Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist, RH-5992, on the Sixth Instar Larva of the Spruce Budworm, Choristoneura fumiferana

J. Insect Physiol. Vol. 43, No. 1, pp. 55–68, 1997 1997 Elsevier Science LtdPergamon Printed in Great Britain. All rights reserved0022-1910/97 $17.00 + 0.00PII: S0022-1910(96)00062-5

Ultrastructural Effects of a Non-SteroidalEcdysone Agonist, RH-5992, on the Sixth InstarLarva of the Spruce Budworm, ChoristoneurafumiferanaA. RETNAKARAN,*† A. MACDONALD,* W. L. TOMKINS,* C. N. DAVIS,* A. J. BROWNWRIGHT,*S. R. PALLI*

Received 12 February 1996; revised 18 April 1996

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moultedsixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera:Tortricidae) initiates a precocious, incomplete moult. Within 6 h post treatment (pt) the larvastops feeding and remains quiescent. Around 12 h pt, the head capsule slips partiallyrevealing an untanned new head capsule that appears wrinkled and poorly formed. By24 h pt, the head capsule slippage is pronounced and there is a mid-dorsal split of the oldcuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this stateand ultimately dies of starvation and desiccation. The temporal sequence of the external andinternal changes of the integument were studied using both scanning and transmission electronmicroscopy. Within 3 h pt, there is hypertrophy of the Golgi complex indicating syntheticactivity and soon after, large, putative ecdysial droplets are seen. Within 24 h, a new cuticlethat lacks the endocuticular lamellae is formed. The formation of the various cuticular compo-nents, the degradation of the old cuticle and changes in the organelles of the epidermal cellsof the mesothoracic tergite are described. The difference between the natural moult and theone induced by RH-5992 are explained on the basis of molecular events that take place duringthe moulting cycle. The persistence of this ecdysone agonist in the tissues permits theexpression of all the genes that are up-regulated by the presence of the natural hormone butthose that are turned on in the absence of the hormone are not expressed. 1997 ElsevierScience Ltd. All rights reserved

RH-5992 Tebufenozide Ecdysone agonist Dibenzoyl hydrazine Ecdysteroids Moult induction Scan-ning electron microscopy Transmission electron microscopy Gene regulation Spruce budworm

INTRODUCTION them being the secretion and release of 20-hydroxyecdy-sone (Steel and Davey, 1985). In response to this hor-The formation of a rigid, exoskeleton by the epidermismone, the epidermis initiates the process of dissolutionlimits the process of continuous growth and forces theof the old and biosynthesis of a new cuticle, concomitantinsect to grow in a saltatory manner interspersed with awith the growth of the insect (Riddiford, 1985; Zacharuk,series of moults (Williams, 1980). During the process of1976). Recently, a new class of compounds, dibenzoylmoulting, a cycle of events occurs as a result of a care-hydrazines, have been shown to act as ecdysone agonistsfully orchestrated programme of gene expression, coordi-that initiate a precocious and incomplete moult. Thenated and controlled by endocrine cues, chief amongmode of action of one of these ecdysone agonists, RH-5849, has been traced to the ecdysteroid receptor (Wing,

*Natural Resources Canada, Canadian Forest Service, 1219 Queen 1988; Wing et al., 1988). An analogue of this compound,Street East, PO Box 490, Sault Ste Marie, Ontario, Canada P6A RH-5992, which also acts in a similar fashion at the mol-5M7.

ecular level, was found to produce phenotypic effects that†To whom all correspondence should be addressed. Tel.: (705) 759-were due to the altered expression of some of the genes5740, EXT. 2465; Fax: (705) 759-5700, E-MAIL: ARETNAKARA

NKGLFC.FORESTRY.CA involved in the moulting cycle (Retnakaran et al., 1995).

55

56 A. RETNAKARAN et al.

The effect of this ecdysone agonist in inducing the moult- and the material was force fed. Larvae that did not regur-gitate the material after recovery were placed in cupsing process in the sixth instar larva of the spruce bud-

worm, Choristoneura fumiferana Clem. (Lepidoptera: containing diet and were used at various time intervals.Larvae were fixed for Scanning Electron MicroscopyTortridicae) was studied. The sequence of ultrastructural

changes that occur in the newly ecdysed last larval instar (SEM) at 11, 23 and 47 h post treatment (pt). For Trans-mission Electron Microscopy (TEM), the treated larvaeas a result of ingesting this ecdysone analogue is

described. The structural and physiological changes were fixed at 1, 2, 3, 5, 7, 9, 11, 15 and 23 h pt.Untreated control larvae were fixed as moulting fifthobserved in the RH-5992 induced moult as compared to

the 20-hydroxyecdysone induced moult are discussed. instars (larvae with slipped heads that would moult intosixth instars 24 h later) and as sixth instars that rangedin age from 1 to 24 h. Although many stages were exam-MATERIALS AND METHODSined, ultrastructural details are reported only for moultingfifth instars and 2, 24, and 48 h old sixth instars.Insects

For examining the external morphology, treated andSpruce budworm larvae were reared on a meridic dietuntreated larvae that were 12, 24, and 48 h old sixth(McMorran, 1965) after the method of Grisdale (1970)instars were frozen and freeze dried. They were cementedat 22°C, 70% RH and a photoperiod of 18 h light andto aluminum stubs with silver paint, coated with6 h darkness. Moulting fifth instar larvae showing headgold/palladium to a thickness of 20 Å and observedcapsule slippage (Retnakaran, 1980) were separated andunder a Hitachi 570 S SEM operated at 15 KY. Themaintained in Petri dishes overnight. Newly moultedimage was magnified 50× and photographed using Ilfordsixth-instar larvae with white, untanned head capsulesPan F film with an ASA rating of 50.were collected and designated as 0 h sixth instars. The

For freeze-fracturing, larvae that were force fed 96 hlarvae thus selected were used at various intervals fromearlier with RH-5992, were fixed in glutaraldehyde/OsO4the 0 h collection time.as described in the next section. They were then washedin water, quick frozen-in liquid nitrogen and freeze-frac-Treatment of larvaetured at the mesothorax with a pre-cooled scalpel blade.An aqueous flowable formulation of RH-5992 (Fig. 1)The pieces were freeze-dried, mounted on aluminumprovided by Rohm and Haas, Philadelphia, PA wasstubs and coated with carbon/gold in an Edwards E-306diluted to 1 mg (active ingredient)/ml in water contain-Vacuum Coater. The fractured portions were examineding a red food colouring as a marker dye. Newly moultedin a Hitachi 570 S SEM at 20 KV and photographed assixth instar larvae with white heads (0 h) were alloweddescribed earlier.to tan and harden for an hour (1 h) and force fed 1 ml

of suspension containing 1 mg of active ingredient usingTEMa micro-applicator mounted under a binocular micro-

The larvae were fixed by injecting ice-cold 5% aque-scope (Percy-Cunningham et al., 1987; Retnakaran,ous glutaraldehyde in 0.05 m cacodylate buffer contain-1983; Retnakaran et al., 1989). A 3-cm glass needleing 0.01 m CaCl2 and 2% sucrose at pH 7.3. The meso-made with a 1-mm diameter glass tube attached to athoracic tergite was dissected 10 min after injection and250 ml glass syringe containing RH-5992, was gentlyfixed in fresh, ice-cold glutaraldehyde solution (same asinserted into the mouth of a cold-immobilized (0°C) larvadescribed earlier) for 3 h with gentle agitation in a rotaryshaker. The tissues were then washed twice with 0.05 mcacodylate buffer containing 10% sucrose at pH 7.3 andfixed in 1% OsO4 in 0.05 m cacodylate buffer containing4% sucrose. After washing in water, the tissues werestained en bloc in aqueous 2% uranyl acetate at 60°C inthe dark, dehydrated in ethanol and embedded in araldite(Locke et al., 1971). Thin, gray sections were surfacestained with lead citrate after the method of Venable andCoggeshall (1965). Sections were observed and photo-graphed in a Jeol 1200 Ex II. TEM operated at 80 kV.

RESULTS

Behaviour and morphology

Larvae that received RH-5992 per os started feedingon the diet, but within 3–4 h pt became lethargic andceased feeding. The anterior end was turned inward andFIGURE 1. Structures of the moulting hormone, 20-hydroxyecdysone

and the dibenzoyl hydrazine analogue, RH-5992. at 12 h head capsule separation was initiated. At 24 h pt

57ULTRASTRUCTURAL EFFECTS OF RH-5992

there was partial slippage of the head capsule with a new Ultrastructural changes induced by RH-5992‘white head’ underneath. At 48 h pt the old head capsule

At 1 h pt, the treated sixth-instar larvae resembled thewas loose and when removed with a needle revealed a

untreated control very closely with a distinct cuticulinwrinkled, soft, new head capsule that was white and

layer, dense epicuticle and approximately 5 endocuticularuntanned. Very little tanning except for the tips of thelamellae and organelles that were representative. [Fig.palps and mandibles was seen [Fig. 2 (A)—(H)].4(C)]. The earliest signs of possible RH-5992 effect wereFreeze-fracturing of the 96 h old, treated larvae in thenoticeable at 2 h pt. There was hypertrophy of the Golgimesothoracic region which had not yet ecdysed (althoughcomplex with numerous vesicles and the rough endoplas-the old head capsule could be removed), revealed the oldmic reticulum was pronounced [Fig. 5(A)]. The firstand new cuticles in the mesothoracic region as well as aclearly discernible and definitive effects of the compoundtrachea showing both the old and new intima [Fig. 2(I)].were present at 3 h pt. Large, putative ecdysial dropletsmade their appearance and, unlike lipid droplets, they

Ultrastructural changes in the untreated larva were found above the apical plasma membrane.Microvilli, dense plaques, coated vesicles, electron denseThe formation of the new, sixth instar integumentvesicles, electron lucent vesicles were all similar to thecould be observed in the moulting (pharate) fifth instarones in the controls. There were numerous Golgi com-larva. At this stage, apolysis had occurred and the oldplexes that were producing small electron dense vesiclesand new cuticles had separated but ecdysis had not takenand also the endoplasmic reticulum was well representedplace. Remnants of the old fifth-instar cuticle werewith an abundance of free ribosomes [Fig. 5(B)]. Somepresent as a relatively thin jacket with the endocuticularelectron dense vesicles as well as limited involutions ofarea being digested by ecdysial droplets revealing thethe basal plasma membrane with the lateral plasma mem-sixth instar, teneral cuticle underneath [Fig. 3(A)]. Thebrane showed some spreading that created intercellularnew sixth instar cuticle showed a well-formed epicuticlespaces (not shown).with its cuticulin and dense epicuticle layers. The apical

At 5 h pt the larvae had ecdysial droplets in a diffusedplasma membrane was beginning to show the formationstate and were less prominent [Fig. 5(C)]. The basal por-of microvilli. Endocuticular lamellae were not yet for-tion of the epidermal cell showed intercellular spaces andmed, but in its place, a fibrous layer could be observedbasal involution of the plasma membrane [Fig. 5(D)]. At[Fig. 3(B)]. The epidermal cells in the 2 h old sixth instar7 h pt, the larvae showed an ecdysial space between theappeared active with a full complement of intracellularapical plasma membrane and the endocuticle, theorganelles, a fully developed epicuticle with a distinctmicrovilli tended to smooth out and there was involutioncuticulin layer and a dense epicuticle with epicuticularof the basal plasma membrane [Fig. 6(A)]. The ecdysialfilaments, and at least five endocuticular lamellae. Thespace had some fibrous material and there were manyapical plasma membrane had numerous microvilli withactive multivesicular bodies. The intercellular spacesdense plaques at their tips. A fibrous material could becontained vesicles and flocculent material [Fig. 6(B)]. Atobserved emanating from the plaques and orienting9 h pt there was an increase in the size of the ecdysialtowards the lamella. Few multivesicular bodies, mito-space and the dissolution of the endocuticle had started.chondria and autophagic bodies but more electron lucentThe ecdysial droplets became diffused and were distrib-vesicles, active rough endoplasmic reticulum with freeuted in various regions of the endocuticle initiating theribosomes than the earlier stage were present. Variousdigestive process. The ecdysial space was filled withtypes of vesicles such as coated, electron dense and elec-fibrous material as a result of the massive dissolution oftron lucent were seen in moderate numbers [Fig. 3(C)].the endocuticle [Fig. 6(C)]. Ecdysial droplets could beNumerous short profiles of rough endoplasmic reticulum,observed in the crypts of the apical plasma membranefree ribosomes, Golgi complexes, some autophagic bod-[Fig. 6(D)]. The organelles were similar to the earlieries and average number of mitochondria were observedstage but the basal involutions and intercellular spacesin the body of the. epidermal cell by 24 h [Fig. 3(D) andwere enlarged [Fig. 7(A)].(E)]. Lateral plasma membrane showed moderate separ-

At 11 h pt the sixth-instar larvae showed massiveation near the base with intracellular spaces, some ofendocuticular dissolution with the ecdysial space beingwhich contained vesicles. The basal plasma membranefilled with fibrous material and the old larval cuticle pro-showed moderate involutions and the resting-stagegressively moving away [Fig. 7(B)]. The apical plasmanucleus was at the base of the cell (not shown).membrane was smooth for the most part with fewThe integument of the 24-h old sixth instar larva didmicrovilli. Coated vesicles sequestering plaque materialnot show any changes except an increase in the numberand moving into multivesicular bodies could beof endocuticular lamellae. By 48 h, the integumentobserved. Copious amounts of rough endoplasmic reticu-appeared fully developed with numerous endocuticularlum and free ribosomes were present [Fig. 7(C)].lamellae [Fig. 4(A)]. Many autophagic bodies and active

At 15 h pt the first signs of apolysis were evident withGolgi complexes were apparent [Fig. 4(B)]. Very fewthe remnant of the old cuticle separating, the ecdysialbasal involutions and few intercellular spaces could be

observed (not shown). space appearing less fibrous and the ecdysial droplets still


FIGURE 2. SEM view of the head capsule and thoracic regions of the untreated and RH-5992-treated sixth instar sprucebudworm. (A) Dorsal view of an untreated 24 h old larva showing a normal, hard and smooth head capsule. (B) Dorsal viewof a treated 48 h old larva with the loose old head capsule removed showing an abnormal, wrinkled and thin head capsule.(C) Ventral view of a treated 12 h old larva showing partial slippage of the old head capsule. (D) Ventral view of an untreated24 h old larva showing the mouth parts. (E) Ventral view of a treated 48 h old showing malformed mandibles. (F) Lateralview of a treated 12 h old larva showing head capsule slippage. (G) Lateral view of an untreated 24 h old larva showingocelli and palps. (H) Lateral view of a treated 48 h old larva showing the buckling of the thin new head capsule that wasexposed by removing the loose old head capsule. (I) Freeze fracture of the mesothoracic region of a 48 h old treated larvashowing the old and the new cuticles as well as the two layers of intima in the trachea. (Magnification: A–H ×100; I ×1400)


FIGURE 3. Ultrastructural changes in the epidermis of the untreated sixth instar spruce budworm. (A) Pharate sixth instar 24h before ecdysis into sixth instar showing remnants of the old cuticle and the epidermal cell with an undifferentiated endocuticle.(B) A close-up of the apical region showing the newly formed cuticulin layer and the microvilli secreting the fibrous cuticle.(C) The cytoplasmic region showing various cytoplasmic organelles such as electron lucent vesicles and multivesicular bodiesin a 2 h old sixth instar. (D) and (E) Cytoplasmic region of the 24 h sixth instar epidermal cell showing inter alia, activeendoplasmic reticulum, Golgi complex and vesicles. (Ab, autophagic body; bi, basal involution of plasma membrane; bm,basement membrane; cj, cell junction; cu, cuticulin; cv, coated vesicle; de, dense epicuticle; dep, dense plaque; des, desmosome;e, endocuticular lamellae; ed, ecdysial droplet; edv, electron dense vesicle; elv, electron lucent vesicle; es, ecdysial space; fc,fibrous cuticle; fm, fibrous material; fr, free ribosome; G, Golgi complex; ic, intercellular space; m, mitochondrion; mv,microvilli; mvb, multivesicular body; n, nucleus; oc, old cuticle; pc, pore canal; pm, plasma membrane; pv, pinocytic vesicle;

rer, rough endoplasmic reticulum.)


FIGURE 4. Ultrastructural changes in the integument of untreated 2 day old and 2 h old treated sixth instars. (A) A 48 h olduntreated epidermis showing a fully developed cuticle with numerous endocuticular lamellae and pore canals. (B) Apicalregion of the epidermal cell showing a large number of vesicles and autophagic bodies. (C) A 2 h old treated sixth instarintegument showing a well formed cuticulin layer, dense epicuticle and about five endocuticular lamellae as well as a fullcomplement of all the representative organelles. The structure was identical to the untreated 2 h old stage. (For abbreviations

see Fig. 3.)


FIGURE 5. Ultrastructural changes in 3–6 h old treated sixth instars. (A) Cytoplasmic region of a 3 h old sixth instar showinghypertrophied Golgi complex, the earliest indication of the possible effect of the ecdysone agonist. (B) Apical region of a 4h old integument showing large, putative ecdysial droplets, the first definitive effect of RH-5992. (C) The apical region of a6 h old integument showing the characteristic ecdysial droplet. (D) The basal portion of the cell showing intercellular spaces

and basal involution of the plasma membrane. (For abbreviations see Fig. 3.)


FIGURE 6. Ultrastructural changes in the 10 h old treated sixth instars. (A) Dissolution of the endocuticle and the formationof an ecdysial space. (B) The ecdysial space is filled with fibrous material and the multivesicular bodies are actively sequesteringmaterial. (C) Massive dissolution of the endocuticle occurs. (D) Ecdysial droplets in the crypts of the microvilli are released

into the endocuticular region. (For abbreviations see Fig. 3.)


FIGURE 7. Ultrastructural changes in the 10–16 h old treated sixth instars. (A) Basal region of a 10 h old larva showingenlargement of the basal involutions of the plasma membrane and intercellular spaces. (B) A 12 h old integument showingan enlarged ecdysial space and the old cuticle moving distally from the epidermal cell. (C) Apical region of the epidermalcell showing the coated vesicles sequestering plaque material and moving into multivesicular bodies. (D) A 16 h integumentshowing further separation of the old cuticle and the plasma membrane reorganizing into microvilli. (For abbreviations see

Fig. 3.)


digesting parts of the old cuticle [Fig. 7(D)]. The apical membrane. The characteristic appearance of these drop-lets, vesicles and Golgi [Fig. 5(C) and (D)] is preciselyplasma membrane was once again organized into

microvilli which were secreting a layer of cuticulin [Fig. as described by Locke and Krishnan (1973). At 7 h ptthere is dissolution of the endocuticular lamellae and the8(A)]. Multivesicular bodies, electron dense vesicles,

electron lucent vesicles and a large number of Golgi were beginning of the disappearance of the microvilli with theappearance of fibrous material in the ecdysial space [Fig.all evident. Basal involutions and intercellular spaces

were few in number (not shown). At 24 h pt the forma- 6(A) and (B)], which is similar to the description in Cal-podes ethlius (Locke, 1976). The process is carriedtion of a new cuticulin layer from the tips of the

microvilli of the apical plasma membrane could be seen further at 9 h pt [Fig. 6(C) and (D); 7(A)), and at11 h pt, the ecdysial space is filled with fibrous material,[Fig. 8(B)]. The old cuticle had separated and the ecdys-

ial space appeared electron lucent. The basal involutions which is probably the partially degraded endocuticularmaterial on its way to the vesicular bodies where it isand intercellular spaces were fewer in number and the

nuclear membrane showed enlarged pores (not shown). broken down further [Fig. 7(B) and (C)] (Locke, 1969;Locke and Sykes, 1975). Apolysis starts at 15 h pt [Fig.At 48 h pt there was wide separation of the new and

old cuticles, with the old one lying outside the field of 7(D)] with the formation of a new cuticulin layer (Locke,1976). At 24 h pt the secretion of the new cuticle is com-observation. The new epicuticle was fully formed with

distinct cuticulin and dense epicuticle layers. The endo- plete and there is further separation of the old cuticlewith an expansion of the ecdysial space [Fig. 8(B)]. Thecuticular lamellae were conspicuously absent and in their

place a fibrous material could be seen [Fig. 8(C)]. The breakdown products are primarily processed in the multi-vesicular bodies (MVB) with the help of enzymes thatmicrovilli appeared to make an occasional abortive

attempt at secreting endocuticular lamellae [Fig. 8(D)]. originate from the Golgi complex [Fig. 7(C)], (Locke,1976). Recently, other autophagic bodies away fromThe basal plasma membrane had little or no involutions.

The Golgi complexes were secreting electron dense ves- endoplasmic reticulum have also been implicated in thecatabolic process (Furuno et al., 1990). At 15 h pt, apo-icles and material was being transferred into the cell by

pinocytosis [Fig. 8(E)]. lysis is well on its way with the ecdysial space becomingelectron lucent and the apical plasma membrane beingreorganized into microvilli that actively secrete the cutic-

DISCUSSIONulin layer. At this stage the Golgi complex and MVB areactive [Fig. 7(D); 8(A)]. At 24 h pt, the process is carriedThe changes in the integument from the initiation of

the moulting cycle at the end of the fifth instar to the further [Fig. 8(B)] and at 48 h pt, a new cuticle is formedbut without the endocuticular lamellae. A feeble attemptformation of a full fledged cuticle in the 48 h sixth instar

are similar to those observed in several other arthropods to secrete an endocuticular lamella could be seen [Fig.8(C) and (D)].(Locke, 1976; Binnington, 1985; Filshie, 1970; Sedlak

and Gilbert, 1979; Mothes-Wagner and Seitz, 1984). The Distinct stages of ecdysis into anecdysis, proecdysisand metecdysis, as described in crustaceans (Stringfellowrapid increase in the formation of the endocuticular

lamellae follows closely the formation of chitin during and Skinner, 1988), are not clearly delineated in thespruce budworm but instead there was a gradual trans-the initial 48 hr (Retnakaran et al., 1989). Force feeding

RH-5992 to newly moulted sixth-instar spruce budworm formation without any distinct landmarks. The RH-5992induced moult closely resembles the early stages of theinduced an incomplete moult resulting in the formation

of a new, untanned larval head capsule in 48 h. Since the natural moulting process induced by 20-hydroxyecdy-sone (Locke, 1976; Quennedey et al., 1983; Sedlak andtreatment was before the commitment peak of ecdystero-

ids (Retnakaran, 1973; Riddiford, 1985), the new cuticle Gilbert, 1979). The major differences are the lack of tan-ning and the absence of endocuticular lamellae forma-secreted was larval [Figs 3–9]. The temporal sequence

of ultrastructural changes brought about by the ingestion tion. Ecdysis, the process in which the old cuticle is shedby peristaltic movement of the larva, is also absent.of RH-5992 was similar to the early stages of moulting.

Since the treatment with this compound was on newly Ecdysteroids appear as a distinct peak during eachinstar and regulate the process of moulting and metamor-ecdysed sixth instar larvae, the effect manifested was at

a time when the 20-hydroxyecdysone-induced moulting phosis by controlling the selective expression of genes(Smith, 1985). Studies on cloning, characterization andcycle was almost complete. There were no discernible

effects for the first 2 h after treatment [Fig. 4(C)] but hormonal regulation of key regulatory moleculesinvolved in moulting and metamorphosis (reviewed byat 2–3 h pt, numerous active Golgi complexes and large

putative ecdysial droplets were evident [Fig. 5(A) and Riddiford and Truman, 1993) can provide explanationsfor some of the differences in the effects of 20-hydrox-(B)]. The presence of these droplets above the apical

plasma membrane and their appearance were quite unlike yecdysone and RH-5992. A summary of the variousevents that occur during the moulting process, based onthe lipid droplets in pheromone glands (Percy, 1979). In

all probability they are ecdysial droplets at the very early the Ashburner model (Ashburner et al., 1974) ispresented in Fig. 9. Recent studies on Drosophila mel-stage of formation. At 5 h pt, these droplets take on a

different shape and move away from the apical plasma anogaster (reviewed by Thummel, 1995) and other


FIGURE 8. Ultrastructural changes in the 16–49 h old treated sixth instars. (A) Apical region of a 16 h old epidermal cellshowing the microvilli secreting the new cuticulin layer. (B) Apical region of the epidermal cell of a 25 h old sixth instarshowing coalescence of the cuticulin precursors to form a continuous layer. (C) A 49 h old integument with a new, RH-5992induced cuticle that has no endocuticular lamellae. (D) Apical region of the epidermal cell showing microvilli making anabortive effort to form an endocuticular lamella. (E) The basal plasma membrane showing transfer of material into the cell

by pinocytosis. (For abbreviations see Fig. 3.)


FIGURE 9. Molecular and physiological events during a normal, 20-hydroxyecdysone-induced moult and a precocious, RH-5992-induced incomplete moult. CHR75, Choristoneura hormone receptor 75 (homologue of E75); CHR3, Choristoneurahormone receptor 3 (homologue of DHR3 and MHR3); DDC, dopadecarboxylase; EcR, ecdysone receptor; LCP 14, larvalcuticle protein -14 kDa; USP, ultraspiracle. (This figure is based on data from Ashburner et al., 1974; Thummel, 1995; Palliet al., 1996a; Hiruma et al., 1991; Hiruma and Riddiford, 1990; Palli et al., 1995, 1996b; Retnakaran et al., 1995; Retnakaranand Oberlander, 1993. The ecdysone titre is based on Palli et al., 1995 and the RH-5992 persistence is based on unpublished

results of Retnakaran, Hiruma and Riddiford.)

insects indicate that 20-hydroxyecdysone binds to the ford, 1990). The mRNAs for most of the larval cuticularproteins are expressed during the intermoult period whenEcR/USP complex to induce the expression of a set of

‘early’ genes such as E74 (Thummel et al., 1990), E75 there is little or no ecdysteroid. When the moulting hor-mone level is high, the expression of these mRNAs is(Segraves and Hogness, 1990) and Broad complex genes

(Guay and Guild, 1991). The products of early genes are suppressed (Riddiford et al., 1986). Thus, ecdysteroidsregulate a moult through a receptor complex to activateecdysteroid regulated transcription factors and therefore

they are capable of regulating their own expression as a number of regulatory genes whose products in turnregulate the expression of genes that are responsible forwell as the expression of other late genes such as the

ones that code for cuticular proteins. Studies on Manduca the formation of the next instar in a cascading fashion(Riddiford and Truman, 1993). Early genes have differ-sexta showed that expression of some of these genes such

as dopadecarboxylase (DDC) require the exposure to ent isoforms that have a different time-course and dose–response to 20-hydroxyecdysone (Karim and Thummel,ecdysteroids during the moult to programme their later

expression, but subsequent decline of ecdysteroids is 1992). Thus, it is not only the ecdysteroid peaks but alsothe rise and fall of these peaks that determine theessential for this expression to occur (Hiruma and Riddi-


hornworm by 20-hydroxyecdysone and juvenile hormone. Dev.expression of specific genes at appropriate times duringBiol. 138, 214–224.moulting.

Karim F. D. and Thummel C. S. (1992) Temporal coordination ofDuring the early stages of the moulting process, RH- regulatory gene expression by the steroid hormone ecdysone.

5992 mimics the effects of 20-hydroxyecdysone and the EMBO J. 11, 4083–4093.Kothapalli R., Palli S. R., Ladd T. R., Sohi S. S., Cress D., Dhadiallaresults of both these compounds on gene expression are

T. S., Tzertzinis G. and Retnakaran A. (1995) Cloning and develop-indistinguishable. These early effects are probably relatedmental expression of ecdysone receptor from the spruce budworm,to the rising phase of the ecdysteroid peak and RH-5992Choristoneura fumiferana. Dev. Gent. 17, 319–330.

mimics this effectively. Examples of these effects include Locke M. (1969) The structure of the epidermal cell during the forma-ultrastructural changes during early stages of the moult- tion of the protein epicuticle and the uptake of the moulting fluiding process (this work), induction of C. fumiferana ecdy- in an insect. J. Morphol. 127, 7–40.

Locke M. 1976. The role of plasma membrane plaques and Golgi com-sone receptor (CfEcR, Kothapalli et al., 1995), Manducaplex vesicles in cuticle deposition during the moult/intermoulthormone receptor 3 (MHR3, Palli et al., 1992; Retnaka-cycle. In: The Insect Integument (ed. Hepburn H. R.), Chap. 13,ran et al., 1995), Choristoneura hormone receptor 3pp. 237–258. Elsevier, Amsterdam.

(CHR3, Palli et al., 1995, 1996a), Choristoneura hor- Locke M. and Krishnan N. (1973) The formation of the ecdysial drop-mone receptor 2 (CHR75, Palli et al., 1996b) and Mala- lets and the ecdysial membrane in an insect. Tissue Cell 5, 441–

450.cosoma hormone receptor 3 (MdHR3, Sohi et al., 1995).Locke M., Krishnan N. and MacMathon J. T. (1971) A routine methodThe difference in the effects of 20-hydroxyecdysone and

for obtaining high contrast without staining sections. J. Cell Biol.RH-5992 probably starts at a stage in the moulting cas-50, 540–544.

cade where the falling phase of the ecdysteroid peak is Locke M. and Sykes A. K. (1975) The role of Golgi complex in theimportant. Cuticular protein genes such as LCP14 and isolation and digestion of organelles. Tissue Cell 7, 143–158.

McMorran A. (1965) A synthetic diet for the spruce budworm. Choris-genes coding for enzymes necessary for cuticular melan-toneura fumiferana (Lepidoptera: Tortricidae). Can. Ent. 97, 58–ization and sclerotization such as DDC are not expressed62.in the presence of RH-5992 (Retnakaran et al., 1995).

Mothes-Wagner U. and Seitz K.-A. (1984) Fine structure of the cuticleUpon ingestion, RH-5992 is metabolized and most of it and structural changes occurring during moulting in the miteis excreted but enough remains in the tissues to cause Tetranychus urticae I. Fine structure of the cuticle. Acaralogia 25,the suppression of genes that require ecdysteroid with- 253–258.

Palli S. R., Hiruma K. and Riddiford L. M. (1992) An ecdysteroid-drawal for their expression (Retnakaran, unpubl. data).inducible Manduca gene homologous to Drosophila DHR3 gene,Chitin is not synthesized (Retnakaran and Oberlander,a member of the steroid hormone receptor superfamily. Dev. Biol.1993) and eclosion hormone is not released in the pres- 150, 306–318.

ence of RH-5992 (Retnakaran and Truman, unpubl. Palli S. R., Primavera M., Lambert D. and Retnakaran A. (1995) Agedata). The result of this failure in the expression of genes specific effects of a non-steroidal ecdysone agonist, RH-5992 on

the spruce budworm Choristoneura fumiferana (Lepidoptera:that require ecdysteroid withdrawal is manifested in theTortricidae). Eur. J. Entomol. 92, 325–332.formation of an incomplete cuticle, absence of ecdysis

Palli S. R., Ladd T. R., Sohi S. S., Cook B. J. and Retnakaran A.and a lack of tanning as shown in this study.(1996a). Cloning and developmental expression of Choristoneurahormone receptor 3, an ecdysone-inducible gene and a member ofthe steroid hormone receptor superfamily. Insect Biochem. Mol.

REFERENCESBiol. (in press).

Palli S. R., Ladd T. R., Sohi S. S., Ricci A. R. and Retnakaran A.Ashburner M., Chihara C., Meltze P. and Richard G. (1974) Temporal(1996b). Cloning and developmental expression of Choristoneuracontrol of puffing activity in polytene chromosomes of Drosophilahormone receptor 75, a homologue of the Drosophila E75. A gene.melanogaster. Cold Spring Harbor Symp. Quat. Biol. 38, 655–662.Dev. Gent. (in press).Binnington K. C. (1985) Ultrastructural changes in the cuticle of the

Percy J. (1979) Development and ultrastructure of sex pheromonesheep blowfly, Lucilia, induced by certain insecticides and biologi-gland cells of the cabbage looper moth, Trichoplusia ni (Hubner)cal inhibitors. Tissue Cell 17, 131–140.(Lepidoptera: Noctuiidae). Can. J. Zool. 57, 220–236.Filshie B. K. (1970) The fine structure and deposition of the larval

Percy-Cunningham J., Nicholson D. and Retnakaran A. (1987) Thecuticle of the sheep blowfly (Lucilia cuprina). Tissue Cell 2,effect of ingested benzoylphenylurea on the ultrastructure of the479–489.cuticle deposited during the last larval instar of Choristoneura fumi-Furuno K., Ishikawa I., Akasaki K., Lee S., Nishimura Y., Tsugi H.,ferana Clem. (Lepidoptera: Tortricidae). Can. J. Zool. 65, 2715–Himeno M. and Kato K. (1990) Immunocytochemical study of the2723.surrounding envelope of autophagic vacuoles in cultured rat hepa-

Quennedey A., Quennedey B., Delbeque J.-P. and Delachambre J.tocytes. Exp. Cell Res. 189, 261–268.(1983) The in vitro development of the pupal integument and theGrisdale D. (1970) An improved laboratory method for rearing largeeffects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera).numbers of spruce budworm. Choristoneura fumiferanaCell Tiss. Res. 232, 493–511.(Lepidoptera: Tortricidae). Can. Ent. 102, 1111–1117.

Retnakaran A. (1973) Hormonal induction of supernumerary instarsGuay P. S. and Guild G. M. (1991) The ecdysone induced puffingin the spruce budworm. Choristoneura fumiferana (Lepidoptera:cascade in Drosophila salivary glands: A broad complex early geneTortricidae). Can. Ent. 105, 459–461.regulates intermoult and late gene transcription. Genetics 129,

Retnakaran A. (1980) Effects of three new moult inhibiting insect169–175.growth regulators on the spruce budworm. Choristoneura fumifer-Hiruma K., Hardie J. and Riddiford L. M. (1991) Hormonal regulationana (Clem.). J. Econ. Ent. 73, 520–524.of epidermal metamorphosis in vitro: control of expression of a

Retnakaran A. (1983) Spectrophotometric determination of larvallarval specific cuticle gene. Dev. Biol. 144, 369–378.ingestion rates in the spruce budworm (Lepidoptera: Tortricidae).Hiruma K. and Riddiford L. M. (1990) Regulation of dopa decarboxy-

lase gene expression in the larval epidermis of the tobacco Can. Ent. 115, 31–40.


Retnakaran A., MacDonald A., Nicholson D. and Percy-Cunningham that are responsive to ecdysone and ecdysone agonists. J. InsectPhysiol. 41, 457–464.J. (1989) Ultrastructural and autoradiographic investigations of the

Steel C. G. H. and Davey K. G. (1985). Integration in the insect endo-interference of Chlorfluazuron with cuticle differentiation in thecrine system. In: Comprehensive Insect Physiology, Biochemistryspruce budworm, Choristoneura fumiferana. Pest. Biochem. Phy-and Pharmacology (eds Kerkut G. A. and Gilbert L. I.) Vol. 8,siol. 35, 172–184.Chap. 1, pp. 1–35. Pergamon, Oxford.Retnakaran A., Hiruma K., Palli S. R. and Riddiford L. M. (1995)

Stringfellow L. A. and Skinner D. M. (1988) Molt-cycle correlatedMolecular analysis of the mode of action of RH-5992, a lepidop-patterns of synthesis of integumentary proteins in the land crab,teran-specific, non-steroidal ecdysteroid agonist. Insect Biochem.Gecarcinus lateralis. Dev. Biol. 128, 97–110.Mol. Biol. 25, 109–117.

Thummel C. S. (1995) From embryogenesis to metamorphosis: theRetnakaran A. and Oberlander H. (1993). Control of chitin synthesis inregulation and function of Drosophila nuclear receptor superfamilyinsects. In: Chitin Enzymology (ed. Muzzarelli R. A. A.). European.members. Cell 83, 871–877.Chitin Society, Ancona.

Thummel C. S., Burtis K. C. and Hogness D. S. (1990) Spatial andRiddiford, L. M. (1985). Hormone action at the cellular level. In: Com-temporal patterns of E74 transcription during Drosophila develop-parative Insect Physiology, Biochemistry and Pharmacology (edsment. Cell 61, 101–111.Kerkut G. A. and Gilbert L. I.), Vol. 8, Chap. 2, pp. 37–84. Perga-

Venable J. H. and Coggeshall R. (1965) A simplified lead citrate stainmon, Oxford.for use in electron microscopy. J. Cell Biol. 25, 407.Riddiford L. M., Baeckmann A., Hice R. H. and Rebers J. (1986)

Wing K. D. (1988) RH-5849, a nonsteroidal ecdysone agonist: effectsDevelopmental expression of three genes for larval cuticular pro-on a Drosophila cell line. Science 241, 467–469.teins of the tobacco hornworm, Manduca sexta. Dev. Biol. 118,

Wing K. D., Slawecki R. A. and Carlson G. R. (1988) RH-5849, a82–94.nonsteroidal ecdysone agonist: effects on larval lepidoptera.Riddiford L. M. and Truman J. W. (1993) Hormone receptors and theScience 241, 470–472.regulation of insect metamorphosis. Amer. Zool. 33, 340–347.

Williams C. M. (1980) Growth in insects. In: Insect Biology in theSedlak B. and Gilbert G. J. (1979) Correlations between epidermal cellFuture (Eds Locke M. and Smith D. S.), pp. 369–383. Academicstructure and endogenous hormone fiters during the fifth larvalPress, New York.instar of the tobacco hornworm, Manduca sexta. Tissue Cell 11,

Zacharuk R. Y. (1976) Structural changes of the cuticle associated with643–653.moulting. In: The Insect Integument (Ed. Hepburn, H. R.), pp. 299–Segraves W. A. and Hogness D. S. (1990) The E75 ecdysone-inducible321. Elsevier, Amsterdam.gene responsible for the 75B early puff in Drosophila encodes two

new members of the steroid receptor superfamily. Genes Dev. 4,204–219.

Smith, S. L. (1985). Regulation of ecdysteroid titer: synthesis. In: Acknowledgements—The gift of RH-5992 by Rohm and Haas Co. isComprehensive Insect Physiology, Biochemistry and Pharma- gratefully acknowledged. This study was supported by the Canadiancology (eds Kerkut G. A. and Gilbert L. I.), Vol. 7, Chap. 8, pp. Forest Service and a grant from Rohm and Haas Co., Spring House,295–341. Pergamon, Oxford. PA, USA. We thank Dr S. S. Sohi and Mrs K. Jamieson for critical

comments on the manuscript.Sohi S. S., Palli S. R. and Retnakaran A. (1995) Forest insect cell lines

Documents

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist, RH-5992, on the Sixth Instar Larva of the Spruce Budworm, Choristoneura fumiferana