Synchrony of juvenile hormone-sensitive periods for internal and external development in last-instar larvae of Oncopeltus fasciatus

J. Insect Physiol. Vol. 28, No. 9, pp. 797 to 803, 1982 Printed

0022-1910/82/090797-06$03.00/O Q 1982 Peryamon Press Ltd

in Great Britain.

SYNCHRONY OF JUVENILE HORMONE-SENSITIVE PERIODS FOR INTERNAL AND EXTERNAL DEVELOPMENT IN LAST-INSTAR LARVAE OF ONCOPELTUS FASCZATII’S WENDY A. SMITH* and H. FREDERIK NIJHOUT Dept. of Zoology,

Duke University,

Durham,

NC 27706. U.S.A.

(Received 9 March 1982) Abstract--Breakdown of the moulting glands in Oncopeltus can be completely inhibited by topical application of a juvenile hormone analogue prior to day 2 of the fifth instar. and partially inhibited by application prior to day 4. The analogue-sensitive period for the inhibition of external metamorphosis is very similar to that for the inhibition of cell death in the moulting glands. A decline in response to the analogue between days 2 and 4 is correlated with rising ecdysteroid levels in the haemolymph. That this rise in ecdysteroids may be responsible for termination of the juvenile hormone-sensitive period is suggested by premature loss of sensitivity to the analogue by the moulting glands following premature exposure to 20-hydroxyecdysone. Kry Word lndrz: Juvenile

hormone

analogue.

cell death.

INTRODUCTION THE ABILITYof juvenile hormone to inhibit metamorphosis is restricted to a specific part of the moulting cycle known as the juvenile hormone-sensitive period or critical period (WILLIS, 1974: NIJHOUT and WHEELER, 1982). In most insects studied to date, termination of the juvenile hormone-critical period is associated with the onset of ecdysone secretion. For example, epidermal sensitivity to juvenile hormone in pupae of Antheraen polyphemus (WILLIAMS,1956) and Hyalophora cecropia (WILLIAMS,1961). and in lastinstar larvae of Oncopeltusjhscicrtus (LAWRENCE,1969) and Bombyx mori (CALVEZ.1981) appear to end soon after the initiation of a new moulting cycle. During the last-larval instar of some insect species, termination of the juveile hormone-sensitive period for epidermal cells has been linked with a brief period of exposure to ecdysone, prior to that which initiates moulting (Manduca sexta, TRUMANet al., 1974; MITSUI and RIDDIFORD,1978; RIDDIFORD,1978; Aeschnc~ cyuneu. ANDRIES, 1979; and Galleria mellonella, HWANG-HSUet al.. 1980). In the absence of juvenile hormone this small pulse of ecdysone also appears to be responsible for a larval-pupal switch in the developmental programme (TRUMAN et al., 1974; MITSUI and RIDDIFORD.1978; RIDDIFORD,1978; LAFONT et al.. 1977; HWANG-HSUet al.. 1980; DEANet al., 1980). Juvenile hormone has been demonstrated to inhibit metamorphic changes in many internal tissues (muscle. LOCKSHINand WILLIAMS,1964; gut, JUDY and GILBERT.1970: fat body, SASSand KOVACS,1975; TOJO et ~1.. 1981; salivary glands, RICHARDS.1978; Malpighian tubules, RYERSE,1980; moulting glands, WIGGLESWORTH, 1955; Gilbert, 1962; OBERLANDER et ul., 1965; OZEKI. 1968; GASSIERand FAIN-MAUREL, 1970; LANZREIN,1975). However, few studies have focused on the timing of juvenile hormone-sensitive * Author to whom correspondence

should

be addressed.

Oncopeltusfusciatus

periods in non-epidermal cells, particularly with respect to changes in circulating ecdysone levels. Juvenile hormone-critical periods for a variety of internal organs in last-instar larvae of Gal/et% me/lone/la (SEHNAL, 1968) and pupae of Hyalophora cecropia (WILLIAMS,1961) occur prior to the initiation of pharate development, in synchrony with the integument. Sensitivity to juvenile hormone in the Malpighian tubules of last-larval Cafpodes ethfius (RYERSE.1980) and the fat body of last-larval Galleria me/lone/la MCCALEBand KUMARAN,1980). Mamestrrr brussicae (SASSand KOVACS,1975), and Bornby mori (TOJO et ul., 1981) clearly decline in conjunction with initial exposure to ecdysone in the absence of the hormone. However, RIDDIFORD(1972) has reported that in Hya/ophoru cecropia, juvenile hormone is most effective in preserving larval viscera when applied during the prepupal stage, long after the epidermal juvenile hormone-sensitive period has ended and presumably coincident with a moult-inducing peak of ecdysone. In the present paper we have examined the ability of juvenile hormone analogue to inhibit cell death in the moulting glands of Oncope/tus,fusciatus during the fifth (final)-larval instar. In particular we were interested in answering the following questions: (a) Does the end of the juvenile hormone-critical period for inhibition of cell death coincide with that for external characters? (b) Is the end of this critical period correlated with a change in haemolymph ecdysteroid levels‘? and (c) Does ecdysone play an active role in altering the sensitivity of the moulting glands to juvenile hormone? MATERIALS

AND

METHODS

Insects Oncopeltus ftisciatus were reared at 27 k 0.5”C under a l6L: 8D photoperiod. Water and dried milkweed seeds were supplied ud libitum. The fifth-nym-

797

WENDY A. SMITHand H. FREDERKNIJHOUT

798 phal instar under these conditions days (SMITH and NIJHOUT.1981).

lasts approx.

75

Hormorws The juvenile hormone analogue ZR-515 (methoprene; isopropyl-11-methoxy-3,7-trimethyl-2.4 didecadienoate; Zoecon Corp., Palo Alto, CA) was dissolved in acetone, and 1~1 aliquots were applied to the prothorax of fifth-instar nymphs at known times following ecdysis from the fourth to the fifth instar. Control animals received a 1 pl dose of acetone alone. 20-Hydroxyecdysone (Sigma Chemical Corp., St. Louis. MO) was dissolved in saline of the following composition: NaCl, 100mM; KCI, 10mM; CaCI,. 160 mM ; PIPES 5 mM; MgCI,. 5 mM: Sucrose. buffer, 15 mM; pH 6.8. Injections were made through the cut end of a metathoracic femur with the use of a Hamilton syringe attached to a 26-gauge needle. Each animal received a 2~1 injection of 20-hydroxyecdysone solution or a similar volume of saline alone.

Dissected moulting glands were fixed overnight in Bouin’s fixative and rinsed for 15-50 min in 709; ethanol to remove the resulting yellow stain. Glands were stained for IO-15 min in lo”,, Delafield’s haematoxylin, dehydrated in ethanol. cleared in cedarwood oil, and mounted on microscope slides in Permount.

RESULTS

We first examined the ability of ZR-515 to inhibit external signs of metamorphosis. Topical applications of 5 pg of the analogue or of acetone were made immediately following ecdysis from the fourth to the fifth instar (day 0). or 24. 48, 72. 96 or 120 hr later (days I. 2. 3, etc.). Effects of the analogue were evaluated at the time of subsequent ecdysis. or in cases where treated animals were unable to escape from the old cuticle, at the time of resorption of the ecdysial fluid. In such cases, the old cuticle was gently removed with forceps prior to the evaluation of external characters. A simple scoring system, based on criteria illustrated by NOVAI< (1951) and BOWERS(1968), was used to evaluate the external effects of ZR-515

Table

0

1

2

3

1. Scoring system for evaluating external juvenile hormone analogue treatment

effects of

Normal adult. Adultoid. Wings black/yellow extending to lastabdominal segment: posterior edge of pronotum concave: thoracic pleurites >f black; ventral abdominal spots diffuse; well developed genitalia. Juvenoid. Wings black extending to fourthabdominal segment or less; posterior edge of pronoturn Rat: scutellum. thoracic pleurites
Day 01 JHA Treatment [Fifth lnstarl Ftg. I. Effects of 5 or 5Opg juvenile hormone analogue (JHA) on external development following application at daily intervals in the fifth instar. External appearance at the time of subsequent ecdysis was evaluated according to criteria described in Table I. Sample sizes days &5 respectively: 5J
12. 10, 20. 30, 10. (Table I). We chose this scoring method, as opposed to the detailed analysis of small areas of cuticle described by LAWRENCE (1969), as we were interested in estimating the length of the juvenile hormone-critical period for a broad sample of cuticular characters. Acetone treatment at any time in the fifth instar had no developmental effects. Results of treatment with 5 pg juvenile hormone analogue at daily intervals in the fifth instar are shown in Fig. 1. All animals exposed to the analogue on day 0 emerged as supernumerary sixth nymphs (Score 3). Nymphal-adult intermediates were obtained following analogue treatment on days 1, 2 and 3 (Scores 1 and 2). Following application on day 4, only one of 16 animals showed any nymphal characters at the subsequent ecdysis and following treatment on day 5, all animals emerged as normal adults (Score 0). We repeated the above series of experiments using a 5Opg dose of ZR-515 (Fig. 1). Results of treatment with this enormous dose were virtually identical to those obtained with the lower dose, indicating that the endpoint of the juvenile hormone-sensitive period during day 4 was a characteristic of the epidermis and not of the dose of hormone employed. 6ffLcf.s 0f’jurenik hormone trrwlogw 011 cell drath irf thr morrltiny glands To determine the effects of ZR-515 on breakdown of the moulting glands, experimental and control animals described in the preceding experiments were maintained for 5-10 hr following evaluation of external characters; moulting glands were then removed and prepared as whole mounts. Moulting glands removed from normal adults at a similar time are in an advanced stage of necrosis (SMITH and NIJHOUT, 1982).

In whole-mount preparations, intact glands consisted of individually distinct cells with large nuclei surrounded by a narrow band of cytoplasm (Fig. 2a). By contrast. degenerate glands consisted solely of darkly stained, irregular cell fragments, held in place

799

Fig. 2. Whole mounts of moulting glands following topical application of 5 pg JHA during the fifth instar. Moulting glands were removed at the time of subsequent ecdysis. mg = moulting gland, tr = trachea. (a) JHA day 0. Intact gland; cell death completely inhibited. (b) JHA day 4. Degenerate gland. Scale as in 2A. (c) JHA day 2. Cell death partially inhibited; note degenerate cells (arrows) interspersed with intact cells. Scale as in 2A.

Juvenile

0

I23

45 Day

hormone-sensitive

012345

of JHA treotment (fifth instar)

Fig. 3. Effects of 5 or 5Opg juvenile hormone analogue (JHA) on cell death in the moulting glands following application at daily intervals in the fifth instar. Moulting glands were prepared as whole mounts at the time of subsequent ecdysis and scored as ~-9096 intact. >50”; intact, or degenerate (DEG.. < 50”; intact). Sample sizes as in Fig. I.

by the basement membrane (Fig. 2b). In some cases, both intact and degenerate cells were present within the same gland (Fig. 2~). In such glands, lengths of degenerate and intact portions were measured with the use of an ocular micrometer, and the glands were designated as > 90% intact, > 50% intact or degenerate ( < 50% intact). The effects of topical application of 5 pg ZR-515 on the moulting glands are shown in Fig. 3. Following application of the analogue on days 0 and 1, all moulting glands remained > 90% intact at the time of subsequent ecdysis. When applications of analogue were made on days 2 and 3, large numbers of partially degenerate (> 50”//,intact) and degenerate glands were obtained. By day 4. only one animal retained partially intact moulting glands at the time of ecdysis to an adult-the same individual that emerged as a nymphal-adult intermediate in Fig. 1. Following application on day 5, all moulting glands were degenerate at the time of emergence. Application of 50 pg ZR-5 15 increased the period of maximal sensitivity by about 1 day, but did not extend the endpoint of juvenile hormone analogue-sensitivity for the moulting glands beyond day 4 (Fig. 3). Eficts of ecdvsone OFItiming analope-sensitive period

of

the

juvenile hormone

Sensitivity of the moulting glands to ZR-515 declined sharply during days 2 and 3 of the fifth instar. Table

periods

As seen in Fig. 4, this decline was correlated with a rise in haemolymph ecdysteroid levels. Sensitivity to the analogue was completely lost by the time peak ecdysteroid levels were attained (day 4)) suggesting that increased ecdysteroid levels in the absence of ZR-5 15 were responsible for terminating subsequent sensitivity to the analogue. To test this hypothesis, we injected animals with 20-hydroxyecdysone on day 1 of the fifth instar, 3 days prior to the time when the moulting glands would normally be exposed to high levels of ecdysteroids (Fig. 4). Twenty-seven animals were injected with 5 pg 20-hydroxyecdysone, while 27 control animals were injected with an equal volume of saline alone. On day 2, both groups received topical application of 50 pg ZR-515. a dose that would normally be highly effective in inhibiting degeneration of the moulting glands. As seen in Table 2, animals injected with saline exhibited a normal sensitivity to the analogue on day 2 of the fifth instar (859; of glands >90”/; intact at the time of subsequent ecdysis). By contrast, animals prematurely exposed to 20-hydroxyecdysone showed a significantly reduced sensitivity to ZR-515 (18% of glands >90:& intact). Thus, premature exposure to 20-hydroxyecdysone resulted in a premature loss of sensitivity to the analogue by the moulting glands. Correspondingly, saline-injected nymphs emerged with nymphal external characters, whereas the external appearance of ecdysone-injected nymphs was adultoid.

DISCUSSION Results of the present study directly demonstrate an inhibitory role of juvenile hormone analogue on involution of the moulting glands in Oncopeltus at the time of metamorphosis. These results complement a previous report of premature breakdown of the moulting glands following precocene-induced destruction of the corpora allata (MASNERet al., 1979). Of additional interest, however, is our finding that the juvenile hormone-critical period for inhibition of cell death in the moulting glands is remarkably similar to that for the inhibition of ‘progressive’ changes in the integument. In both tissues ZR-515 is most effective when applied during days 0 and 1 of the fifth instar. A decline is seen in analogue-sensitivity during days 2 and 3, and the hormone becomes completely ineffective by day 5. regardless of the dose employed. These results suggest that cellular re-programming in both

2. Effects of exposure to 20-hydroxyecdysone moulting glands to juvenile hormone Treatment Day 1

20-hydroxyecdysone (5 Pg/2 PI) Saline (2 fll)

Treatment Day 2 JHA (50 fig) JHA (50 fig)

801

in Oncopeltus

on subsequent sensitivity analogue (JHA)

State of moulting glands at emergence > 509, intact > 90% intact Degenerate 18”/”

26”,

85Pb

1lU;

56% 4”/’0

of the

n 27 27

Fifth nymphs were injected with 20-hydroxyecdysone or with saline on day 1 of the instar; both groups received 5Opg ZR-515 on day 2. At emergence moulting glands were removed, prepared as whole mounts, and scored as in Fig. 3. Chi-square analysis indicates significantly fewer animals with >90% intact glands (P
802

WENDY

A.

SMITH and H. FREDERIKNIJHOUT

Fig. 4. Inverse correlation of juvenile hormone analogue-sensitivity with haemolymph ecdysteroid levels. Solid line represents haemolymph ecdysteroid concentrations at the time of analogue application (IO-hydroxyerdysone equivalents; data from SMITH and NIJHOUT.1981). Circles indicate percentage of glands >90”,, Intact at the time of subsequent ccydsis (data from Fig. 3). Closed circles: 5 fig ZR-5 15: open circles. 50 pg ZR-5 15.

tissues is carried out between days 2 and 4 of the last-larval instar. with commitment to the adult stage being completed by day 5. The temporal agreement between the juvenile hormone-critical periods for external and internal tissues is similar to that reported for last-larvae of Gollrric~ mellonella (SEHNAL. 1968; MCCALEB and KUMARAN, 1980) and Calpnrles etl~lius (RYERSE, 1978: DEAN et al., 1980), and pupae of HJW lophora cem~piu (WILLIAMS. 1961). However. in larvae of H. cecropiu. the juvenile hormone-critical period

for preservation of larval viscera occurs quite late in the last-larval instar (post spinning) in contrast to the early sensitivity of the epidermis (prior to gut evacuation) (Riddiford. 1972). Riddiford has suggested that the extended diapduse undergone by cecropia pupae may obviate the need for simultaneous commitment of internal and external organs in this species. ZR-515 application during days 2 and 3 of the fifth instar frequently led to a mosaic of degenerate and intact cells in the moulting glands at the time of ecdysis to an adult. Such a response indicated that changes in the commitment of moulting gland cells occurred on an individual basis over the course of several days. Similar examples of mosaic development within a single tissue type have been reported for the hindgut of Hy&&m crcropiu following injection of near-threshold levels of juvenile hormone (JUIIY and GILBERT, 1970). and in the integument of Rhodni~r.s proli.ws (WIGGLESWORTH, 1940). Hpcrbphwo wcmpia (WILLIAMS. 196 I ). On~~~pt’lr~s fil.s&tu.s (LAWRENCE, 1969), and Munducu sextu (TRUMAN et ul., 1974) fol-

lowing exposure to juvenile hormone near the end of a juvenile hormone-critical period. A detailed study by LAWRENCE (1969) clearly showed that individual epidermal cells could respond to juvenile hormone in a mosaic manner. leading to the deposition of cuticle that was larval in form but adult in pigmentation. These results suggest that genes coding for specific characters may be independently regulated by juvenile hormone. In the present study, individual cells of the moulting glands respond to juvenile hormone in an all-or-none fashion, supporting a concept of the

hormone as a master gene repressor, inhibiting the expression of all stage-specific characters (WILLIAMS and KAFATOS, 1972). It is likely, however, that individual moulting gland cells could simply not tolerate a mosaic form of cell death. Loss of sensitivity by the moulting glands to ZR-515 was clearly correlated with increased levels of circulating ecdysteroids. That ecdysone plays a causative role in terminating sensitivity to juvenile hormone analogue was suggested by premature loss of sensitivity to ZR-515 following premature exposure to increased levels of 20-hydroxyecdysone. Injection of ecdysone may have initiated the expression of juvenile hormone-sensitive characters (differentiation), thereby rendering subsequent exposure to the analogue irrelevant. Alternatively. early exposure to ecdysone may have caused some change in the developmental potential of moulting-gland cells. We are unable from results of the present study to clearly distinguish between these two explanations. However, during the last-larval instar of several insect species, it appears that irreversible loss of sensitivity to juvenile hormone (commitment) and the onset of differentiation itself are controlled by two separate periods of exposure to ecdysone (Manduw .wxtu, TRUMAN rt cd.. 1974: RILIDIFORD and MITSUI. 1978; Arschnu L_VUMI. ANDRIES, 1979; Gtrllrrio mrllonella, HWANG-HSU et ul., 1980: MCCALEB and KIJMARAN, 1980). These results

indicate that ecdysone plays two discrete roles in the regulation of metamorphic development-alteration of the developmental potential of target cells and stimulation of the expression of new characters. Whether ecdysone acts in two similarly discrete steps during instars characterized by a single ecdysone peak (e.g. larval Oncopeltus: larval Bomhy.u, CALVEZ et al., 1976; and all pupal stages, DEAN et ul., 1980) is a question which remains unresolved. rl(,~12011’/~~1~1t,)~;~t1f.\ This work was supported in part by NSF Grant PCM79-I 1779 to HFN and by a Cocos Foundation Fellowship to WAS. REFERENCES ANDRIESJ. C. (1979) Effect of exogenous JHI on imaginal determination in Aeschnu cyrwa. J. fnsecf Physiol. 25, 621-627. BOWERSW. S. (1968) JH : Activity of natural and synthetic synergists. Science, N. I’. 161, 895-897. CALVEZ B. (1981) Progress of developmental programme during the last larval instar of Botwh~z nlori. relationships with food intake, ecdysteroids and juvenile hormone. J. Iwrt’t Physiol. 27, 233. 239. CALVU B.. HIRN M. and DE REGGI M. (I 976) Ecdysone changes in the haemolymph of two silkworms (Bomhy~ rtlori and Philosamia c_wthio) during larval and pupal development. FEBS Lert. 71, 57-61. CASSKR P. and FAIN-MAUREI.M. A. (1970) Controle plurifxtoriel de I’evolution post-imaginale des glandes ventrales chez Locusts migrtrtoriu L.. donnees experimentales et infrastructurales. J. Inwt Physiol. 16. 301 318. E&AN R. L.. BOLLENBACHERW. E.. LOCKE M.. GILBERT L. 1. and SMITH S. L. (1980) Haemolymph ecdysteroid levels and cellular events in the intermoultimoult sequence of Calpodes. J. Insect Physiol. 26. .?67-280. GILBERT L. 1. (1962) Maintenance of the prothoracic glands by juvenile hormone in insects. .Noture, Land. 193, 1205- 1207.

Juvenile

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SMIIIITH W. A. and NIJHOUT H. F. (1982) Ultrastructural changes accompanying secretion and cell death in the molting glands of the milkweed bug. Oncopeltus ,fasciutus.

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TOJO S.. KIC%JCHI K. and KIMURA S. (1981) Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm. Bomhy.~ mar;. J. Inswt Ph~siol. 27, 491-497. TR~JMAN J. W.. RIDDI~ORD L. M. and SAFRANE~ L. (1974) Temporal patterns of response to ecdysone and juvenile hormone in the epidermis of the tobacco hornworm, Manducu se.yta. Deul Biol. 39. 247-262. WIGGLESWORTH V. B. (1940) The determination of characters at metamorphosis in Rhodrks pro/iw.\ (Hemiptera), J. e.xp. Biol. 17, 201-222. WIGGLESWORTH V. B. (1955) The breakdown of the thoratic gland in the adult insect Rhodniu.s pro/i.ws J. ezp. Biol. 32. 485.-49 1. WILLIAMS C. M. (1956) The juvenile hormone of insects. Nuture, Land. 178, 212-213. WILLIAMS C. M. (1961) The juvenile hormone. II. Its role in the endocrine control of molting, pupation. and adult development in the cecropia silkworm. Biol. Bull. mur. hiol. Lab.. Woods Hole 121. 572-585. WILLIAMS C. M. and KAFATOS F. C. (1971) Theoretical aspects of the action of juvenile hormone. &I/. Sw. cnt. Sui.w. 44, 151p162. WILLIS J. H. (1974) Morphogenetic action of insect hormones. .4. Rev. Ent. 116, 323-338.