The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera)

Cell Tissue Res (1983) 232:493-511 Cell and Tissue Research �9 Springer-Verlag 1983

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera)

Andr6 Quennedey, Brigitte Quennedey, Jean-Paul Delbecque, and Jean Delachambre Laboratoire de Zoologie, Universit6 de Dijon, Dijon, France, (Equipe de Recherche Associ6e au CNRS No. 231)

Summary. In order to study the pupal-adult metamorphosis of Tenebrio in vitro, pupal sternites of different ages were cultured in Landureau's medium and their development systematically observed by electron microscopy. In hormone-free medium, explants taken from young pupae do not secrete pupal postecdysial cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle. On the contrary, explants taken from pharate adults continue to secrete adult preecdysial cuticle in vitro, and the epidermis never detaches from the cuticle. Ecdysterone in physiological concentrations (0.2 to 4 lag/ml) induces the secretion of a new cuticle in explants from young pupae but the epidermis remains undifferentiated. Ecdysone is necessary for the induction of some adult differentiation. Moreover, the quality of the cuticle secreted in vitro is increased by the addition of 2% foetal calf serum; the best results have thus far been obtained in a medium containing 0.2 ~tg/ml ecdysone, 1 ~tg/ml ecdysterone, and 2% foetal calf serum.

K e y w o r d s : Ecdysteroids - Epidermis - In vitro differentiation - Tenebrio - Ultrastructure

Hormonal requirements for the development of several insect tissues have been intensively studied in vitro during the past 15 years (see Marks 1976, 1980, for review). Most of the data concern endocrine processes in moulting of dipteran and lepidopteran wing disks and have emphasized the primordial role of ecdysteroids in triggering moulting and morphogenesis. Though other holometabolous species have been almost completely ignored, several studies in the beetle Tenebrio molitor have shown that ecdysteroids are also required for the differentiation of adult organs in the last larval instar (geni- tal anlagen, Huet 1971; legs, Lenoir-Rousseaux and Huet 1975) and their

Send offprint requests to: Dr. A. Quennedey, Laboratoire de Zoologie, Universit6 de Dijon, Facult6 des Sciences, 6, Boulevard Gabriel, 21100 Dijon, France

494 A. Quennedey et al.

development during pupal-adult metamorphosis (ovaries, Laverdure 1970; reproductive accessory glands, Szopa and Happ 1982).

Tenebrio molitor shows typical ecdysteroid fluctuations in the pupal stage, temporally correlated with the initiation of adult cuticle deposition (Delbecque et al. 1978a); however, several observations suggest that the endocrine control of metamorphosis in this insect may differ from that in Lepidoptera and Diptera. Prothoracic glands degenerate in fact very early (Srivastava 1960; Glitho et al. 1979) and isolated abdomens, ligated at pupal ecdysis, are able to develop autonomously into the adult form (Janda 1933); they even show a quite normal ecdysteroid peak (Delbecque et al. 1978b). All these data, together with similar findings in two other beetles (Leptinotarsa, Hsiao et al. 1975; Dermestes, Slfima 1975; Delbecque and Slfima 1981) cast doubt on the exact significance of the ecdysteroid peak during pupal-adult development and suggest that at least some events of the moulting cycle either can proceed autonomously, without any hormonal stimulation, or are triggered long before, i.e., in the last larval instar.

The aim of the present paper is thus to define the role of ecdysteroids in the pupal-adult development of Tenebrio. Toward this end, we have first studied in vitro the development of epidermal explants, taken from pupae at different periods of the cycle, under hormone-free conditions. Then we have tested the effects of various ecdysteroid administrations on the development of young pupal epidermal explants in order to reproduce metamorphosis in vitro. All experiments of this study were carried out at the electron microscopic level.

Materials and methods

Animals. Tenebrio molitor larvae were reared at 25~ on flour containing 1% yeast and prepupae were removed regularly. Dating of pupae was carried out by determination of the time from pupal ecdysis, and also by use of physiological stages as previously described (Del- becque et al. 1978a; Delachambre et al. 1980).

Culture conditions. Pupae were disinfected by the application of merfene (Rikes Laboratory) followed by two rinses with sterile water. Six sternal explants, corresponding to contiguous half-sternites, were cut off from each pupa; for each experimental situation (age or hormone presence) at least three pupae were used, i.e., a total of 18 explants. (All experiments of this study involved more than 200 pupae.) Each explant of pupal integument, clean of fat body and muscle, was washed twice for 5 min in culture medium. In culture, residual fat body soon disappears. Culture was carried out on the surface of 2 ml medium renewed every 5 days in culture dishes (Falcon 3037), either for a short period (5-10 days with daily sampling) or for a longer time (about a month with five-days sampling). With 2 ml medium 6 explants (one pupa) can be cultured for 5 days at 27.5 ~ C.

Landureau 's S 20 medium (Landureau and Grellet 1972) was used slightly modified according to the Na/K ratio (65/42) and osmolality (380 mOsm) of Tenebrio haemolymph. The medium was used alone or supplemented by 2% foetal calf serum (FCS, Gibco). Ecdysteroids, i .e, ecdysone and ecdysterone (Simes Laboratory, Italy) were dissolved in the culture medium at different concentrations.

Electron microscopy. At the end of culture, explants were fixed for 16 h in a cold 2% parafor- maldehyde - 3% glutaraldehyde mixture in 0.2 M, pH 7.4 cacodylate buffer, postfixed 1 h in cold 1% osmium tetroxyde in the same buffer, and embedded in Epon - Araldite. Ultrathin

Tenebrio epidermis in vitro 495

sections were stained in a solution saturated with alcoholic uranyl-acetate and in lead citrate, and examined in a Hitachi HU-11 E electron microscope.

Results

The sternite cuticle of pupae has a characteristic regular helicoidal structure (Bouligand 1965; Neville 1970). The monolayer of epidermal cells has scattered epidermal oenocytes (Fig. 1 A). In adults (Fig. 1 B) the sternal integument is very different (Wigglesworth 1948; Delachambre 1971). The cuticle has an apical scute-shaped surface.The helicoidal preecdysial cuticle is interrupted by vertical columns, and the postecdysial cuticle is composed of large lamellae with preferred orientation (Caveney 1970; Delachambre 1970,

Fig. 1A, B. Diagrams of pupal (A) and adult (B) abdominal sternite integuments. A Pupal integument composed of monolayered epidermal cells (1) and scattered oenocytes (2). Smooth- surfaced cuticle composed of epicuticle (ep), preecdysial mesocuticle (me) and postecdysial helicoidal endocuticle (en). B Adult sternite integument with epidermal cells (1), oenocytes (2), differentiated adult exocrine glands : pit-glands (3) opening at cuticle surface via perforated plate, associated with mechanosensory bristle (arrow), and dermal glands (4) opening via nar- row aperture. Cuticle with its typical scute-shaped surface composed of epicuticle (ep), helicoidal preecdysial exocuticle (ex) interrupted by vertical columns, and posteedysial mesocuticle (me) with preferred orientation

496 A. Quennedey et at.

I . . . . . . . . . . . . . . . . . . . . i ........... "," ......................................... 1 0 70 100 250 hr

~ l II1[1III II [lIIll Illl J II II~tl|]lflt IlJlt| r I[11 PPOC APOLYSIS APC

Fig. 2. Diagram of pupal-adult cycle in Tenebrio: ecdysteroid titres in haemolymph, apolysis and phases of cuticular secretion (PPOC pupal postexuvial cuticle; APC adult preecdysial cuticle) in relation to age of pupae expressed in hours (modified from Delachambre et al. 1980)

1975; Delachambre et al. 1980). Epidermal polyploid oenocytes are differentiated already in young pupae (Besson-Lavoignet and Delachambre 1981). Two types of newly differentiated exocrine cells arising from epidermal mitoses are observed in great number in the epidermis: dermal glands (Dela- chambre 1973) and pit-glands (Wigglesworth 1948) also called setiferous sex patches (Faustini and Halstead 1982) which are associated with mechanosensory bristles.

In vivo epidermal development without exogenous hormones

Sternites were removed during different phases of the moulting cycle (pupal postecdysial cuticular secretion, apolysis and synthesis of preecdysial adult cuticle, see Fig. 2) to be grown in culture.

In explants taken during the deposition of pupal postecdysial cuticle (0-70 h pupae), detachment of the cuticle from the epidermis (Figs. 3, 4) occurs in about 95 per cent of the cases observed (31/33 pupae) and there is always degeneration of epidermal oenocytes (Fig. 5). The detachment of the cuticle is clearly observable 24 h after the beginning of the culture (Fig. 3). The apical microvilli of the epidermis are poorly developed, and an abundant fibrillar material is secreted forming loose layers covering the epidermis (Fig. 4). A reduction in the thickness of the epidermis is observed throughout the culture. Such a detachment is never observed in larval and adult explants at the same time of postecdysial cuticle secretion. Epidermal cells contain the same cellular organelles as in vivo, i.e., rough endoplasmic reticulum in great abundance, mitochondria with a dense matrix mainly located in the upper part of the cell, dense vesicles, and an ovoid nucleus. Dense chromatin is located at the periphery of the nucleus.

In explants taken during the pupal-adult apolysis (70-100 h pupae), microvilli are well developed, and after 4-5 days of incubation the in vitro tissue shows no further development (Fig. 6). In about 100 h pupae, electron-dense, disorganized material appears above the microvilli (Fig. 7). Such material, rarely observed in vivo could be interpreted either as ecdysial droplets (Locke and Krishnan 1971) or as epicuticle patches, unable to build a continuous layer.

Figs. 3-5. Sternites from 15-24 h pupae during deposition of pupal postecdysial cuticle, cultured without exogenous hormone. Scales : 3 ~tm

Fig. 3, After 1 day of culture, detachment of pupal cuticle (arrow) clearly visible. Note loose material (asterisk) between microvillous border and cuticle, x 5200

Fig. 4. After 10 days of culture, loose material (asterisk) more abundant, epithelium decreases in thickness, x 3600

Fig. 5. Culture for 5 days. Note degeneration (asterisk) of epidermal oenocyte, x 6500

Figs. 6-11. Sternites from 70-250 h pupae during pupal-adult apolysis (70-100 h) and during secretion of adult preecdysial cuticle (100-250 h), cultured for 5 days in medium without exogenous hormone. Scales : 1 ~tm


CULTURE EVOLUTION

~ . _ cuticle detachment Q ~ oenocyte degeneration

~ _ ~ no evolution Q ~ oenocyte degeneration

c o o

Q oenocyte degenerati on �9 differentiations continue

Fig. 12A-C. Diagrams summarizing in vitro development in hormone-free medium of sternites taken during A pupal postexuvial cuticle secretion (0-70 h), B apolysis (70-100 h), C adult preexuvial cuticle secretion (100-250 h)

In explants taken during the secretion of adult preecdysial cuticle (100-250 h pupae), the secretion of cuticle continues in vitro. In pupae of about 120 h, in which the epicuticular layer has been deposited before the onset of culture, a loose fibrillar and less abundant material corresponding to a poorly structured endocuticle is secreted (Fig. 8). In 185 h pupae, beneath the adult cuticle secreted in vivo, cuticular layers are secreted in vitro (Fig. 9). However, this material does not show the typical columnar organization observed in vivo (Fig. 10). In older pupae (230 h) an in vitro

Fig. 6. 70 h pupal sternite. Cuticle apolysed before culture, loose material (asterisk) produced in vitro above erect microvilli (arrows). x 16000

Fig. 7. 100 h pupal sternite. Abundant electron-dense material (arrows) secreted, x 16000

Fig. 8. 120 h pupal sternite. Beneath in vivo secreted epicuticle (small arrow), little structured cuticular material deposited (asterisk). Note also epicuticular duct (open arrow) of dermal gland differentiated before culture, x 10000

Fig. 9. 185 h pupal sternite. Beneath adult preecdysial cuticle (open arrow) deposited in vivo, another type of cuticle (small arrow) secreted in vitro. Note also degenerating oenocytes (aster- isks), x 4000

Fig. 10. High magnification of same sternite as in Fig. 9 showing organization of in vitro secreted cuticle (asterisk). Typical columnar appearance (arrow: column) of overlying adult cuticle not preserved, x 8500

Fig. 11. 230 h pupal sternite. Two less dense lamellae of adult cuticle secreted in vitro (arrows). x 14000

Figs. 13-16. Sternites from 0-40 h pupae cultured in medium with exogenous ecdysterone

Fig. 13. 1 h pupal sternite cultured for 8 days with ecdysterone (0.2 gg/ml). Epidermis remains apolysed but in few cases a new cuticle composed of interrupted outer epicuticle (small arrow) and poorly structured endocuticle (open arrow) secreted after at least 6 days. Note large hole (asterisk) following oenocyte degeneration. Scale: 3 I.tm. x 5500

Fig. 14. 1 h pupal sternite cultured for 3 days with ecdysterone (1 pg/ml). Above microvilli, outer epicuticular patches (arrows) with typical triple-layered structure. Scale: 0.25 gin. x 60000

Fig. 15. 1 h pupal sternite cultured for 4 days with ecdysterone (4 pg/ml). Beneath outer epicuticle (black arrow), inner epicuticle (open arrow) secreted during fourth day of culture. Scale: 0.5 pm. x 32500

Fig. 16. 40 h pupal sternite cultured for 5 days with ecdysterone (0.2 ~tg/ml). Endocuticular lamellae (open arrow) of new cuticle secreted during fifth day of culture. Note numerous pore-canal filaments (black arrow) hanging beneath old pupal cuticle. Scale : 3 Ixm. x 5500


Fig. 17. 2 h pupal sternite cultured for 10 days with ecdysterone (4 gg/ml). New cuticle (black arrow) about 4 gm thick, with serrated surface, as in adult cuticle; columnar organization lacking. Above, pupal cuticle (open arrow) and loose material (asterisk). Scale : 3 gm. x 4200

Fig. 18. 1 h pupal sternite cultured for 15 days with ecdysterone (4 gg/ml). New cuticle (black arrow) secreted in vitro detached. Note atrophy of epidermis (open arrow). Scale: 3 gm. x 4200

cuticular secretion is recognizable (Fig. 11). It is interesting to note that, unlike in explants taken during the deposition of postecdysial pupal cuticle, explants taken during the deposition of preecdysial adult cuticle do not show any separation between epidermis and cuticle. Epidermal oenocytes always degenerate (Fig. 9), whereas glandular structures such as dermal glands are preserved (Fig. 8). The addition of 2% foetal calf serum (FCS)


to Landureau's medium had the same results. These observations in hormone-free conditions are summarized in Fig. 12.

Epidermal development with exogenous ecdysteroids

Ecdysteroids (ecdysterone and/or ecdysone) were added to the culture medium in order to stimulate a new epidermal-cuticular cycle. Explants were taken from pupae before the in vivo ecdysteroid peak (i.e., 0 to 85 h pupae) and different hormonal concentrations were used, according to our previous data (Fig. 2), i.e., 0.2 ~tg/ml corresponding to the pupal basal titre, 1 ~tg/ml (intermediate) and 4 Ixg/ml corresponding to the ecdysteroid peak.

a) Ecdysterone effects. In young pupae (less than 15 h) incubated with 0.2 ~tg/ml ecdysterone in the medium, as in hormone-free experiments, the detachment of pupal cuticle and the secretion of a loosely fibrillar material are observable but no new cuticular cycle occurs. In a few cases an interrupted outer epicuticle and a poorly structured helicoidal endocuticle are produced after a culture period of at least 6 days (Fig. 13).

In 0-15 h pupae incubated with 1 to 4 ~tg/ml ecdysterone and in 15-85 h pupae with 0.2 to 4 ~tg/ml, detachment of the pupal cuticle occurs and secretion of a new cuticle begins at the 3rd day: outer epicuticular patches appear at the tip of oriented microvilli (Fig. 14). The inner epicuticle is deposited during day 4 (Fig. 15) and several endocuticle lamellae are secreted during day 5 (Fig. 16). When the incubation is longer the thickness of the cuticle increases. After a culture of 10 days, it can reach a thickness of 4 ~tm (Fig. 17) and after 15 days the cuticle is detached from the atrophied epidermis (Fig. 18). The cuticle shows both pupal and adult characteristics, i.e., the lack of columnar organization as in pupae and the presence of a scute-shaped pattern as in adults (Figs. 17, 18).

Addition of 2% FCS to the medium (0.2 to 4 ~tg/ml ecdysterone) allows secretion of a well-structured, denser and thicker cuticle (Fig. 19) and the adult pavement pattern is more marked. Moreover, cuticle detachment is delayed and occurs after 25-30 days of culture (Fig. 20).

b) Ecdysone effects. Though ecdysone represents less than 10% of the ecdysterone detected in vivo during pupal-adult development of Tenebrio (Del- becque et al. 1978a, b) two different concentrations of ecdysone have been

Fig. 19. 1 h pupal sternite cultured for | 0 days with ecdysterone (1 ~tg/ml) and 2% FCS. Addi- tion of FCS allows well-structured new cuticle with several adult characters, serrated outline (black arrows), density, dual structural organization with thicker inner lamellae (asterisk) tra- versed by columns and enlarged pore-canals (open arrow). Scale: 3 txm. x 6200

Fig. 20. 90 h pupal sternite cultured for 31 days with ecdysterone (1 ~tg/ml) and FCS. As in vivo lateral part of sternite shows regular helicoidal structure and pore-canals without columns. Detachment of thick cuticle (asterisk). Scale: 3 pm. x 6200

Figs. 21 and 22. Sternites from 24 h pupae cultured in medium with ecdysone (4 ~tg/ml)


Fig. 21.10-day culture. Secretion of new cuticle induced only by long exposure to extraphysio- logical ecdysone. Only epicuticle (black arrow) and first endocuticular lamella (open arrow) secreted. Scale: 0.5 ~tm. x 29000

Fig. 22. 3-day culture. As for lower hormone concentration (0.2 gg/ml), sternal epidermis cyto- logically distinct but oenocytes degenerating (asterisk). Some cells with dense cytoplasm, arranged one around another (arrows), suggesting early stages of differentiation. Scale: 2 gm. x 5500

Figs. 23-26. Sternites from 24 h pupae cultured in medium with ecdysone (0.2 lag/ml) + ecdysterone (1 I~g/ml)+ FCS; differentiation of sensory organs and dermal glands. Scales: 2 pm

Fig. 23. 3-day culture. Two groups of dense cells (large black arrows) with ciliary processes (open arrows) among epidermal cells secreting epicuticle (small black arrow), x 4500

Fig. 24. 5-day culture. Longitudinal section of mechanosensory bristle (arrow). Associated secretory cells of pit-glands undifferentiated, x 11200

Fig. 25. Sections of efferent ductule (arrows) of dermal gland beneath newly secreted cuticle. x 8000

Fig. 26. Among epidermal cells, proximal cell (arrow) of newly formed dermal gland. In cytoplasm, note evaginations and secretory vesicles (asterisk). Note also thickness of epidermis. x 8000


~ l I I

no evolution with 0.2 9g/ml for less 15 hr

pupae

pupal- l ike cut icle

epicut icle ~ .~ .

,d:2,.21i: ~

IN VIVO cuticle detachment

oenocyte degenerati on

I 5 m I0

detachment

mitoses

epicut icle

adult d i f ferent ia t ion

no more D evolution

20 i 30

detachment

I

:.jm, ! ECDYSTERONE ]

MEDIUM + I - 0 , 2 - i - 4 pg/mz

ECDYSTERONE - 2 % FCS

MEDIUM + I - 0 , 2 p g / = l

EUDYSONE

MEDIUM + I - 4 ~g/ml

ECDY$ONE

MEDIUM + - 0.2 pg/ml ECDYSONE - ip~/ml ECDYSTERONE

- 2 % FCS

Fig. 27. Summary of development of sternites from 0-70 h pupae, cultured under different conditions

tested in vitro, 0.2 and 4 ~g/ml. With 0.2 l~g/ml, detachment of the cuticle occurs; after 20 days of culture the epidermis has not secreted any cuticle but only loose fibrillar material as in hormone-free condition. Epidermal cells present a better cytological appearance than in ecdysterone cultures but oenocytes continue to degenerate. With 4 I~g/ml the same development is observed at the beginning of the culture, but after 10 days a typical epicuticle and the first endocuticle lamella are secreted (Fig. 21). The cytological appearance of the epidermis is much diversified; some cells have a denser cytoplasm and are arranged one around another (Fig. 22). Such figures may represent early stages of glandular or sensory-organ differentiations and suggest that epidermal mitoses have occurred.

c) Ecdysone+ecdysterone effects. FCS medium supplemented with hormones (0.2 I~g ecdysone + 1 I~g ecdysterone/ml) have been used for 5 days. After 3 days, a typical outer epicuticle is secreted. Groups of lengthened cells with dense cytoplasm are seen. They differentiate an outer ciliary pro- cess protruding apically which is covered by a cuticular sheath (Fig. 23). These units are typical of differentiating sensory organs (see Ernst 1972, Sanes and Hildebrand 1976). On day 5, they are fully developed with a mechanosensory bristle (Fig. 24). On account of their relative number and tegumental arrangement, such organs correspond undoubtedly to sensory bristles associated with pit-glands (cf. Fig. 1 B). However, the sensory appa-


ratus is not associated with the glandular part of a pit-gland which is not differentiated.

On the contrary, dermal glands are well developed after 5 days of culture. Sections of efferent ductules are observed between epidermal cells and in the cuticle (Fig. 25). Each ductule is connected with a proximal secretory cell. Lucid secretory vesicles are abundant in the cytoplasm of a proximal cell pushing the nucleus aside, and in its middle an extracellular space is observed. Numerous finger-like extrusions with microtubules are present within this space (Fig. 26). Such glandular features are reminiscent of type A dermal glands (Delachambre 1973) belonging to class 3 gland cells (Noirot and Quennedey 1974). The results obtained with ecdysteroid supplemented media are summarized in Fig. 27.

Discussion

From these in vitro experiments several interpretations and conclusions on the endocrine control of pupal-adult development in Tenebrio can be drawn.

Postecdysial cuticle secretion and apolysis

Though the secretion of pupal postecdysial cuticle normally takes 2 days in vivo, explants taken from young pupae never secrete this cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle, whatever the culture conditions, i.e., presence or absence of ecdysteroids or of serum. The loose fibrillar material between epidermis and cuticle resembles that described by Marks and Leopold (1971) in Leucophaea. Sowa and Marks (1975) did not identify chitin in such material, which thus cannot be considered as a true cuticular coating (Marks and Sowa 1976). In Tenebrio, its ultrastructure calls to mind the in vivo moulting gel (cf. Delbecque et al. 1978a) and not a cuticular secretion.

According to this hypothesis, the spontaneous detachment of the epidermis could be considered as true apolysis. Our data in pupal cultures suggest a link between this apolysis and the absence of postecdysial cuticular secretion. (Is apolysis the cause or the consequence of this lack of secretion?) However, similar cultures from last instar larvae or adults failed to show apolysis in spite of a similar arrest in postecdysial cuticular secretion, thus indicating that these two events are independent, but demonstrating also that these observations cannot be considered as a culture artifact.

The absence of effects of various ecdysteroid additions on postecdysial cuticular secretion undoubtedly means that postecdysial secretion is not controlled by these hormones but needs continuous stimulation either by nutrients, absent in our culture medium, or more probably by other hormones; the intervention of cephalothoracic factors for this secretion has been demonstrated by Locke (1965) in Calpodes. More recently, Wielgus and Gilbert (1978) have established that the brain and the fat body, but not ecdysterone, are necessary for postecdysial cuticular secretion in Man- duca larvae. The tanning hormone bursicon has also been implicated in


this control (Fogal and Fraenkel 1969, Sarcophaga; see also Grillot et al. 1976, Tenebrio).

In vitro induction of apolysis by ecdysteroids has been demonstrated by several authors (e.g., Agui etal. 1969, 1972; Mandaron 1970, 1973; Mitsui and Riddiford 1976; Caruelle and Cassier 1979). However, the spontaneous apolysis of Tenebrio pupal explants in hormone-free medium is apparently not affected by various ecdysteroid additions, and this means that apolysis is either independent of ecdysteroids or triggered by hormones received in vivo before culture; this last possibility cannot be dismissed, though apolysis normally occurs later in vivo. This hypothesis can be sub- stantiated by the observation that young sternal explants liberate ecdysteroid-like molecules in the culture medium (in preparation; see Cassier et al. 1980, for similar findings in Schistocerca). These ecdysteroids, detectable by radioimmunoassay after 1-day culture, were probably stored in undetect- able form in Tenebrio integument before culture; they could be sufficient for spontaneous in vitro apolysis, but not for a new cuticular cycle. It must also be stressed that in our experiments a supplementary apolysis can be seen in long term culture with ecdysteroids; this observation supports the concept of hormonal induction of apolysis.

Adult preecdysial cuticular secretion

As demonstrated in numerous other in vitro studies (see Marks 1976, 1980) the epidermal explants of Tenebrio never undergo a new cuticular cycle without hormones in vitro. However, in explants taken from pupae that have already begun the adult preecdysial cuticular secretion in vivo, secretion continues in vitro, even in the absence of ecdysteroids, as already described by Szopa and Happ (1982), and in other species (Caruelle et al. 1979; Caruelle 1980; Fristrom et al. 1982). In our observations; it must be noted that the typical adult architecture is lost.

Addition of various concentrations of ecdysterone to the medium con- firms its well-documented effect on the induction of cuticle secretion; a concentration as low as 0.2 ~tg/ml, which corresponds to the in vivo pupal basal concentration in the haemolymph, is able to induce a new cuticular cycle, provided that explants are taken from pupae older than 15 h. In the case of younger pupae, a higher concentration is needed to obtain the same effect. However, the increase in ecdysterone concentration does not accelerate cuticle deposition; in all cases, epicuticle is deposited after two days of culture.

The results obtained with ecdysone confirm the observations of several authors (Oberlander et al. 1973; Marks and Sowa 1976; Riddiford 1980) who noted that ecdysone is far less active than ecdysterone in inducing a new cuticular cycle; only a concentration of 4 ~tg/ml culture medium trig- gers a new cycle, and that with an important delay. If this concentration corresponds to the maximal titer of ecdysteroids detected in the haemolymph, only less than 10% is represented by ecdysone while ecdysterone is the major product (Delbecque et al. 1978 a, b).


Ultrastructurally the new cuticle secreted in chemically defined Landur- eau's medium is very similar to that secreted in vivo. Outer and inner epicuticle shows the typical in vivo lamellation, but the special structure of the adult preecdysial chitinous cuticle is not observed; a 2% FCS supplementa- tion of the medium can bring about the typical columnar structure and a more pronounced secretion. Thus, it seems that in the chemically defined medium some nutritional and/or hormonal factors are lacking which pre- vents the complete expression of the adult phenotype. Similar conclusions have been drawn by Ryerse and Locke (1978) in Calpodes tracheae cultured in the chemically undefined Grace medium.

Differentiation

The differentiation of the dermal glands of Tenebrio in vitro has been obtained only when ecdysone is added to the medium; no differentiation is observed in hormone-free medium or in media containing ecdysterone only. The specific effect of ecdysone in triggering cellular modification and differentiation in several in vitro systems has been discussed by numerous authors (e.g., Oberlander 1969; Fristrom et al. 1973; Mandaron 1973; Oberlander et al. 1973; Bulli6re and Bulli6re 1977; Lafont etal. 1977; Caruelle and Cassier 1979; Riddiford 1980 etc). In Tenebrio, the absence of differentiation with ecdysterone alone does not appear to be a matter of concentration; lower ecdysterone doses (e.g., 10 and 20 ng/ml) have been tested (not shown in the results) without any effect on differentiation and on cuticle synthesis in vitro; moreover such concentrations are under the pupal basal titer in vivo. Nevertheless ecdysterone seems to have a synergistic effect with ecdysone and their coupled action, together with the presence of 2% FCS, gave the best results, i.e., more complete differentiation of dermal glands and formation of mechanoreceptors associated with the pit-glands. However, as in Schistoeerca (Caruelle and Caruelle 1978a, b; Caruelle and Cassier 1979) the dermal glands obtained with both ecdysteroids are probably non- functional because of the lack of well differentiated microvilli. In Tenebrio the pit-glands themselves, which develop in vivo together with the dermal glands, have never been obtained in vitro. They probably need more differ- entiative mitoses (Wigglesworth 1948) since in vitro the number of mitoses is far less important than in vivo (Besson-Lavoignet, personal communica- tion).

Oenocytes

A striking observation made during this study is the degeneration of epidermal oenocytes after a few days of in vitro culture, whatever the culture conditions. This rapid degeneration suggests that they are not strictly necessary for the deposition of a well-structured preecdysial cuticle though they secrete in vivo some paraffin components added to the basic cuticular material (Wigglesworth 1948; Diehl 1975). The cause of this degeneration is unknown; it is possibly due to the lack of some nutrient or hormonal factor


in the medium, though previous data, obtained on another kind of oenocytes, the segmentary oenocytes of Tenebrio and Schistocerca (see Landur- eau 1976) indicate that they remain alive in the same medium. The degeneration observed here appears thus to be a characteristic of epidermal oenocytes.

In conclusion, these data demonstrate that both ecdysone and ecdysterone are required for pupal-adult development in Tenebrio, in spite of the low levels of ecdysone detected in vivo and in spite of the lack of prothoracic glands. The in vitro development under our culture conditions is incomplete and several experiments are presently under way (e.g., pulses, mimicking of in vivo ecdysteroid fluctuations) to achieve better differentiation.

Acknowledgements. The authors are indebted to Prof. J.C. Landureau for adjustment and gift of culture medium, and to Drs. C. Blais and J.P. Caruelle for their helpful suggestions during the culture experiments. We thank Mrs. S. Mariot and Mr. Mitchell Mruk for their help with the manuscript.

References

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Accepted March 9, 1983

Documents

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera)