Role of the brain and ring gland in regulation of pupal diapause in the flesh fly Sarcophaga crassipalpis

J. fnwcr Phviol. Vol. 32. No. 2. pp. 161-166. 1986 Printed in Great Britain. All rights reserved

0022-1910/86 $3.00 + 0.00 Copyright C 1986 Pergamon Press Ltd

ROLE OF THE BRAIN AND RING GLAND IN REGULATION OF PUPAL DIAPAUSE IN THE FLESH FLY SARCOPHAGA CRASSIPALPLS J. M. Department

and D. L.

GIE~UL~~WICZ

of Entomology,

DENLINGER*

Ohio State University, Columbus, OH 43210, U.S.A.

(Received 2 MaJl 1985; revised 28 June 1985) Abstract-Larvae of Sarcophaga cras~ipafpisphotoperiodically programmed for pupal diapause pupariate later than larvae programmed for continuous development. Pupariation time is determined by the brain-ring gland complex as evidenced by transplantation experiments in which the timing of pupariation was transferred from one larva to another by transplantation of the brain-ring gland complex. The developmental commitment (diapause or nondiapause) of the larva also can be transferred with the brain-ring gland complex if the recipient’s own neuroendoctine system is suppressed by ring gland extirpation. Thus, photoperiodic programming of the brain-ring gland complex is not only responsible for developmental commitment but also for determining the duration of the prepupal period. Surgical experiments with pupae indicate that an intact brain-ring gland complex is required for diapause termination and initiation of adult development. Pupae fail to break diapause if either the brain or the ring gland is removed or if their nervous connections are severed. Key Word Index: Brain, ring gland, pupal diapause, Sarcophaga

INTRODUCTION

individuals

of temperate zone flesh flies is initiated in response to short daylength perceived during embryonic and early larval development (Denlinger, 1971; Saunders, 1971; Gnagey and Denlinger, 1984). These photoperiodic cues are translated into specific hormonal information that halts development early in the pupal stage. The brain-ring gland complex is presumed to play the central role in dictating the development fate of the fly (diapause or nondiapause), and in addition, it is assumed to mediate the events of diapause development and to provide the signal used to initiate adult development. But, in Diptera, the roles of the brain and ring gland in diapause regulation have not been critically examined. Structurally, the ring gland is unique to larvae and pupae of the Diptera. It is a composite of the corpora cardiaca, corpora allata and prothoracic gland and is richly innervated by axons projecting from the medial and lateral regions of the brain (Giebultowicz and Denlinger, 1985). In these experiments with the flesh fly Sarcophuga emsipalpis, we evaluate the roles of the brain and ring gland in the initiation and termination of pupal diapause. The hormonal milieu of diapause-destined flies clearly differs from that of flies destined for continuous development: the release of ecdysone that initiates adult development is absent in flies committed for diapause (Ohtaki and Takahashi, 1972; Walker and Denlinger, 1980), cyclic pulses of juvenile hormone activity are unique to diapause-destined *To whom correspondence

(Walker and Denlinger,

1980; Denlinger of cyclic AMP are much lower in diapause-destined flies (Gnagey and Denlinger, 1983). The commitment to pupal diapause, however, is not immutable. A high temperature shock following pupariation can reverse the decision to enter diapause (Gibbs, 1975; Denlinger, 1976), and hormonal manipulations that elevate the titre of ecdysteroids (Fraenkel and Hsiao, 1968; Zdarek and Denlinger, 1975; Gibbs, 1976) or cyclic AMP (Denlinger, 1976) can very effectively promote continuous development in a fly photoperiodically programmed for diapause. Likewise, transplantation of brain-ring gIand complexes from non-diapause-destined larvae into diapause-clestined larvae successfully prevents pupal diapause (Giebultowicz and Saunders, 1983). Our present study utilizes recipient larvae that have had their ring gland removed and demonstrates that transplanted brain-ring gland complexes can change the timing of pupariation and induce diapause in nondiapausedestined larvae. We also show that an intact brain-ring gland complex is essential for initiating adult development in diapausing pupae. et al., 1984), and brain concentrations

Flesh flies, like many insects, circumvent inimical seasons of the year in diapause. The pupal diapause

should be addressed. 161

MATERIALS AND METHODS insects

Cultures of Snrcophuga crassfpalpfs were reared as previously described (Denlinger, 1972). To prevent diapause, adult flies were m&ained at 25 + l”C, 15L:9D (light-dark cycle) and their progeny at 20 & O.S”C, lSL:9D. To induce pupal diapause, adults were kept at 25 rt l”C, 12L: 12D and progeny at 20 + O.S”C, 12L: 12D. Operated larvae and pupae were kept at 20 + 0.5”C and the photoperiod from

162

J. M. GIEEIULTOWICZand D. L. DENLINGER

which they originated.

Age of larvae used for operations was defined by collecting individuals that left the food at 6 h intervals.

a’

A

15:9

60 40

Surgery on larvae Brain-ring gland complexes used for implantation were dissected from post-feeding larvae (second day after departure from the food, prior to release of prothoracicotropic hormone) and stored in cold, sterile saline (Ephrussi and Beadle, 1936) for no longer than 1 h. Recipients of the same age were chilled on crushed ice for 1-2 h, washed, and submerged in cold, sterile saline. A V-shaped incision was made through the integument on the dorsal surface of the 4th segment. The crop was pulled out through the incision to expose the ring gland, and the ring gland beyond the corpus cardiacum (i.e. the part consisting of the corpora allata and prothoracic gland) was extirpated. Brain-ring gland cotiplexes were inserted through the same opening, the crop was carefully reinserted, and the cut integument was aligned. Larvae were immediately placed on ice for at least 1 h to permit the wound to heal. Approximately 80% of the operated larvae formed normal puparia and 40-50% survived to the adult stage. Shamoperated controls were treated identically, except their ring gland was left intact and they received no implants. Surgery on pupae

In preparation for surgery, puparia of diapausing pupae 4-5 weeks old were washed in detergent, water and 95% ethanol. After removal of the anterior cap of the puparium, the pupae were submerged in sterile saline containing a mixture of penicillin and streptomycin. An incision was made in the dorsal region of the head and the brain-ring gland complex was exposed by flushing away the fat body with saline. The ring gland or brain (without optic lobes) was extirpated or nervous connections between the brain and ring gland were severed. Cut cuticle was realigned, a few crystals of penicillin and streptomycin were placed on the wound, and excess moisture was absorbed with a tissue. Sham-operated pupae were treated in the same manner except the brain-ring gland complex remained intact. After a few hours all pupae received a 5 ~1 topical application of hexane, an agent known to break diapause (Denlinger et al., 1980). About 50% of the operated pupae survived for at least 2 weeks at which time they were scored for signs of adult developmental (antenna1 development, red pigmentation of the eyes, dark bristles on the thorax). Lack of such developmental markers was considered evidence for diapause. RESULTS

Eflect of photoperiod on pupariation time

Larvae of S. crassipalpis reared at 20°C pupariate later in a diapause-promoting photoperiodic regime (12L: 12D) than in a photoperiodic regime (15L:9D) that promotes uninterrupted development (Figs IA and B). Though duration of the feeding period was the same in both group of flies, the post-feeding, wandering phase of the third instar lasted only

20 $j

0 i+

f

: .i

So

12: 12-l

D.

5:9

60

20 40 0 i+ 012345676

Days

after

leaving

food

Fig. I. Time of pupariation in larvae of S. crussipalpis reared at 20°C with (A) continuous exposure to LD 15:9, (B) continuous exposure to LD 12: 12, (C) transfer from LD 15:9 to LD 12:12 one day after departure from the food, and (D) transfer from LD 12: 12 to LD 15:9 one day after departure from the food. Each N = 20-40.

2.2 + 0.1 days (mean + SE, n = 57) at 15L:9D but was extended to 4.3 + 0.2 days (n = 37) at 12L: 12D. To determine whether the difference was a direct response of the post-feeding larvae to the photoregime or an effect programmed at an earlier stage, groups of 20 larvae were transferred to the opposite photoregime after feeding. This treatment did not alter time of pupariation: larvae transferred from 15L:9D to 12L:12D pupariated 2.3 rt 0.2 days (mean + SE) later (Fig. lC), while larvae transferred from 12L: 12D to 15L:9D pupariated 4.2 k.O.1 days later (Fig. ID). In both cases, pupariatlon time remained similar to that of untransferred controls. Thus, the differences in pupariation time are photoperiodically determined before the onset of the postfeeding wandering stage. Effect of brain-ring gland transplantation on pupariation time and developmental commitment

If pupariation time is determined earlier than the post-feeding stage, the information should be “transferable” from one larva to another by transplantatoin of the brain-ring gland complex. To detect a clear effect from the implant, this experiment requires suppression of the recipient’s own hormonal system. We achieved this type of suppression by removing the ring gland from 2-day old, post-feeding larvae that were programmed for continuous development. After ring gland extirpation, 3 out of 18 larvae formed puparia but never pupated. The remaining 15 individuals survived for several days as wandering larvae but never pupariated.

Regulation of diapause

163

.z

a

._ 2

100

D-0

ND-ND

80

D-ND

23456

r

ND-O

01

Days after operation Fig. 2. Pupariation time in (A) long day larvae (programmed for non-diapause, ND) or (B) short-day larvae (programmed for diapause, (D) that were sham-operated or received implants of 2 brain-ring gland complexes from either non-diapause or diapause-destined donors. Surgical manipulations were completed on day 2 of the wandering stale. Except for sham-operated groups, recipients’ own ring gland was removed. Letters to left of arrows refer to developmental commitment of donors and letters to the right describe commitment of recipients. Each N = 20-38.

Figure 2 summarizes experiments in which larvae with their ring gland removed were supplied with 2 brain-ring gland complexes from donors programmed photoperiodically for diapause or nondiapause. Non-diapause sham-operated larvae and non-diapause larvae which received implants of nondiapause brain-ring gland complexes pupariated at nearly the same time (1.9 zb 0.1 and 2.3 +_0.2 days respectively, Fig. 2A). In contrast, non-diapause larvae which received 2 brain-ring gland complexes from diapause larvae pupariated much later (3.9 f 0.1 days) and at a time similar to that of diapause sham-operated larvae (3.5 + 0.2 days). If the ring gland was not removed, non-diapause larvae receiving diapause implants showed no delay in pupariation (1.8 f 0.2 days, n = 20). When diapause larvae deprived of their own ring gland were supplied with brain-ring gland complexes from diapause donors, they pupariated in 3.6 f 0.1 days (n = 33), similar to pupariation time in diapause sham-operated controls. But, diapause larvae receiving non-diapause transplants pupariated in 1.9 &-0.1 days (n = 28). a time characteristic of nondiapause sham-operated controls. Flies used in the above experiments were subsequently scored for diapause 3 weeks after pupariation (Fig. 3). All but one of the recipients of non-diapause brain-ring gland complexes were developing at that time. regardless of their own nondiapause

or diapause

commitment.

Among

diapause

DONOR

-

-

NO

ND

D

D

RECIPIENT

ND

D

ND

D

D

ND

Sham

Sham

Fig. 3. Incidence of continuous (non-diapause) pupal development observed in long-day (programmed for nondiapause, ND) and short-day (programmed for diapause. D) flies that were sham-operated or deprived of the ring gland and received implants of 2 brain-ring gland complexes from either diapause or nondiapause donors on the second day after departure from the food. Numbers above each bar indicate sample sire. Responses of non-diapause sham and nondiapause implants are not significantly different (Pearson x2, P > 0.05); diapause shams differ from diapause implants but differences between the two diapause implants are not significant.

larvae receiving diapause transplants, 68% initiated development and the remainder were in diapause. More importantly, a portion (30%) of nondiapause larvae entered diapause after receiving implants from diapause donors. No cases of diapause were observed in 20 other nondiapause recipients that retained their own ring gland. Thus, nondiapause larvae supplied with implants from diapause-destined larvae can enter diapause, but only if their own hormonal system is suppressed. Pupae which were scored as being in diapause were subsequently treated with a 5 ~1 topical application of hexane, an agent known to break diapause (Denlinger er al., 1980). In response to this treatment, all but one of the pupae initiated adult development. This implies that a true diapause state had been induced and eliminates the possibility that the implanted brain-ring gland complexes were unable to survive and differentiate. Larvae which had their own ring gland removed and received implants of 2 brain-ring gland complexes developed to the adult stage, but emergence from the puparium was usually unsuccessful. Seven flies of this type were dissected and examined under a dissecting microscope. The adult corpora allata were missing in all flies and 4 flies were also lacking corpora cardiaca. In all 7 flies, implanted brains were recovered in the thorax or abdomen. The brains had Tabk 1. Effect of brain or ring gland mnoval on the ability of pupae to terminate dispausc after application of 5pl hexanc Stwry

N

Unopctatcd shaqKqJcrated Cut between brain

14 19

% Development 100 63.2

and ring gland 17 0 Ring gtand removed 20 0 Brain remand 13 0 N represents number of pupae which survived for more than 2 weeks after operation.

J.

164

M.

GIEBULTOWKZ

fully differentiated to the adult shape with corpora cardiaca and corpora allata clearly visible. Results presented in this section indicate that transplanted brain-ring gland complexes can control the timing of pupariation and the developmental commitment of the recipients. Role of the brain and ring gland in termination of pupal diapause

Diapausing pupae 3-4 weeks old which had their brain or ring gland removed were treated with a 5 ~1 topical application of hexane and monitored for adult development. Over 60% of sham-operated pupae initiated development within 1 week in response to hexane application (Table 1). In contrast, none of the pupae that had the connection between the brain and ring gland severed, the ring gland removed, or the brain removed showed any signs of development within 2 weeks of hexane application. Apparently, an intact brain-ring gland complex is essential for initiation of adult development in diapausing pupae of S. crassipalpis. DISCUSSION

Larvae of S. crassipalpb reared in a diapauseinducing short photoperiod pupariate much later than siblings reared in long daylength. The feeding phase is the same duration in both groups of larvae. The difference appears only in the post-feeding, wandering phase of the third-instar larva. Switching photoperiods at the onset of the post-feeding stage does not affect pupariation time, thus pupariation time has been photoperiodically determined at an earlier stage of development. We demonstrated that timing of pupariation can be “transplanted” between the two group of larvae. Brain-ring gland complexes from nondiapause-destined larvae implanted into diapause-destined recipients (which had their own ring gland removed) accelerated pupariation while implants from diapause-destined larvae consistently delayed pupariation. Implanted brain-ring gland complexes from nondiapause-destined larvae not only accelerated pupariation but also consistently promoted continuous (non-diapause.) development in their hosts. Though implantation of brain-ring gland complexes from diapause-destined larvae also stimulated development in many individuals, about 30% of the hosts entered diapause in response to an implant from diapause-destined larvae. As suggested for several species of Lepidoptera (McDaniel and Berry, 1967; Wilson and Larsen, 1974), we suspect that surgical trauma is responsible for stimulating many of the diapause brains to initiate development. We have previously shown that heat shock (Denlinger, 1976), physical shaking (Denlinger, 1981), and a wet pupariation site (Giebultowicz, unpublished observation) can also reverse the diapause programme in wandering larvae of S. crassipafpis. But, in spite of surgical manipulations, many diapause brains retained their original programme. We conclude that the programme for pupariation time and diapause both reside within the brain-ring gland complex and can be transferred to other individuals by transplantation. Though Saunders (1976)

and D. L. DENLINGER argues that the timing of pupariation and the decision to enter diapause are independent photoperiodic responses in S. argyrostoma, it is clear from our results with S. crassipalpis that pupariation time is a programmed response closely linked to diapause. As in other insect examples (Beck, 1980; Denlinger, 1985), a delay in prediapause development is likely to be a component of the diapause syndrome. As such, it is one of the lirst indicators of the hormonal differences that distinguish diapause and nondiapause-destined larvae. A study with Man&a sexta (Lepidoptera) did not show differences in ecdysteroids or juvenile hormone production in differently programmed larvae (Bowen et al., 1985) but in this species length of larvae life is not affected by developmental commitment. Experiments with S. argyrostoma (Giebultowicz and Saunders, 1983) also demonstrated that brainring gland implants could stimulate development in diapause-programmed flies. However, in the experiments with S. argyrostoma, recipient’s brain and ring gland were kept intact, thus it was not known if the response resulted from direct action of the donor’s brain or its hormonal influence on the neuroendocrine system of the recipient. Our results with S. crassipalpis indicate that the brain-ring gland complex from nondiapause-destined larva can induce development in diapause-destined recipients which have their own hormonal system suppressed by removal of the ring gland. Clearly, a brain-ring gland complex implanted in the body of a diapausedestined recipient is capable of initiating adult development and promoting differentiation to the adult stage. A similar result was recently reported in M. sexta. Brains programmed for continuous development can override diapause commitment of the recipient larvae (Bowen et al., 1984). There are no previous reports of successful induction of diapause by implanting diapausecommitted brains. If the recipient’s own neuroendocrine system remains intact, it prevails and diapause can not be induced. But, by suppressing the recipient’s neuroendocrine system by ring gland extirpation, we were able to initiate diapause in some of the recipients. Larvae committed to continuous development are clearly different from diapausecommitted larvae as demonstrated by cold hardiness attributes and stores of metabolic reserves (Adedokun and Denlinger, 1984, 1985), yet late in the third-larval instar the developmental fate of the larva remains mutable and dependent upon the programme of the brain-ring gland complex. The decision to terminate pupal diapause and initiate adult development has classically been attributed to the brain. Clearly this is the case in ffyafophora cecropia (Williams, 1946, 1952). But, several recent investigations with other Lepidoptera (Kono, 1977; Meola and Adkisson, 1977; Kind, 1978) suggest that the brain capacitates the prothoracic gland very early in diapause and the regulatory decision to terminate diapause then resides strictly within the prothoracic gland. In these cases, the brain may be essential for only a few days after pupation. Our results with S. crassipalpis are similar to H. cecropia: the brain is essential at the actual time of diapause termination.

Regulation of diapause

Flesh flies, like many other insects, are known to produce ecdysteroids in the adult stage, long after the prothoracic gland has degenerated (Briers and deLoof, 1980). This suggests the possibility that sites other than the pupal ring gland may produce the ecdysone used to initiate adult development. Redfem (1983) argues for alternative sources of ecdysteroids based on the low capacity of the pupal ring gland of Drosophila melanogaster to synthesize ecdysteroids. Though alternative synthetic sites may exist in pupae of S. crassipalpis, it is clear from our results that development can not be initiated in the absence of a ring gland. Moreover, nervous connections Ftween the brain and ring gland are essential for dtapause termination and initiation of adult development. Cobalt back-fills (Giebultowicz and Denlinger, 1985) indicate that several neurones from medial and lateral regions of the brain send axons that branch profusely in the ring gland. Acknowledgements-This

research was supported in part by the Science and Education Administration of the U.S. Department of Agriculture under Grant No. 8300051 from the Competitive Research Grants Office.

Denlinger 6. L., Shukla M. and Faustini D. L. (1984) Juvenile hormone involvement in pupal diapause of the flesh fly Sarcophaga crassipalpis: regulation of infradian cycles of O2 consumption. 1. exp. Biol. 109, 191-199. Ephrussi B. and Beadle G. W. (1936) A technique for -transplantation in Drosophila. Am. Nat. 70, 218-225. Fraenkel G. and Hsiao C. (I%81 Moroholotil and endocrinological aspects of pupai dia$use In a flesh fly, Sarcophaga argyrostoma. 1. Insect Physiol. 14, 707-718.

Gibbs D. (1975) Reversal of pupal diapause in Sarcophaga argyrostoma by temperature shift after puparium formation. J. Insect Phvsiol. 21, 1179-l 186. Gibbs D. (1976) The*initiation of adult development in Sarcophaga argyrosroma by j-ecdysone. J. Insect Physiol. u,

1195-1200.

Giebultowiu J. M. and Denlinger D. L. (1985) Identification of neurons innervating the ring gland of the flesh fly larva. Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). Int. J. Insect Morphol. Embryol. 14, 155-161.

Giebultowin J. M. and Saunders D. S. (1983) Evidence for the neurohormonal basis of commitment to pupal diapause in larvae of Sarcophaga argyrostoma. Experientia 3& 194196. Gnagey A. L. and Denlinger D. L. (1983) Brain and ring gland cyclic AMP and cyclic GMP levels during initiation and termination of pupal diapause in flesh flies. Comp. Biochem. Physiol. 76c;

REFERENCES Adedokun T. A. and Denlinger D. L. (1984) Cold hardiness: a component of the diapause syndrome in pupae of the flesh flies, Sarcophaga crassipalpis and S. bullara. Physiol. En!. 9. 361-364. Adedokun T. A. and Denlinger D. L. (1985) Metabolic reserves associated with pupal diapause in the flesh fiy, Sarcophaga crassipalpis. J. insect Physiol. 31, 229-233. Beck S. D. (1980) Insecr Photoperiodism. 2nd edn. Academic

Press, New York. Bowen M. F., Saunders D. S., Bollenbacher W. E. and Gilbert L. I. (1984) In vitro reprogramming of the photoperiodic clock in an insect brain-retrocerebral complex. Proc. nam. Acad. Sci. U.S.A. 81, 5881-5884.

Bowen M. F., Irish R., Whisenton L. R., Granger N. A., Gilbert L. 1. and Bollenbacher W. E. (1985) Endocrine pm-diapause and non-diapause events during larval-pupal development of the tobacco homworm, Manduca sexta. J. Insect Physiol. 31, 83-90.

Briers T. and De Loof A. (1980) The moiting hormone activity in Sarcophaga bullata in relation to metamorphosis and reproduction. Int. J. Invert. Reprod. 2, 363-372.

Denlinger D. L. (1971) Embryonic determination of pupal diapause in the flesh fly Sarcophaga crassipalpis. J. Insect Phwiol. 17, 1815-1822. Denlinger D. L. (1972) Induction and termination of pupa1 diapause in Sarcophaga (Diptera: Sarcophagidae). Biol. Bull. mar. biol. Lab., Woods Hole 142, 11-24. Denlinger D. L. (1976) Preventing insect diapause with hormones and cholera toxin. Li/e Sci. 19, 1485-1490. Denlinger D. L. (198 I ) The physiology of pupal diapause in flesh flies. In Current Topics in Insect Endocrinology and Nutrition (Ed. by Bhaskaran G., Friedman S. and Rodriguez J. G.), pp. 131-160. Plenum Press, New York. Denlinger D. L. (1985) Hormonal control of diapause. In Comprehensiae Insect Physiology Biochemistry and Phar mucolog!, (Ed. Kerkut G. A. and Gilbert L. I.). Pergamon

Press. Oxford. Dcnlinger D. L., Campbell J. J. and Bradfield J. Y. (1980) Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly, Sarcophaga crassipalpis. and the tobacco homworm, Manduca sexta. Pk.vsiol. Ent. 5, 7-15.

iP i::--E

165

l21-li5.

Gnagey A. L. and Denlinaer D. L. (1984) Photoneriodic induction of pupal diapause in the fresh ‘fly, Sar>ophaga crassipalpl: embryonic sensitivity. J. camp. Physiol. B. 154, 91-96. Kind T. V. (1978) A study of the reactivation of diapausing pupae of Acronycta rumicis L. (Lcpidoptera, Noctuidae). I. Cold activation of the prothoracic glands in brainless pupae. Ear. Rev. 56, 13-16. Kono Y. (1977) Ultrastructural changes of neurosecretory cells in the pars intercerebralis during diapause development in Pieris rapae. J. Insect Physiol. 23, 1461-1474. Me& R. W. and Adkisson P. L. (1977) Release of prothoracicotropic hormone and potentiation of development ability during diapause in the bollworm, Heliothis zea. J. Insect Physiol. 23, 683-688.

McDaniel C. N. and Berry S. J. (1967) Activation of the prothracic glands of Antheraea polyphemus. Nature 214, 1032-1034.

Ohtaki T. and Takahashi M. (1972) Induction and termination of pupal diapause in relation to the change of ecdysone titer in the flesh fly, Sarcophaga peregrina. Jap. J. med. Sci. Biol. 25, 369-376.

Redfem C. P. F. (1983) Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval, prepupal and pupal development. J. Insect Physiol. 29, 65-71.

Saunders D. S. (1971) The temperature-compensated photoperiodic clock “programming” development and pupal diapause in the flesh fly. Sarcophaga argyrostoma. J. Insect Physiol. 17, 801-812.

Saunders D. S. (1976) The circadian eclosion rhythm in Sarcophaga argyrosroma: some comparisons with the photoperiodic clock. J. camp. Physiol. 110, II l-133. Walker G. P. and Denlinger D. L. (1980) Juvenile hormone and moulting hormone titres in diapause and nondiapause destined flesh flies. J. Insect Physiol. 26, 661-664. Williams C. M. (1946) Physiology of insect diapausez the role of the brain in the production and termination of pupal dormancy in the giant silkworm Platysamla cecropia. Biol. Bull. mar. biol. Lab., Woo& Hole 93, 89-98.

Williams C. M. (1952) Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Blol. Bull. mar. biol. Lab., Woods Hole 103, 120-138.

J. M.

166

GIEBULTOWKZ

Wilson G. R. and Larsen J. R. (1974) Debraining and diapause development in Man&co sexru pupae. J. Insect Physiol.

20, 2459-2474.

and D. L. DENLINGER Zdarek J. and Denlinger D. L. (1975) Action of ecdysoids, juvenoids, and non-hormonal agents on termination of pupal diapause in the flesh fly. J. Insecr Physiol. 21. 1193-1202.