J. exp. Biol. (1980), 88, 339-349 339With 3 figures

tinted in Great Britain

PHYSIOLOGY OF PUPAL ECDYSIS IN THETOBACCO HORNWORM, MANDUCA SEXTA

II. CHEMISTRY, DISTRIBUTION, AND RELEASE OF ECLOSION HORMONEAT PUPAL ECDYSIS.

BY PAUL H. TAGHERT, JAMES W. TRUMANAND STUART E. REYNOLDS*

Department of Zoology, University of Washington,Seattle, WA 98195, U.S.A.

(Received 20 March 1980)

SUMMARY

Eclosion hormone activity was found in the brain and ventral gangliaof pharate pupae of Manduca sexta. No activity was detected in the corporacardiaca-corpora allata complex. At the time of ecdysis the store of activitydropped by 50-75 % in the ventral cord whereas the hormone level in thebrain remained unchanged. Also, larvae whose brains were removed at thewandering stage subsequently showed pupal ecdysis behaviour and also hadessentially normal levels of hormonal activity in their blood at the start of thebehaviour. It was concluded that at pupal ecdysis the hormone responsiblefor the initiation of the behaviour is released from the ventral nerve cordrather than from the brain.

The chemical characteristics of the pharate pupal eclosion hormonewere determined. The factors from the brain and ventral nerve cord wereboth active in a number of adult and pupal eclosion hormone bioassays.Both showed an apparent molecular weight of 8500 daltons and an iso-electric point of about 5-0, values essentially the same as that seen for theadult form of the hormone. We concluded that pupal ecdysis and adulteclosion are triggered by the same hormone but for the former it is releasedfrom the ventral nerve cord and, for the latter, from the brain. The choiceof release site may depend on whether or not the release is under circadiancontrol.

INTRODUCTION

The adult eclosion of large moths from their pupal case is triggered by a peptidehormone, the eclosion hormone (Truman & Riddiford, 1970). The previous paperin this series (Truman, Taghert & Reynolds, 1980) presented evidence that theeclosion hormone might also initiate pupal ecdysis in the tobacco hornworm, Manducasexta. It was shown that exogenous eclosion hormone can trigger pupal ecdysisbehaviour and that a factor with eclosion hormone-like activity appears in the

* Permanent address: School of Biological Sciences, University of Bath, Claverton Down, Bath,Wbz 7AY, U.K.

34° P- H. TAGHERT AND OTHERS

blood of pharate pupae about 30 min before ecdysis. The activity in the blood w ^sufficient to cause ecdysis when injected into another pharate pupa.

This evidence is sufficient to show that a hormone with eclosion hormone-likeproperties is involved in pupal ecdysis, but it does not prove that this factor isidentical with eclosion hormone. This paper reports on the presence of an eclosionhormone-like factor in the pharate pupal central nervous system (CNS), its distri-bution within the CNS, the dynamics of its release, and the factor's biochemicalcharacteristics and activity in a number of different bioassays. We conclude that thehormones controlling pupal ecdysis and adult eclosion are the same.

MATERIALS AND METHODS

Experimental animals

The rearing and staging of Manduca sexta larvae were as described in the previousreport (Truman et al. 1980). Diapausing pupae of Hyalophora cecropia were obtainedfrom dealers. Stimulation of development and the methods of abdomen isolationfrom pharate adults were as described in Truman (1978).

Preparation of tissues and separation techniques

Tissues were homogenized in distilled water containing a few crystals of phenyl-thiourea in ground-glass homogenizers, heated to 80 °C for 5 min and centrifugedat iooog at room temperature for 20 min. The resultant supernatant was usedimmediately or stored at — 20 °C for at most a few days before use.

For gel filtration, tissues were treated as above (50 tissues homogenized in 0-9 mldH2O) and acidified by addition of o-1 ml 1 -o N-HAc. Following centrifugation at17 500 g for 20 min at 4 °C, supernatants were chromatographed immediately onSephadex G-50 fine (Pharmacia). One ml samples were layered on a 1-6x30 cmcolumn, eluted at room temperature with o-i N-HAc with a mechanically controlledflow rate of approximately 8 ml/cm2/h. The column was calibrated using bluedextran (Pharmacia), cytochrome C (Sigma), insulin (CalBioChem), bacitracin (Sigma)and KC1.

For electrofocusing the relevant fractions from replicate runs through Sephadexwere pooled, lyophylized, and resuspended in o-i N-HAC and o-i% bacitracin (toenhance the stability of the peptides; Mumby, Truman & Reynolds, in preparation)and layered over pre-cast electrofocusing gels (0-5 x 10 cm) containing wide-rangeampholines (pH 3-9; Bio-Rad). The gels were run for 22 h at 4 °C at 200 V. Gelswere then sliced (3 mm thickness) and the slices eluted for 48 h in o-i N-HAc ando-1 % bacitracin at 4 °C.

Calibrations were performed by eluting slices from a simultaneously run blankgel in freshly boiled dH2O for 2-3 h at room temperature, then measuring the pHof the eluents.

Pupal ecdysis in the tobacco hornworm 341

biological assays

A number of biological assays were used in the present study. For the pupalecdysis assay described in the preceding paper (Truman et al. 1980), anteriorshrinkage (AS) stage pharate pupae were injected with tissue extracts or aliquots ofunbuffered column fractions. Positive scores were calculated as the reciprocal ofthe latency (min) x 10*. As described previously, negative assays were scored as

Si-A second assay utilized abdomens isolated from pharate adult H. cecropia. The

responses of the abdomen to injections of various fractions were recorded as describedin Truman (1978) and were scored according to the criteria in Mumby, Truman &Reynolds (in preparation). Briefly, these were: (1) a weak, drawn-out pre-eclosionbehaviour; (2) a normal pre-eclosion behaviour but no eclosion behaviour; (3) a weakeclosion behaviour; (4) a strong eclosion behaviour with shedding of the pupalcuticle. The assay based on this scoring system is about 10-fold less sensitive thanthe pupal ecdysis assay and is roughly linear with the log of hormone concentration.

The third assay utilized wings from pharate adult Manduca sexta. Fractions inacid were buffered (Reynolds, 1977) before injection of 10 jul into the wing veins.The score (in mm) is based on the difference in plasticity in the test wing comparedwith a paired control and measured according to the technique of Reynolds (1977)as modified by Reynolds & Truman (1980).

In titering the amount of eclosion hormone activity in various organs, activitywas expressed in terms of 'units'. One unit is equivalent to the amount of activityextracted from a CC-CA pair from a pharate adult.

Surgical techniques

Some experiments involved removing the brain from animals on the night ofwandering (Truman & Riddiford, 1974), i.e. 3-4 days since the last larval ecdysisand 4 days before the larval-pupal ecdysis. Animals were anaesthetized by immersionin water, then suspended by a loose neck clamp over a stream of carbon dioxide.A small rectangle of cuticle was completely removed just lateral to the invertedY suture of the head. The brain was excised and either discarded or reimplanted toits original position. The excised piece of cuticle was replaced over the hole andsealed in place with melted Tackiwax (Cenco).

RESULTS

(1) Distribution of eclosion hormone activity in the prepupal CNS

The nervous systems were removed from pharate pupae that were approximately4-8 h before ecdysis. Ganglia from various regions were combined, extracted asdescribed in the Materials and Methods and assayed for their eclosion hormoneactivity using the pupal ecdysis assay described in the previous paper (Trumanet al. 1980). The distribution of eclosion hormone activity is summarized in Table 1.The largest store of the hormone appeared in the abdominal ganglia with lesser•nounts present in the brain and the thoracic ganglia. The assay detected no hormone

342 P. H. TAGHERT AND OTHERS

Table i. Distribution of eclosion hormone activity in the nervous systemof prepupal Manduca sexta

Tissue

BrainCC-CA complexSubesophageal ganglionThoracic gangliaAbdominal ganglia

No.

131 0

91 0

1 0

Assay score(X±SM.)

n6±7Si ±051 ±085± 11

133 + 5

Eclosion hormone(units)

0 0 7< OOI

< OOI

0 0 3o n

. ±

S .E

.

CO

oa>.

ecd

Pupa

l

140

120

100

80

60

A

M\l

•VT

- ki1 N '10 OS 0-25 012 10 OS 0-25 012

Fraction of organ

Fig. 1. Dose-response curves for brains (A) and abdominal nerve cords (B) taken fromanimals as AS stage pharate pupae (open circles) or as newly ecdysed pupae (filled circles).Each average is based on 15-20 assays.

in the subesophageal ganglion or the corpora cardiaca-corpora allata (CC-CA)complex. The lack of eclosion hormone activity in this last structure is in strikingcontrast to the pharate adult stage in which the majority of the cephalic store ofeclosion hormone is stored in the CC-CA complex (Truman, 1973).

(2) Depletion of hormonal activity during ecdysis

Pupal ecdysis is accompanied by the abrupt appearance of substantial eclosionhormone activity in the blood (Truman et al. 1980). The results described aboveshowed that eclosion hormone activity is widely distributed in the pharate pupalCNS and consequently there are a number of potential sources of this blood-borneactivity. We examined the two major sites of activity, the brain and the abdominalnerve cord, from AS stage pharate pupae (about 3 h before release) and pupae thathad ecdysed less than 1 h before sacrifice (i.e. less than i£ h after hormone release).The relative amounts of eclosion hormone activity in these two regions of the CNSis illustrated in Fig. 1. The brains from AS stage pharate pupae and from ^

Pupal ecdysis in the tobacco hornworm 343

Table 2. The effect of 'debraining' on the ability to perform pupal ecdysis

Successful ecdysisN (%)

Sham operated 5 100Reimplanted brain 7 57Brainless 34 62

showed similar amounts of extractable activity. By contrast, the activity in theabdominal nerve cord dropped by approximately 50-75 % during this 4^ h period.Thus, the hormonal activity that appears in the blood before ecdysis apparentlycomes primarily from stores in the abdominal ganglia. The thoracic ganglia probablyalso contribute to this release but this was not tested.

(3) Effect of brain removal on pupal ecdysis and hormone titres

In order to confirm that the prepupal brain was not the source of the blood-borneeclosion hormone activity, we removed brains from larvae that had just entered thewandering stage (about 4 days before the scheduled time of pupal ecdysis). Theseanimals were somewhat delayed in their subsequent timetable of pupal developmentbecause the brain extirpation also removed the source of the prothoracicotropichormone that normally drives ecdysone secretion (Williams, 1952). As seen in Table 2,over 60% of the operated animals went on to show a complete pupal ecdysis behaviourand shed the old larval cuticle. These animals that underwent successful ecdysis didso at the proper stage of pupal development as measured by the state of tanningof the pupal cuticle (see timetable in Truman et al. 1980). Of the remaining debrainedanimals, a number showed at least the initiation of the ecdysis programme as seen bythe pulling out of the tracheal linings in the posterior abdominal segments. Interest-ingly, even the animals that did not show a complete ecdysis nevertheless eventuallyturned ventral-side-up thereby assuming the characteristic pupal posture.

To verify the assumption that debrained animals performed the behaviour inresponse to blood-borne hormone, we bled five such animals just as they beganecdysis movements. The individual blood samples from each of these animals scoredstrongly positive in the pupal ecdysis assay; the mean score was 143 ± 10 ( + S.E.).This compares favourably with the mean score of 171 obtained from blood ofecdysing intact animals (Truman et al. 1980). Therefore although the pharate pupalbrain contains substantial amounts of eclosion hormone activity, it appears to havelittle or no function in the secretion of the hormone that triggers pupal ecdysis.

(4) Biochemical characteristics of eclosion hormone-like activities from the pharate pupa

The ecdysis stimulating activity in the pharate pupal brain and abdominal nervecord was characterized as to its apparent molecular weight and charge. Heat-treatedextracts of brains and abdominal nerve cords were subjected to gel filtration chroma-tography through a Sephadex G-50 column. The fractions were then assayed foreclosion hormone activity using the pharate adult Manduca isolated wing assaytf Reynolds, 1977). As seen in Fig. 2, both tissues showed a large peak of activitythat centred around a VJV^ ratio of about i-6, which corresponded to an apparent

344 P. H. TAGHERT AND OTHERS

30 Ki

12-5 Kl

6KI

1-5 KI

2 -

ISO

100

50 L

3 I

I

10 1-5 20

Fig. 2. Profiles of biological activity obtained by Sephadex G-so chromatography of extractsprepared from pharate pupal brains (A) or abdominal nerve cords (B). Fractions were testedfor their ability to induce extensibility in Manduca wings (top), to elicit behaviours of theemergence sequence from isolated abdomens of H. cecropia (middle), and to stimulate ecdysisof pharate pupal Manduca (bottom). Wing assay scores greater than i mm were consideredpositive; negative scores (ranging from — o-i to —07 mm) were plotted on the zero line.Numbers at the top of the figure represent the elution positions of various molecular-weightmarkers.

Pupal ecdysis in the tobacco hornworm

A

345

150

100

50 L

3 I

2 I

'pH

Fig. 3. Profiles of biological activity obtained by subjecting the active fractions from Fig. 2to electrofocusing on polyacrylamide gels. Each point represents the activity eluted froma 3 mm gel slice. Material was prepared from pharate pupal brains (A) or abdominal nervecords (B). See Fig. 2 for further details.

346 P. H. TAGHERT AND OTHERS

molecular weight of 8000-9000 daltons. Assay of these active fractions on H. cecropiAisolated abdomens showed that those with substantial wing plasticising activity alsowere capable of releasing various behaviours of the adult emergence sequence.Fig. 2 also shows that the extracts from both tissues showed only one peak of pupalecdysis stimulating activity and that in both cases it coincided with the eclosionhormone activities revealed by the other two assays.

The active fractions from the Sephadex G-50 column were then subjected towide band (pH 3-9) isoelectrofocusing. In both the brain (Fig. 3 A) and the abdominalnerve cord (Fig. 3 B) the various biological assays revealed only one peak of activityat approximately pH = 5. Thus, the eclosion hormone activities present in thebrain and abdominal CNS of pharate pupae have common molecular properties inso far as they have similar molecular weights and isoelectric points.

DISCUSSION

In pharate adult moths eclosion is triggered by the release of eclosion hormonethat had been made in brain neurosecretory cells during adult development andtransported down the axons to terminals in the CC for subsequent release (Truman,1973). An initial survey of larvae and pupae for eclosion hormone activity (Truman,1973) concluded that the brain-CC-CA complexes from these stages contained atmost 10% of the amount of hormone found in the pharate adult. This estimate wasmade using a whole-animal behavioural assay which could only detect amounts ofhormone as small as this with some degree of uncertainty. The conclusion drawn atthe time from this finding was that eclosion hormone was unlikely to be importantin the control of ecdysis in larvae and pupae.

The results of the'present study require that this conclusion be reevaluated. Theinitial estimate of eclosion hormone content in the pharate pupal brain has beenconfirmed here (0-07 units of eclosion hormone). In addition we also find 0-14 unitsstored in the CNS outside of the brain, in the thoracic and abdominal ganglia. Thetotal amount of eclosion hormone in the pharate pupa is thus about 21 % of that inthe pharate adult CC, but this smaller quantity of hormone assumes increasedsignificance with the finding that the pharate pupal CNS is approximately 10 timesmore sensitive to the eclosion hormone than is that of the pharate adult (Trumanet al. 1980).

The distribution of activity in the pharate pupal CNS posed two questions: arethe activities in the brain and the ventral nerve cord attributable to the same factor,and is either, or both, the same as the eclosion hormone of the pharate adult? Theresults of gel filtration chromatography and electrofocusing showed that both tissuescontained only one biologically active species and that the apparent molecular weightand charge of the active factor were the same in each case. Both factors were activein all three bioassays. The total amount of activity in the active fractions after gelfiltration chromatography (Fig. 2) was calculated using the standard dose responsecurves for each assay (Reynolds & Truman, 1980; Truman et al. 1980; Mumby,Truman & Reynolds, in preparation). A value was then obtained for the number of50% dosages that were present in each extract for each assay. The ratio of S°°/mdosages for adult abdomen eclosion:pupal ecdysis:adult wing plasticization was

Pupal ecdysis in the tobacco hornworm 347

f ^-5:1:27 for prepupal brains and 0-37:1:30 for the abdominal nerve cord. Thus,e activities from the 2 parts of the pharate pupal CNS were very similar in twoaspects of their molecular properties and in their relative potencies in three differentbiological assays. We conclude that it is highly likely that these activities are due tothe same molecule.

Eclosion hormone purified from heads of pharate adult Manduca (Reynolds &Truman, 1980) or from CC-CA complexes from the same stage (Mumby, Truman& Reynolds, in preparation) has an apparent molecular weight of 8500 daltons. Wideband electrofocusing (pH 3-9) shows one band of activity centring around 4-9(Reynolds & Truman, 1980). When the ratios of 50% dosages for pharate adultCC-CA extracts, or for electrofocused eclosion hormone from the same source arecomputed for the three assays listed above, ratios of 012:1:13 and 006:1:16respectively are obtained (Reynolds & Truman, 1980; Truman et al. 1980; Mumby,Truman & Reynolds, in preparation). Thus, purified adult eclosion hormone pre-parations have a spectrum of biological activity that is similar to the material obtainedfrom pharate pupae. Also, the molecular properties of hormone from the two stagesare very similar. Consequently we conclude that the prepupal activity is probablyidentical to the eclosion hormone of the pharate adult and that the same hormone isused to trigger both pupal ecdysis and adult eclosion. Definitive confirmation of thisconclusion will require amino acid sequence determinations of hormone purifiedfrom each of the two stages.

We have presented two lines of evidence to suggest that, in the pharate pupa,eclosion hormone is released from the ventral nerve cord rather than from thebrain, as is the case in the pharate adult. First, the store of hormone in the pupalabdominal CNS is depleted during the course of ecdysis, whereas that in the brainis not. Second, debrained insects release eclosion hormone into their blood inapproximately normal quantities and undergo pupal ecdysis at the proper develop-mental stage. It is significant that although the pharate pupal brain contains appre-ciable amounts of eclosion hormone, we could find no activity in the brain's neuro-haemal organ, the corpora cardica. In the pharate adult on the day of eclosion, theCC contains the majority of the total cephalic store of eclosion hormone (Truman,

1973)-Why does the pharate pupal brain contain a store of eclosion hormone which is

not destined for release at pupal ecdysis? The eclosion hormone present at thistime may simply represent an early stage in the process whereby hormone is accu-mulated for future use at adult ecdysis. Alternatively, the store of hormone in thebrain may not be destined for release into the blood but may represent a local, non-hormonal, peptidergic pathway within the brain. A number of studies have shownthat hormones which are secreted into the blood may also be released locally in theCNS to modulate neuronal activity. Examples include the egg-laying hormone ofthe mollusc Aplysia (Branton et al. 1978) and the luteinizing hormone releasinghormone of amphibians (Jan, Jan & Kuffler, 1979).

A distribution of hormonal activity between the brain-CC-CA complex and thechain of ventral ganglia has been reported for a number of different insect hormones

raenkel & Hsiao, 1965; Gersch & Sturzebecher, 1967; Hartmann, Wolf & Loher,). In the case of the last example, a hormone that promotes oocyte maturation

348 P. H. TAGHERT AND OTHERS

in a grasshopper, Gomphocerus rufus, it was suggested that the ventral ganglioncentre supplemented the brain centre and could partially compensate for damage tothe pars intercerebralis (Hartmann et al. 1972). The present study shows thateclosion hormone has a broad distribution in the CNS but that the two centres arecontrolled independently and are used at different times in the life history of theinsect.

The existence of two distinct sites for eclosion hormone release presents thequestion of what are the factors responsible for selection of the brain or the ventralnerve cord sites. Adult eclosion in silkmoths and in the sphinx moth, Manduca, iscontrolled by a circadian clock that is sensitive to light (Truman, 1972 a) or tem-perature cycles (Lockshin, Rosett & Srokose, 1975) respectively. In the silkmothsthe circadian system comprised of a photoreceptor and clock is contained withinthe brain (Truman, 1972 a) and directs the release of eclosion hormone from itsbrain centre. Other neurohormones in insects that are released under photoperiodiccontrol are likewise released from endocrine centres in the brain (Williams &Adkisson, 1964; Steele & Lees, 1977; Truman, 1976). In contrast to the aboveexamples, pupal ecdysis in Manduca is similar to larval ecdysis (Truman, 19726) inthat the event itself is not gated but rather is developmentally triggered. In thisrespect it is of interest that for this ecdysis, the hormone is released from the ventralnerve cord rather than from the brain. From this limited set, one would predictthat the brain centre presides over photoperiodically gated releases of hormonewhereas the ventral nerve cord centre is reserved for those triggered by endogenous,developmental factors. It will be of interest to determine if this association betweenthe site of hormone release and a gated or developmental control over release willhold true for ecdyses of other stages and of other insects.

We thank Mr Oliver Dominick for advice on the surgical techniques. Thesestudies were supported by grants from NSF (PCM 77-24878) and from NIH(Roi NS 13079 and K04 NS 00193). P.H.T. was supported by a NIH traininggrant (GM 0710805) and by an NSF Predoctoral Fellowship (63-6665).

REFERENCES

BRANTON, W. D., MAYERI, E., BROWNELL, P. & SIMON, S. (1978). Evidence for local hormonal com-munication between neurons in Aplysia. Nature, Lond. 274, 70—72.

FRAENKEL, G. & HSAIO, C. (1965). Bursicon, a hormone which mediates tanning of the cuticle in theadult fly and other insects. J. Insect Physiol. 11, 513-556.

GERSCH, M. & STURZEBECHER, J. (1967). Zur Frage der Idcntitat und des Vorkommens von Neuro-hormon D in verschiedenen Bereichen des Zentralnervensystems von Periplaneta americana. Z.Naturforsch. B 22, 563.

HARTMANN, R., WOLF, W. & LOHER, W. (1972). The influence of the endocrine system on reproductivebehavior and development in grasshoppers. Gen. comp. Endocr., Supp. 3, 518-528.

JAN, Y. H., JAN, L. Y. & KUFFLER, S. W. (1979). A peptide as a possible transmitter in sympatheticganglia of the frog. Proc. natn. Acad. Sci. U.S.A. 76, 1501—1505.

LOCKSHIN, R. A., ROSETT, M. & SROKOSE, K. (1975). Control of ecdysis by heat in Manduca sexta. J.Insect Physiol. ax, 1799-1802.

REYNOLDS, S. E. (:977). Control of cuticle extensibility in the wings of adult Manduca at the time ofeclosion: effects of eclosion hormone and bursicon. J. exp. Biol. 54, 705—814.

REYNOLDS, S. E. & TRUMAN, J. W. (1980). Eclosion hormone. In Insect Neurohormones (ed. T. A.New York: Springer-Verlag. (In the Press.)

Pupal ecdysis in the tobacco horn-worm 349

•TEELE, C. G. H. & LEES, A. D. (1977). The role of neurosecretion in the photoperiodic control ofpolymorphism in the aphid Megoura viciae.J. exp. Biol. 67, 117-135.

TRUMAN, J. W. (1972a). Physiology of insect rhythms. II. The silkmoth brain as the location of thebiological clock controlling eclosion. J. comp. Physiol. 81, 99-114.

TRUMAN, J. W. (19726). Physiology of insect rhythms. I. Circadian organization of the endocrineevents underlying the moulting cycle of larval tobacco hornworms. J. exp. Biol. 57, 805—820.

TRUMAN, J. W. (1973). Physiology of insect ecdysis. II. The assay and occurrence of the eclosionhormone in the Chinese Oak Silkmoth, Antheraea pernyi. Biol. Bull. mar. biol. Lab., Woods Hole144, 200-211.

TRUMAN, J. W. (1976). Extraretinal photoreception in insects. Photochem. Photobiol. 23, 215-225.TRUMAN, J. W. (1978). Hormonal release of stereotyped motor programmes from the isolated nervous

system of the Cecropia silkmoth. J. exp. Biol. 74, 151-174.TRUMAN, J. W. & RIDDIFORD, L. M. (1970). Neuroendocrine control of ecdysis in silkmoths. Science,

N.Y. 167, 1624-1626.TRUMAN, J. W. & RIDDIFORD, L. M. (1974). Physiology of insect rhythms. III. The temporal organ-

ization of the endocrine events underlying pupation of the tobacco hornworm. J. exp. Biol. 60,371-382.

TRUMAN, J. W., TACHFRT, P. H. & REYNOLDS, S. E. (1980). Physiology of pupal ecdysis in the tobaccohornworm, Manduca sexta. I. Evidence for control by eclosion hormone. J. exp. Biol. 88, 327-337.

WILLIAMS, C. M. (1952). Physiology of insect diapause. IV. The brain and prothoracic glands as anendocrine system in the Cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole 103, 120-138.

WILLIAMS, C. M. & ADKISSON, P. L. (1964). Physiology of insect diapause. XIV. An endocrinemechanism for the photoperiodic control of pupal diapause in the oak silkworm, Antheraea pernyi.Biol. Bull. mar. biol. Biol. Lab., Woods Hole 127, 511-525.

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