Modulation of the ecdysteroid-induced cell death by juvenile hormone during pupal wing development of Lepidoptera

Females of the tussock moth Orgyia recens have only vestigial wings, whereas the males have normal wings. We previously found that ecdysteroid induces both apoptotic events and phagocytotic activation in sex-specific and region-specific manners. To investigate whether different responses to ecdysteroid are controlled at the receptor level, we cloned ecdysteroid receptor isoforms, EcR-A and EcR-B1, in O. recens. In both male and female wings, EcR-A signal was detected in the distal region of the bordering lacuna (BL), whereas EcR-B1 signal was detected in the proximal region of the BL. The similar expression patterns of both EcR isoforms suggested that molecules other than EcR should be involved in different ecdysteroid responses between male and female of O. recens. We next tested juvenile hormone (JH) effects on pupal wing morphogenesis in O. recens. Interestingly, both JH and 20E addition induced wing degeneration not only in females but also in males. In addition, higher concentration of JH pre-treatment of the pupal wings of the silkworm, Bombyx mori, also caused wing degeneration under ecdysteroid treatment. These results indicate that JH modulates the ecdysteroid action to induce the cell death on pupal wings, generally in Lepidoptera.     

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 μg/ml) and 20E (1 μg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.     

Transition from larva to pupa: morphogenesis,cell proliferation and protein synthesis in Bombyx wing disc

Summary

In the present study, an attempt was made to clarify the timing of the transition from proliferation stage to pupal preparation stage of Bombyx wing discs and the correlation of its timing and ecdysteroid release. An histological study was carried out during the period of ecdysteroid increase in hemolymph in the last larval instar. The number of mitoses in wing discs from the feeding stage to the spinning stage was counted. During the feeding stage, the number of mitoses was about 200/disc, with the number increasing up to about 850/disc on the day of the beginning of spinning (S0); it then decreased to about 250/disc at the S1 stage, and finally mitotic figures were absent at S2. Comparing the mitotic number with hemolymph ecdysteroid titer, ecdysteroid during the high mitotic period is close to 100ng/ml, while ecdysteroid during the low mitotic period is about 10ng/ml at day 5 and 500ng/ml at S1, respectively. Two-dimensional gel electrophoresis was carried out to identify the ecdysone-induced polypeptides or the polypeptides synthesized during the spinning stage. Five ecdysteroid-inducible polypeptides were observed during the spinning stage or after 20-hydroxyecdysone (20E) treatment. From these findings, low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation and induce new protein synthesis.     

The costae presenting in high-temperature-induced <Emphasis Type="Italic">vestigial</Emphasis> wings of <Emphasis Type="Italic">Drosophila</Emphasis>: implications for anterior wing margin formation

It has long been noted that high temperature produces great variation in wing forms of the vestigial mutant of Drosophila. Most of the wings have defects in the wing blade and partially formed wing margin, which are the result of autonomous cell death in the presumptive wing blade or costal region of the wing disc. The vestigial gene (vg) and the interaction of Vg protein with other gene products are well understood. With this biochemical knowledge, reinvestigations of the high-temperature-induced vestigial wings and the elucidation of the molecular mechanism underlying the large-scale variation of the wing forms may provide insight into further understanding of development of the wing of Drosophila. As a first step of such explorations, I examined high-temperature-induced (29°C) vestigial wings. In the first part of this paper, I provide evidences to show that the proximal and distal costae in these wings exhibit regular and continuous variation, which suggests different developmental processes for the proximal and distal costal sections. Judging by the costae presenting in the anterior wing margin, I propose that the proximal and distal costal sections are independent growth units. The genes that regulate formation of the distal costal section also strongly affect proliferation of cells nearby; however, the same phenomenon has not been found in the proximal costal section. The distal costal section seems to be an extension of the radius vein. vestigial, one of the most intensely researched temperature-sensitive mutations, is a good candidate for the study of marginal vein formation. In the second part of the paper, I regroup the wing forms of these wings, chiefly by comparison of venation among these wings, and try to elucidate the variation of the wing forms according to the results of previous work and the conclusions reached in the first part of this paper, and provide clues for further researches.     

Morphological and histological examination of short‐wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera,Geometridae)

Winter geometrid moths exhibit sexual dimorphism in wing length and female‐specific flightlessness. Female‐specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short‐winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short‐winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval‐pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two‐thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.     

Programmed cell death in the wing of Orgyia leucostigma (Lepidoptera: Lymantriidae)

Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.     

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.     

Developmental profile of annexin IX and its possible role in programmed cell death of the Bombyx mori anterior silk gland

During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

Female-specific wing degeneration is triggered by ecdysteroid in cultures of wing discs from the bagworm moth, Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, are completely wingless; by contrast, the male adults have functional wings. Sex-specific differences in the development of wing discs appear to arise during the 8th (penultimate) larval instar. We have previously found that the wing discs of female E. variegata terminate development and disappear during the prepupal period, whereas the wing discs of males continue to develop fully into adult wings. We have investigated the effects of ecdysteroid (20-hydroxyecdysone, 20E) when cultured with larval wing discs, which are normally attached to the larval integument of both male and female larvae. Male wing discs cultured with 20E undergo a remarkable transformation: the discs undergo apolysis and then differentiation. Female wing discs cultured with 20E also undergo apolysis; however, the disc cells enter apoptosis. We have observed condensed chromatin, fragmented nuclei, and secondary lysosomes in the epithelial cells of these female discs. This report establishes that the reduction of female wing discs arises through apoptotic events triggered by ecdysteroid in vitro.     

Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.     

Ecdysteroid promotes cell cycle progression in the Bombyx wing disc through activation of c-Myc

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle–related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.     

Ecdysteroids control eyespot size and wing color pattern in the polyphenic butterfly Bicyclus anynana (Lepidoptera: Satyridae)

The strongly polyphenic African butterfly, Bicyclus anynana, shows conspicuous ventral eyespots and a transverse band in the wet-season form and small eyespots and no band in the dryseason form. These forms are produced when larvae are reared at high and low temperatures, respectively. Truncation selection was applied to a stock population (UNSELECTED-LINE) to produce lines which, at a constant intermediate temperature of 20 °C, always produced the dry season form (LOW-LINE) and the wet-season form (HIGH-LINE) in addition to a line of fast development (FAST-LINE). A relationship between wing pattern and development time was apparent: the FAST-LINE displayed larger eyespots and HIGH-LINE pupae developed faster (mean = 12.5 days) than LOW-LINE pupae (14.1 days). Differences were found among the lines in ecdysteroid titers after pupation. Hemolymph ecdysteroids in HIGH-LINE pupae increased earlier and reached twice the level of those in LOW-LINE pupae during the first 3 days after pupation. FAST-LINE pupae developed faster (11.7 days) than UNSELECTED-LINE pupae (12.8 days) and ecdysteroids in the FAST-LINE increased more quickly and reached higher levels. In the four LINES, ecdysteroid titers in 3 day old pupae were in the order UNSELECT ≈ LOW ⪡ FAST ⪡ HIGH. Thereafter the titers overlapped.An injection of 20-hydroxyecdysone (20E) inhibited pupal development at a dose between 2.5 and 5 μg when it was injected into pupae within 24 h after pupation. At lower doses (0.25–0.5 μg 20E) 22–100% of the pupae in different experimental groups in the LOW- as well as in the HIGH-LINE developed successfully. The pupal stage was significantly shortened, especially in the LOW-LINE. Additionally, 0.25 and 0.5 μg 20E injected into 0–12 h LOW-LINE pupae shifted the wing color pattern towards the wet season form: eyespots increased in size and the transverse wing band appeared in the more conspicuous pattern characteristic of the wet season form. The results demonstrate that ecdysteroids appearing early in the young pupa produce the wet season form of the wings. The same hormonal system mediates both developmental time and wing pattern determination.     

The polarity of axon growth in the wings of Drosophila melanogaster

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.     

Restoration of wing margins in Lyra mutants of Drosophila melanogaster

Lyra is a dominant, homozygous lethal mutation of Drosophila melanogaster; in heterozygotes the wings lack portions of the anterior and posterior margins including the characteristic bristles. We have found that, in addition to the loss of bristle forming cells, there is a decrease in the number of wing surface cells that varies between 10 and 20%. However, we observed no histological evidence of excessive cell death in either the larval discs or the pupal wing precursors in Lyra flies. Restoration of all or part of the normal wing margins occurs in some, but not all, cases of morphogenetic mosaics, in which there were patches of wild-type cells in Lyra wing margins due to irradiation-induced mitotic recombination. Analysis of these restorations, using margin bristles as indicators, shows that the Lyra wild-type gene is not involved in bristle formation per se and further that its expression is not cell autonomous. Instead the effect of the Lyra mutation appears to be associated with development of a margin forming subpopulation of cells and to influence the characteristic pattern of cells and bristles in the wing margin via an inductive interaction. The dorsal-ventral boundary can be demonstrated in the de facto wing margins of Lyra mutants suggesting that its origin is independent of any function Lyra might have in normal wing margin morphogenesis. In wing margin restorations the dorsal-ventral boundary is clearly delimited by trichomes and somewhat less rigorously shown by the margin bristles. Further, in these restorations ventral clones induce dorsal bristles, as well as ventral ones, and vice versa, indicating that the influence of Lyra is not restricted by the dorsal-ventral boundary.     

Color pattern formation on the wing of the butterfly Pieris rapae. 1. Cautery induced alteration of scale color and delay of arrangement formation

Experimental approaches to color pattern formation of lepidopteran insects have been made exclusively by analyzing pattern alterations in adult wings induced by operations. We microcauterized the presumptive black region of the dorsal forewing of the butterfly Pieris rapae and analyzed not only the resultant color pattern in the adult wing but also the cell behavior in the pupal wing epidermis around the injury. Cautery induced color alterations were as follows: (i) cautery up to 49.5 h after pupation resulted in white regions appearing within the black region while later cauteries induced larger white regions; (ii) cautery between 50 and 59.5 h resulted in the white regions induced by the cauteries being dramatically decreased; (iii) cautery after 60 h resulted in white regions that had almost disappeared. The examination of the cell behavior in the pupal wing epidermis after cauteries showed that the row formation of scale precursor cells was delayed. This delayed area varied with the time of cautery, in the same manner as that in the induced white area in the adult wing ((i) – (iii) above). The relationship between scale color alteration and the developmental delay of the scale row formation is discussed.     

The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.     

Commencement of pupal commitment in late penultimate instar and its hormonal control in wing imaginal discs of the silkworm, Bombyx mori

Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis. Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E, indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene (JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to begin the process and 20E is the factor that drives the discs to enter the reversible state.     

Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial–mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation.     

Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.