The ecdysteroid titres during the last-larval instar of Ephestia kuehniella Z. (Lepidoptera: Pyralidae)

J.

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1983 Pergamon

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THE ECDYSTEROID TITRES DURING THE LAST-LARVAL INSTAR OF EPHESTIA KUEHNIELLA Z. (LEPIDOPTERA: PYRALIDAE) H. G.

KALLENBORN

FB Biologie/Zoologie, Universitlt

and G. C.

MOSBACHER

des Saarlandes, 6600 Saarbrticken I I, F.R.G.

(Received 31 January 1983; revised 13 April 1983)

ecdysteroid titre and the body weight during the last-larval instar of Ephestia kuehniella Slightly elevated ecdysteroid titres occur during the first 12 h following the last larval-larval ecdysis (38 rig/g))and again some 120 h later, lasting about 48 h (33 ngfg). A high ecdysteroid peak (750 rig/g))with a maximum in prepupae of the eye-class A4 precedes the larval-pupal ecdysis. The

Abstract-The

were determined.

basal levels between these increased ecdysteroid titres are between 13rig/g and 15rig/g..Compared with the body weight, the first slightly increased ecdysteroid titre 12 h after ecdysis is associated with the beginning of food intake, the second increase at 144 h after ecdysis with reduced gain in body weight. The prepupal ecdysteroid peak occurs whilst the body weight remains constant. Correlations between the varying ecdysteroid titre and morphological and physiological events accompanying the progress in larval-pupal development are discussed. Key Word Index: Ephesria kuehniella, ecdysteroid titres. RIA

INTRODUCTION

For the past few decades Ephescia has been one of the most studied objects for genetic and developmental research e.g. the pattern formation of the wing (Kuhn, 1965) the growth of wing discs (Pohley, 1965) and the development of the male genitalia (Dewes, 1980). Investigations into many species of insects show the postembryonic development to be controlled by the endocrine system i.e. the neurohormones, the juvenile hormones, and the ecdysteroids (for review see Richards, 1981). The ecdysteroids are significant for the initiation of the moult, larval growth, and cell differentiation during metamorphosis. In Ephestiu, correlations between changes in the ecdysteroid titres and some morphogenetic and physiological events during the last-larval instar have been observed (Pohley, 1961; Dewes, 1980; Colln, 1973) although no ecdysteroid titre determinations have been made so far. Indications of the presumed titres have been given by histochemical studies of the endocrine glands (Rehm, 1951; Vitz, 1967) and by extirpation and ligation experiments in Ephestia (Kuhn and Piepho, 1936). Since suitable antisera have become available quite recently, radioimmunoassays are favoured to determine ecdysteroid titres in insects. In the present paper, direct measurements of ecdysteroids using this method have been attempted in Ephestia for the first time. Moreover discussion will centre on whether the changes in the ecdysteroid titres agree with the results of the studies mentioned above and to what extent the endocrine events in different lepidopterans can be compared to Ephestia. MATERIAL A

wild-coloured

AND METHODS

inbreeding

strain

of the meal

moth, Ephestia kuehniellu Z., was tested. Animals were reared under standard conditions at 21°C (Dewes, 1980) and fed with shredded wheat. For age determinations, penultimate-instar larvae were collected about 12 h prior to the last larval-larval ecdysis. Freshly moulted last (6th)-instar larvae were transferred twenty each to Petri dishes (diam. 9cm) with food and kept at 25°C in an incubator. In comparison with the rearing methods of Pohley (1960) the last-larval instar was clearly prolonged: our larvae required 9-10 days to mature (in contrast to 7 to 8 days) and the following prepupal phase lasted 3 to 4 days. According to the method of Kiihn and Piepho (1936) this prepupal phase was divided in the eye-classes Al up to A5 (A5 = pharate pupa). Sexing was rendered possible by the dark pigmented testes shining through the epidermis of male larvae. For direct measurements of ecdysteroids, five specimens of the same age were grouped to provide a sample. Each sample was homogenized in 4 ml of methanol-water 60:40 (v/v). The combined supernatants were washed with chloroform and dried. The extract was dissolved in 6ml of water and then in 6 ml of butanol and washed. The dried butanol phase was dissolved in 1.5 ml of pure methanol and cooled. The efficiency of the extraction method as tested by the recovery of known amount of labelled ecdysone added to homogenized samples from representative development stages was about 55 f 7% (SD). According to the amount of individual ecdysteroid levels, l/75 up to l/5 of the final extract volume was investigated by radioimmunoassay (RIA). RIA was performed according to Spindler et al. (1978). The the labelled (anti-ICT 1) and antiserum [23,24-‘Hlecdysone (specific activity: 68 Ci/mmol) were donated by Dr. C. Beckers, TH Darmstadt. The dried extracts were suspended in 100 ~1 of a 0.1 M borate buffer (PH 8.4) containing 2.5% of normal 749

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H. G. KALLENBORN and G. C. MOSBACHER

rabbit serum, 0.157; of specific antiserum and labelled ecdysone (ca. 4000cpm). This solution was incubated for 1 h at 37°C. The haptene-antibody conjugate was then precipitated by the addition of an equal volume of ammonium sulphate solution (saturated 4°C) and centrifuged. The pellet was dissolved in 20~1 of water and the radioactivity was counted in 500 ~1 aqualuma (Baker, GroB-Gerau) on an Isocap 300 scintillation counter (Zinsser Analytic). Each sample was assayed in duplicate. Individual ecdysteroids were not separated, our data do not distinguish between ecdysone, 20-hydroxyecdysone and their metabolites. The RIA activity is expressed as 20-hydroxyecdysone equivalents. The affinity of the polyhydroxysteroid-specific antiserum was about x 3.6 greater towards ecdysone than towards 20-hydroxyecdysone. For further information about cross-reactivity towards other ecdysteroids see Spindler er al. (1978. Table 1). The binding curve for 20-hydroxyecdysone was log-linear between 0.25 and 2 ng. Student’s t-test was used to determine significant differences in ecdysteroid levels per fresh weight between the times tested.

not significant), later declining to a basic level of 13 rig/g 36 h after ecdysis (P < 0.01). The ecdysteroid titre increases again 84 h after ecdysis and reaches a maximum between 132 h and 156 h (33 ng/g). 180 h after ecdysis the titre is reduced once more to 15 rig/g.. There are significant differences registrated between the titre point at 84 h after ecdysis and the peak at 144 h (P < 0.02), and between this peak and the point at 180 h (P < 0.05). During the wandering phase of the larva from 204 h to 240 h after ecdysis the ecdysteroid titre remains low. The titre rises again (54 rig/g;; P < 0.01) only in the young prepupa which has reached the eye-class A2 and reaches the maximum of the prepupal peak in prepupae of the eye-class A4 (750 rig/g;; P < 0.01 compared to A3). This peak is far higher (about x 20) than the titres 12 h and 144 after ecdysis both of which are at about the same lower level. Subsequently the titre declines once more to reach a level of 304 rig/g in prepupae of the eye-class A5 (pharate pupae). No significant sex-specific differences in the ecdysteroid titres during the last-larval instar were found. The body weight of last-instar larvae (Fig. 1) shows a moderate gain after the larval eclosion and an increased and nearly linear gain from 84 h to 132 h after ecdysis. Afterwards the gain in weight is reduced up to the point of maximal weight (31 mg at 204 h). During the following wandering phase and the eyeclass Al weight decreases down to a steady level (22 mg) beginning in prepupae of the eye-class A2. It is obvious that the ecdysteroid titre is slightly increased when the larval cuticle hardens and food intake begins, and that the maximal gain in weight occurs during reduced ecdysteroid titres. When the middle ecdysteroid peak has been reached the gain in weight decreases. The prepual ecdysteroid peak appears while the body weight remains constant. Ephestia’s ability to pupate is dependent upon a brain critical period in the first half of the prepupal

RESULTS

During the last-larval instar of Ephestiu there is a slight increase in the ecdysteroid titre of whole animals shortly after the larval moult and another six or seven days later. A third and drastic increased titre precedes the larval-pupal moult (Fig. 1). About 12 h before the last-larval ecdysis, the ecdysteroid titre as a part of the peak preceding this moult is still relatively high (89 ng/g). Freshly moulted larvae have an ecdysteroid titre significantly lower than before (33 rig/g;; P < 0.01). During the following 12 h, when the head capsule darkens, the titre remains on this level or even shows a slight increase (38 rig/g;;

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Fig. I. The ecdysteroid titres and growth in Ephestia during the last-larval instar. Each titre point is the mean k SEM of five to ten separate determinations. For each determination, five specimens were extracted. Arrow indicates post-cephalic critical period (V) and post-thoracic critical period (v). respectively. Abscissa: Age in the last (6th)Jarval instar (hours after ecdysis). The prepupal eye-classes Al up to A5 are marked by the progress of stemmata-pigment migration (see Kiihn and Piepho. 1936).

Ecdysteroid titres of Ephestia kuehniella eye-class A2 and a prothoracic gland critical period in the second half of A2 (Kuhn and Piepho, 1936). The arrows in Fig. 1 mark the points where 50% of the animals with the brain extirpated and those with thoracic ligation were still able to pupate whilst the remaining treated animals did not moult. The prepupae of Ephestia remain only within a few hours in the eye-class A2 and these two critical periods lie close together. It is obvious that they coincide with the drastic rise of the ecdysteroid titre in the prepupal peak.

751

which reach a maximum in prepupae of the eye-class A3 (Pohley 1959). The changes in the S-phase duration within wing-disc cells may also be connected with the existence of distinct ecdysteroid levels during the last-larval instar. Lobbecke (1969, Table 1) noticed a short phase of DNA synthesis on the 1st (8.2 h), 4th (8.8 h) and from the 8th day of the stadium (6.6 to 5.5 h) and a long phase between them. Transferred to our rearing conditions and inbreeding strain the short synthesis phases can as to time be correlated with increased ecdysteroid titres. The fact that DNA synthesis lasts a bit longer during the first two slightly increased titres than in young prepupae DISCUSSION with a drastic increased titre probably indicates a We found that the ecdysteroid titre of whole quantitative correlation between ecdysteroid level Ephestia larvae is increased during three distinct and the duration of DNA synthesis in the wing discs. ecdysone and periods between the last-larval moult and the moult DNA synthesis, Concerning to the pupa. The first slightly increased level is to be 20-hydroxyecdysone are assumed to play different found during the first 12 h following the moult. This roles. Oberlander (1972) showed by in uitro assays condition has also been found in different lepidopterthat DNA synthesis in Galleria wing discs may be ans but has either not been mentioned expressively or and finished by initiated by ecdysone has been considered as part of the declining premoult 20-hydroxyecdysone. In Pieris, the studies of Lafont peak in the penultimate-larval instar (e.g. Calvez, et al. (1977) led to similar results. The temporal 1981). The ecdysteroid titre in Ephestia at the lastcoincidence of the middle ecdysteroid peak and the larval eclosion is lower than 12 h before, but is reduction of food intake gives rise to the conclusion decreased only 36 h after ecdysis. This fact gives rise that some behaviour patterns are regulated by ecdyto the interpretation that at least during the first 12 h steroids as well. Similar events take place in Calpodes after moulting the prothoracic glands continue to (Dean et al., 1980) and in Pieris and Manduca (see produce ecdysone at a slightly increased level or else Lafont et al., 1977). As to Galleria at least spinning there is a reduced ecdysteroid degradation. Up to is also initiated by this ecdysteroid peak; if the peak now a pronounced ecdysteroid peak shortly after is delayed the larvae enter diapause (Sehnal et al., moulting has been decribed only in Calpodes as an 1981). In Ephestia the formation of the inner (meta“intermoult peak” (Dean et al., 1980). According to morphosis specific) cocoon envelope only starts in these authors the first slightly increased ecdysteroid prepupae of the eye-class A3 i.e. when the ecdysteroid titre might regulate intermoult activities. titre increases drastically. Indeed, this inner envelope The ecdysteroid titre is increased again towards the construction is closely dependent on the action of middle of the intermoult period for about 48 h (that 20-hydroxyecdysone, whereas the first. the outer enis 120 h to 168 h after ecdysis). At a relatively early velope is considered to be under environmental conperiod Kuhn and Piepho (1936) assumed that a trol (Giebultowicz et al., 1980). secretion of moulting hormone in Ephestia precedes As in other species, the ecdysteroid titre increases the prepupal phase and causes the gradually indrastically during the prepupal phase in Ephestia. tensifying determination of epidermal cells to pupatThe position of this peak confirms the results of ion. The results of brain extirpations reveal that this Rehm (1951). Her histochemical investigations assumed peak cannot induce any pupation. Vitz showed a high synthesis rate of the prothoracic (1967) concluded from histochemical studies that the glands in prepupae of the eye-class Al and a modprothoracic glands of Ephestia release ecdysone beerate rate in A2 whereas later it was no longer fore the larvae enter the prepupal phase. As in detectable. The hormone release was moderate in A 1, Ephestia, a middle ecdysteroid peak was also found increased in A2 and reached a high level in A3, by titre determinations in Manduca (Bollenbacher et thereafter declining. This prepupal peak is generally al., 1981), Pieris (Lafont et al., 1977) Galleria (Sehnal responsible for apolysis, cuticle secretion and indiet al., 1981) and Calpodes (Dean et al., 1980). This rectly for ecdysis. In Ephestia, apolysis of larval middle ecdysteroid peak is interpreted as a signal for cuticle (Rehm, 1951) and evagination of wing discs the change of commitment (reprogramming) causing (Pohley, 1960) and male genital disc (Dewes, 1980) the epidermal cells to synthesize a pupal cuticle when begins in A2. At this stage the haemolymph protein the high ecdysteroid peak preceding the pupal moult storage in the fat body increases simultaneously. occurs (Riddiford 1976, Bollenbacher et al., 1981). Colln (1973) has shown that the correlated metamorThe question, however, as to whether the moulting phic changes in spectra of haemolymph and fat body hormone itself or else the lack of juvenile hormone at proteins are controlled by ecdysone. Similar to the this time is responsible for the reprogramming of the epidermal cells concerning cuticle, the fat body cells genome remains to be answered (Hwang-Hsu et al., are considered to be already reprogrammed from 1979; Sehnal et al., 1981; McCaleb and Kumaran, protein synthesis to protein storage by the “commit1980; Calvez, 1981). Moreover this middle ecdyment peak” before the prepupal phase (Tojo er al., steroid peak effects morphological changes in the 1981). According to these events occurring prior to the imaginal discs. The proliferation of the male genital disc in Ephestia increases at this time (Dewes 1980) peak maximum in A4, the eye-class A2 is marked by and the wing discs show increasing mitotic rates the post-cephalic critical period and the post-thoracic

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H. G. KALLENBORN and G. C. MOSBACHER

critical period (Kuhn and Piepho, 1936). Ligation behind the head or brain extirpation prevent the activation of the prothoracic glands by PTTH and ligation behind the thorax prevents the distribution of ecdysone in the haemolymph. Nevertheless, when A2-prepupae and older prepupae are treated in such a way, pupal cuticle is secreted by post-ligation epidermal ceils as well as by pro-ligation epidermal cells. In prepupae of the eye-class A2 the ecdysteroid titre is not visibly higher than in the “commitment peak”. This means that not all of the ecdysteroids in the prepupal peak are necessary for the initiation of the pupal moult. In Manduca (Truman and Riddiford, 1974; Bollenbacher et al., 1975). Pieris (Lafont et al., 1977) and Calpodes (Dean et al., 1980) the post-thoracic critical period also lies some hours before the titre reaches its maximum. Unlike Pieris and Cufpodes, in which the first critical period is at the beginning of the “commitment peak”, the postcephalic critical period in Ephestiu immediately precedes the post-thoracic one. The reason for this may be that the basic ecdysteroid level in Ephestiu is too low to be effective in an accummulative way. Therefore, a repeated ecdysone release is necessary to initiate moult. The cuticle secretion in Ephestiu takes place only at the beginning of A5. at a time when the titre has already passed its maximum. The ecdysteroid titre decrease preceding the actual pupal ecdysis presumably is essential for the subsequent tanning of the newly formed pupal cuticle (Mitsui and Riddiford, 1976). As mentioned before, different ecdysteroids can play different roles during the developmental progression form larva to pupa. In fact, shifts in ecdysone to 20-hydroxyecdysone ratios have been noted e.g. in Munducu (Bollenbacher et al., 1981) Bombyx (Calve2 et al., 1976) and Pieris (Lafont et al.. 1977). Therefore we are now investigating the components of the shown ecdysteroid titres in Ephestia in detail. Acknowledgements-The

authors wish to thank Dr. C. Beckers for donating the antiserum and the labeled ecdysane and Dr. E. Dewes for useful discussions and critical reading of the manuscript. REFERENCES Bollenbacher W. E., Smith S. L.. Goodman W. and Gilbert L. I. (1981) Ecdysteroid titer during larval-pupal-adult development of the tobacco hornworm. Manduca sexta. Gen. Comp. Endocr. 44, 302-306. Calvez B. (1981) Progress of developmental programme during the last larval instar of Bombyx mori: relationships with food intake, ecdysteroids and juvenile hormone. J. Insect Phvsiol. 27, 233-239. Calvez B.. Him M. and De Reggi M. (1976) Ecdysone changes in the haemolymph of two silkworms (Bombyx mori and Philosamia Cynthia) during larval and pupal development. FE&S Lett. 71, 57-61. Cohn K. (1973) Uber die Metamorphose der Proteinspektren von Hiimolymphe und Fettkijrper bei Ephestia kiihniella Z. Wilhelm Rouxs Arch. Entw Mech. Org. 172, 231-257. Dean R. L., Bohenbacher W. E., Locke M., Smith S. L. and Gilbert L. I. (1980) Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius. J. Insect Physiol. 26, 267-280.

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