Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina,in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development.As well as morphogenetic effects, a proper concentration (3 × 10^?5 M to 3 × 10^?7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10^?9 M seemed to be rather toxic to both development and metabolic activity.     

Microtubules and tracheole migration in wing disks of Galleria mellonella

Tracheoles migrate into the lacunae of wing imaginal disks that are cultured in vitro in medium containing α-ecdysone. Colchicine and vinblastin prevented tracheole migration in vitro under conditions which did not affect the viability of the disks. Cytochalasin B also inhibited tracheole migration, but only at concentrations which were deleterious to the disks. Our ultrastructure findings were in accord with the hypothesis that microtubules, but perhaps not microfilaments, are required for tracheole migration.     

Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

The development of wing imaginal disks of Drosophila virilis after culture in vitro

Drosophila virilis wing disks cultured in vitro in Schneider medium and then transplanted into mature larvae for metamorphosis revealed a shift from successful development of the thoracic area accompanied by poor development of the wing area, to the opposite condition when the period in vitro was increased to 7 days. Cultures in adult hosts for the same length of time maintained the former condition. Labelling experiments with H³-thymidine revealed a substantial decrease in uptake during the first 24 hr in vitro followed by a slight increase after 4 days.     

RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

The influence of larval fat body on wing disk development in vitro

The effects are noted of various fat body treatments on betaecdysone-induced cuticle deposition in cultured wing disks of Plodia interpunctella. The percentage of disks which responded to hormone treatment in vitro depended upon the age of the disk, the amount of fat body, the time the medium was incubated with fat body, and the time the disks were exposed to beta-ecdysone in fat body-conditioned medium. Fat body from pharate pupae did not stimulate disk development.     

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robb's tissue culture medium incorporate ³H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of ³H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

Effects of Weismann ring gland on RNA synthesis in imaginal wing disks of Calliphora erythrocephala

Imaginal wing disks of Calliphora erythrocephala have been studied in normal animals and in permanent larvae surgically deprived of the ring gland. By the end of larval life, uridine incorporation in the nuclear RNA and the ribosomal RNA increases for a brief period. This phenomenon does not occur in permanent larvae. A quantitative study suggests that renewal of a part of the ribosomal RNA might precede wing evagination.     

In vitro differentiation ofPieris brassicae imaginal wing discs: Effects and metabolism of ecdysone and ecdysterone

Summary Imaginal wing discs ofPieris brassicae can be cultured in vitro for extended periods. Their ultrastructural development was investigated after culture in the presence of various concentrations of ecdysone and ecdysterone. When ecdysone or low concentrations of ecdysterone (2×10^–7 M) were added to culture media, larval discs secreted a pupal cuticle and they subsequently differentiated scales; prepupal discs completed their imaginal development. With a higher concentration of ecdysterone (2×10^–6 M), all discs produced abundant but fragmentary cuticular material.Prepupal discs were able to metabolize both hormones in vitro. Ecdysterone was mainly converted into a polar compound detectable after a short period of incubation. Ecdysone was transformed, at a slower rate, forming a polar compound and 26-hydroxyecdysone but no ecdysterone.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Interactions between beta-ecdysone and fat body during wing disk development in vitro

We have investigated the actions of beta-ecdysone and fat body on wing disks of Plodia interpunctella in a series of sequential incubations in vitro. These experiments revealed that extended treatment times of beta-ecdysone at concentrations of 0·5 μg/ml or greater inhibited development of disks, and confirm that the presence of a fat body factor in the culture medium prevents this inhibition.     

Effects of beta-ecdysone and juvenile hormone on the Na+/K+-dependent ATPase in imaginal disks of Drosophila melanogaster.

The evagination of imaginal disks of Drosophila melanogaster is induced in vitro by β-ecdysone and inhibited by juvenile hormone. The possibility that these hormones act by changing intracellular Na⁺ and K⁺ levels was investigated by studying their effects on the sodium-potassium dependent adenosinetriphosphatase (NaK ATPase), an enzyme with a major rôle in regulating Na⁺ and K⁺ levels in cells. We find that β-ecdysone has no effect on this enzyme and can induce evagination even when intracellular Na⁺ concentrations are increased 2 to 3 fold by ouabain. Juvenile hormone stimulates the enzyme, but still acts to inhibit evagination when NaK ATPase activity is inhibited by ouabain. We conclude that the actions of β-ecdysone and juvenile hormone on imaginal disk evagination do not directly involve the NaK ATPase or require specific changes in Na⁺ and K⁺ concentrations.     

Cuticle deposition in imaginal disks of three species of Lepidoptera: Effects of ecdysones in vitro

We report the effects of three ecdysone analogues in the presence and absence of fat body on the wing imaginal disks of Cadra cautella, Paramyelois transitella, and Plodia interpunctella cultured in vitro in a modified Grace's medium. Alpha-ecdysone, unlike beta-ecdysone, did not stimulate cuticle deposition in Plodia disks at low doses and was only slightly effective at high doses. Also, the response of wing disks of Cadra and Paramyelois to beta-ecdysone and alpha ecdysone with and without fat body was similar to that of Plodia. Combinations of alpha-ecdysone and beta-ecdysone were more effective than beta-ecdysone alone in Plodia disks, but a synthetic analogue, 22-iso-ecdysone, had no effect on cuticle deposition when it was used alone or with beta-ecdysone in the presence or absence of fat body. Thus, the results with alpha-ecdysone and beta-ecdysone were not non-specific steroidal effects.     

Control of cuticle formation by wing imaginal discs in vitro.

Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [³H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro.     

Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro

Fragments of imaginal discs of the fruitfly Drosophila undergo growth and pattern regulation when cultured in vivo in adult female hosts for several days prior to metamorphosis in host larvae. Pattern regulation results in either regeneration of excised pattern elements or duplication of elements whose fate map positions lay within the fragment. Initial wound healing along the cut edge of a fragment is thought to be a crucial first step in the process of pattern regulation. We have examined the capacity for wound healing and pattern regulation of fragments (distal halves) of the wing disc cultured in vitro, using the culture system recently reported to support extensive growth and transdetermination of slightly wounded whole imaginal discs in vitro. Our results suggest that disc fragments and whole discs apparently respond differently in the culture system. With disc fragments, wound healing did not occur in vitro. When fragments were first cultured overnight in adult female hosts to allow initial wound healing prior to explantation in vitro, then some volume increase and regeneration of excised portions occurred during 2–3 weeks of culture in vitro. The extent of apparent growth was much less than that reported for whole discs, and the frequency of regeneration in vitro (19%), while highly significant relative to controls not cultured in vitro (0%), was much less than that observed for fragments cultured in vivo (84%). Furthermore the extent of regeneration which occurred in vitro was considerably smaller than that which occurs during regeneration in vivo.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

Plant regeneration from immature seeds of Eugenia myrtifolia Sims.

Eugenia myrtifolia Sims. is an evergreen shrub, native to temperate and tropical rainforests of Australia, which is becoming an important containerized ornamental plant in the US and Mediterranean nursery industries. To satisfy the growing market demands for this new ornamental plant, development of an accelerated propagation method is required. The goal of this study was to investigate the in vitro regeneration potential of E. myrtifolia Sims. seeds at different stages of development, towards establishment of an in vitro multiplication system. Maximum regeneration of adventitious shoots was achieved from immature seeds cultured in the dark on half-strength Murashige and Skoog (MS) macronutrients and full-strength MS micronutrients and vitamins (MS/2) medium supplemented with 2.5 μM thidiazuron (TDZ). Induction of regeneration occurred after at least two successive subcultures on TDZ-enriched medium, followed by subcultures on expression medium (hormone free MS/2) or multiplication medium [MM; MS medium enriched with 4.4 M 6-benzyladenine and 0.05 M α-naphthaleneacetic acid], where complete development of shoots occurred. The regenerated shoots were excised and transferred again onto MM for micropropagation, where a proliferation rate of 1:4 was achieved, and finally the shoots were transferred to a hormone-free MS medium for rooting. Following ex vitro transplanting, acclimatization over a period of 15 d was sufficient to establish greenhouse plants. The regenerated plants grown in the field for more than 2 yr showed the same phenotype as that of mother plants. The adventitious regeneration and micropropagation carried out in this study can be used for a large-scale propagation and genetic engineering of E. myrtifolia Sims.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.     

Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella

Fat body promoted ecdysone induced morphogenesis in Galleria wing disks cultured in vitro. Medium preincubated with fat body and α-ecdysone from any of the first 5 days of the final larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the final larval instar responded equally well to the fat body and α-ecdysone. We suggest a physiological rôle for Galleria fat body as an intermediary in the stimulation of wing disk development by α-ecdysone.