligatured adult locusts. Site 1.

ac

al

le

el

5gx

Ic

c

ICV

ICI

FOR

FIGS.

0

i

FIG. 4. As for Fig. 3, but site 2.

TABLE

2--ENDOCUTICLE

THICKNESS:

F-VALUES

3-5

Fig.

Expt. value

P 0.01

P OWM

P (expt. 1)

3 4 5

F 8,a2= 4407 F 8,3,= 8.72 F,,,, = 8.508

3.13 3.09 3.09

4.48 440 440

0.01 > P > om1 0.001 > P 0.001 > P

632

J. F.V.

VINCENT

If a locust is ligatured between the fourth and fifth abdominal ganglia at the start of ecdysis and then dissected an hour or two later, the tracheae behind the ligature still contain fluid, regardless of whether the old tracheae remain within the new ones or not. 40-

ac 0

al

le

el

5gx

Ic

c

ICV

Ic I

FIG. 5. As for Fig. 3, but site 3.

The effects of handling and damage on the cuticular wax layer Normally emerged adult locusts were taken when about l-day-old. Five were ligatured in front of the last abdominal ganglion in the normal way and five were sham ligatured in that they were handled as usual but the ligature was not pulled tight and was subsequently removed. Fig. 6 shows that the rate of weight loss returns to the normal value of 0.15 to O-2 per cent/hr after 10 to 15 hr. The sham ligatured insects can lose water through the rectum. This could not be corrected for and probably accounts for the higher rate of weight loss by this group. DISCUSSION

The mechanical disturbance of locust larvae delays ecdysis. This is in agreement with casual observation: a locust larva when apparently ready to ecdyse and showing the pre-ecdysis behaviour of hanging inverted and making pumping movements with the abdomen, can easily be delayed in its ecdysis by disturbance It is common for a locust showing either by another locust or by the experimenter. signs of incipient ecdysis while still in the breeding cage to lose completely its pre-ecdysis behaviour when being transferred to the laboratory and to ecdyse only hours later. The insects can, it seems, choose their own time to ecdyse within a period of about 1 day. Since this variation in the time of ecdysis is possible, the necessity arises for a closer control of the hardening than could be provided for by ecdysone, which has been secreted some days earlier. So if the insect is to harden its cuticle as soon after ecdysis as possible it must have a separate mechanism to

EFFECTSOFBURSICON ON

LOCUSTA MIGRATORIA

633

activate the tanning system which is closely associated with the events at ecdysis. This mechanism is mediated by bursicon. It is readily apparent why the fly, which may have to dig upwards for a matter of hours, should need a separate hormone to activate the tanning system since it cannot expand its wings until it has the space to do so. But the choice of place in which it emerges has been taken 0.5

t

0.1

t

0

I

I

IO

I

I

30

20 Time,

I

40

hr

FIG. 6. Weight loss of ligatured and sham-ligatured starved young locusts. 0, Sham-ligatured; 0, ligatured. Means and standard errors from five readings each.

even before the fly has pupated, so there is no other patent reason why the fly should need a separate tanning hormone. A separate tanning hormone should be needed only when there is the possibility that ecdysis might be delayed, as in the locust, or when there is the necessity to delay the tanning process as in the fly. Ligaturing the abdomen of an emerging locust such that the last abdominal ganglion is isolated from the rest of the body influences the amount of endocuticle subsequently deposited in the femur, the rate of water loss and the histological appearance of the cuticle. Ligaturing also causes a reduction in the amount of bursicon in the blood after ecdysis has started (VINCENT,1971). The conclusion is that the effects noted as due to ligaturing are as a result of the reduction of the amount of bursicon available over ecdysis. A change in the staining properties of endocuticle from aniline blue positive to acid fuchsin positive is indicative of tanning (DENNELL and MALEK, 1955). This change has been noted in the sections of femur indicating that the insects do not tan the endocuticle within 100 hr of ecdysis if the supply of bursicon from the last abdominal ganglion is cut off. Also bursicon seems to affect the rate of endocuticle

634

J. F. V.

VINCENT

deposition within the experimental period. Whether this is an effect of lower incorporation rate into the cuticle by the epidermal cells, or whether bursicon affects the metabolism to cause protein to be released from the fat body for incorporation into the cuticle is not known. Preliminary experiments on the amount of protein in the blood over ecdysis have proved inconclusive. The percentage of endocuticle is the same in both sexes as noted by MALEK (1958). The experimental groups have only about two-thirds the thickness of the endocuticle of the control groups, and the LC group falls midway between the two. So it is possible that, over the experimental period at least, bursicon is acting as a modulating hormone rather than a simple switch. FOGAL and FRAENKEL (1969) found a similar relationship. There is also the implication that bursicon is still being released in significant amounts 20 to 25 min after the start of ecdysis when the LC insects were ligatured. This is of interest in connexion with the time scale of release of bursicon (VINCENT, 1971). The higher rate of water loss noted in the experimental insects cannot be due to the thinner endocuticle, since both the untreated control and the ligatured control groups show a low rate of water loss from very soon after emergence (VINCENT, 1969). Also HILL et al. (1968) h ave shown that the deposition of endocuticle in the abdomen of the adult is not very great until 4 to 6 days after ecdysis. Since the insects in the present experiments were killed after only 4 days, and since most of the water loss could be expected to occur via the abdomen, the thickness of the cuticle is probably not involved in the water loss differences noted. The higher rate of water loss in the experimental groups cannot be due to removal of wax whilst handling, since this effect lasts for only 15 hr or so (Fig. 6) by which time the wax has presumably been replaced. However the ligatures are made at the time when wax is presumably being secreted onto the surface of the cuticle, so this effect might be expected to be minimal. The observed difference in water loss also lasts for much longer than 15 hr. However the observation that tracheal emptying does not occur in the ligatured end of the abdomen of the experimental insects indicates that the wax layer is not inverting (BEAMENT, 1960). This suggests that that cuticle has not been tanned. It is possible then that the wax layer does not invert properly on the cuticle. This is unlikely since the tracheae in the rest of the body of a locust ligatured before bursicon release show normal emptying, so the cuticle has been tanned sufficiently for the wax molecules to invert normally. But the fact that the endocuticle is thinner in the experimental insects may, as noted above, indicate that the epidermal cell is functioning at something less than its normal rate. It is probable that sclerotization is not completely stopped under the conditions of the experiment, but merely slowed down. This would account for the staining differences noted in the endocuticle of ligatured and control locusts. If this were so, then the increased rate of water loss could be due to inefficient or incomplete dehydration of the cuticle prior to sclerotization, resulting in less efficient water retention by the cuticle. The following points lend some support for this idea: (1) When a blowfly puparium starts to tan, the water content of the cuticle drops by a third over the first 8 hr. It then continues to drop at a

EFFECTS OF BURSICON ON LOCUSTA

MIGRATORIA

635

slower rate until the chemical processes of sclerotization harden the cuticle (FRAENKELand RUDALL, 1940). (2) 0 ver this ‘period the percentage drop in weight in 4 hr in dry air drops from 1.5 to 0.1 (WOLFE, 1955). (3) The chitinprotein portion of the cuticle can become highly impermeable in areas where it becomes dry (LAFON, 1943). (4) Th ere is a correlation between darkening and impermeability in wounded untanned cuticles (LAI-FOOK, 1966). (5) In the normal intact cockroach the epidermis is involved in drawing liquid water through the cuticle and so is presumable actively dehydrating the cuticle (BEAMENT,1964). In this same paper Beament proposes that, ‘an epidermal cell can create changes in parts of the cuticle with which it makes direct contact, and these changes can Dehydration of the cuticle under the lipid can include or control hydration. produce sufficient force to cause an inward current of water at quite a high rate’. It seems possible that the locust has a hydrated cuticle when it first emerges to allow it to expand with ease. However in the blowfly the cuticle softens for expansion only after sclerotization has started (COTTRELL, 1962d) suggesting that cuticular softening is dependant upon, rather than prior to, the release of bursicon. Such a phenomenon could well occur in the locust, since it would not wish to start expanding until it was quite clear of the old cuticle. In Rhodnius, the expansion of the cuticle when the insect takes a large blood meal could be explained by hydration of the untanned &&in-protein complex by the epidermal cell with a consequent change in the binding of this complex (BENNET-CLARK, 1962; BEAMENT,1964). The control in this instance would appear to be directly nervous, though perhaps mediated by neurosecretion. In the emerging locust dehydration at sclerotization would render the cuticle more impermeable. If the cuticle is not dehydrated, as in the abdominal ligatured locust, water is lost. The reduced dehydration of the cuticle could also possibly affect the subsequent sclerotization: in the blowfly pupa&m dehydration is associated with orientation and packing of the protein and chitin (FRAENKELand RUDALL, 1940). If this is necessary before tanning can take effect, then subsequent addition of chitin and protein might also be affected. If this were so, then bursicon would not need to be directly involved in the hardening and darkening process. The presence of bursicon in the cc but not in the nerve cord of the adult Locus& (VINCENT, 1971) may now be rationalized. If bursicon is necessary to the epidermal cell for it to dehydrate the cuticle, it might be imagined that the epiderma1 cell is in constant need of dehydrating the cuticle, and that this needs a constant titre of bursicon. For such a steady-state system a large amount of bursicon would not be necessary at any one time so that the high-rate release system of the nerve cord (VINCENT, 1971) would not be needed since the corpus cardiacurn could release the material fast enough. It is thus possible that bursicon is the ‘other half’ of an antagonistic pair of hormones controlling the hydration of the cuticle. The ‘first half’ would be a ‘plasticization’ hormone as typified by the one proposed for Rhodnk. Such an antagonistic pair of hormones could account for the changes in plasticity shown by the blowfly cuticle over the period of expansion.

636

J. F. V. VINCENT

Acknowledgements-I am grateful to Professor I. C. JONES, in whose Department this work was carried out, for facilities provided; and to Dr. K. C. HIGHNAMfor his help and supervision. REFERENCES Bm

J. W. L. (1960) Wetting properties of the insect cuticle. Nutwe, Land. 186, 40%

409. BEAMENTJ. W. L. (1964) Active transport of water in insects. Adw. Insect Physiol. 2, 67129. B--CLARK H. C. (1962) Active control of the mechanical properties of insect cuticle. J. Insect Physiol. 8, 627-634. COTTRELLC. B. (1962a) General observations on the imaginal ecdysis of blowflies. T~uns. R. ent. Sot. 114, 317-333. COT-WILLC. B. (1962b) The imaginal ecdysis of blowflies : the control of cuticular hardening and darkening. J. exp. Biol. 39, 395-412. COT-IXELLC. B. (1962~) The imaginal ecdysis of blowflies: detection of the blood-borne factor and determination of some of its properties. J. exp. Biol. 39, 413-430. COTTRELLC. B. (1962d) The imaginal ecdysis of blowflies: evidence for a change in the mechanical properties of the cuticle at expansion. J. exp. Biol. 39, 449-458. DBNNJZLLR. and MALEK S. R. A. (1955) The cuticle of the cockroach, Periplunetu americana -V. The chemical resistance of the impregnating material of the cuticle and the ‘selftanning’ of its protein component. Proc. R. Sot. (B) 144, 545-555. FOCAL W. and FRAENKELG. (1969) The rSle of bursicon in melanization and endocuticle formation in the adult fleshfly, Surcophugu bullutu. J. Insect Physiol. 15, 1235-1247. FRAENKELG. and HSIAO C. (1965) B ursicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11, 513-556. FRAENKELG. and RUDALLK. M. (1940) A study of the physical and chemical properties of insect cuticle. PYOC. R. Sot. (B) 129, l-34. HILL L., LIJNTZ A. J., and STEELE P. A. (1968) The relationships between somatic growth and feeding activity in the adult desert locust. J. Insect Physiol. 14, l-20. LAI-FOOK J. (1966) The repair of wounds in the integument of insects. 7. Insect Physiol. 12, 195-226. MALEK S. R. A. (1958) The appearance and histological structure of the cuticle of the desert locust, Schistocercu gregaria. PYOC. R. Sot. (B) 146, 557-569. MILLS R. R., MATHURR. B., and GUERRAA. A. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. y. Insect Physiof. 11, 1047-1053. UVAROVSIR B. (1966) Grasshoppers and Locusts. Cambridge University Press, London. VINCENT J. F. V. (1969) Bursicon, the hardening and darkening hormone, in Locusta migrutoria migratorioides and other Orthoptera. Ph.D. Thesis, Sheffield University. VINCENTJ. F. V. (1971) The dynamics of release of bursicon in Locusta migrutoria migratorioides and the possible identity of bursicon. r. Insect Pkysiol. To be published.