Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

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.     

Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm, Manduca sexta

During the fifth larval instar of Manduca sexta the commitment of the epidermis to the synthesis of pupal cuticle is presumably affected by a small increase in ecdysteroid titre when juvenile hormone levels are minimal. Two sequential rounds of DNA synthesis without an intervening mitosis occur at about this time, resulting in polyploidy of the epidermis. There is a definite temporal correlation between the first peak of ecdysone and the second round of DNA synthesis and indirect evidence has been presented which suggests that this small increase in ecdysteroid titre actually initiates the second period of DNA synthesis. Further, it appears that large doses of ecdysteroids do not elicit the same response as smaller doses at a specific developmental stage, indicating that the different physiological effects of ecdysteroids (reprogramming and apolysis) may be dependent upon the relative concentration of the hormone. Following mitosis which takes place on approximately day 6 of the last instar, the epidermis undergoes apolysis and secretes pupal cuticle, expressing the commitment made 4.5 days earlier. These results support the ‘quantal mitosis’ theory of cytodifferentiation since the covert differentiative event occurs during a period of DNA synthesis and since mitosis precedes the expression of that event.     

Reprogramming in the absence of DNA synthesis in Galleria larval epidermis.

Larval epidermal cells from a day-1 penultimate instar Galleria larva on implantation into day-5 last instar larva metamorphose and deposit a pupal cuticle at the same time as the host pupates. DNA synthesis in the implanted larval cell was monitored with 3-H-thymidine. Various regimens of 3-H-thymidine application were used and under no conditions did the larval cells incorporate label during the period from implantation to deposition of pupal cuticle. This suggests that a wax moth larval ectoderm cell can reprogram its genome to secrete a pupal cuticle without a precedent cell division.     

The ecdysoid RH5849 induces yolk polypeptide synthesis in male flies

Summary

RH5849 is a benzoyl hydrazine analog which has been reported to mimic several effects of the arthropod steroid hormone ecdysone to which it is chemically totally unrelated. In adult Diptera, ecdysone is the hormone that triggers vitellogenin synthesis. We report here that RH5849, upon oral ingestion, is able to induce vitellogenin synthesis in male Drosophila, Neobellieria, Phormia and Lucilia. This contrasts to data in the literature which showed that RH5849 could not mimic the pupariation-inducing effect of ecdysone in last instar fly larvae. RH5849 neither exerts a juvenile hormone mimicking effect nor behaves as an anti-juvenile hormone in both the Colorado potato beetle and Galleria.     

Inhibition of fatty acid desaturation impairs cuticle differentiation in Drosophila melanogaster

Previously, we showed that inhibition of the activity of fatty acid desaturases (Desat) perturbs signalling of the developmental timing hormone ecdysone in the fruit fly Drosophila melanogaster. To understand the impact of this effect on cuticle differentiation, a process regulated by ecdysone, we analysed the cuticle of D. melanogaster larvae fed with the Desat inhibitor CA10556. In these larvae, the expression of most of the key cuticle genes is normal or slightly elevated at day one of CA10556 feeding. As an exception, expression of twdlM coding for a yet uncharacterised cuticle protein is completely suppressed. The cuticle of these larvae appears to be normal at the morphological level. However, these animals are sensitive to desiccation, a trait that according to our data, among others, may be associated with reduced TwdlM amounts. At day two of CA10556 feeding, expression of most of the cuticle genes tested including twdlM is suppressed. Expression of cpr47Eb coding for a chitin‐binding protein is, by contrast, highly elevated suggesting that Cpr47Eb participates at a specific compensation program. Overall, the cuticle of these larvae is thinner than the cuticle of control larvae. Taken together, lipid desaturation is necessary for a coordinated deployment of a normal cuticle differentiation program.     

Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius

The haemolymph ecdysteroid titre of the last larval and pupal stadia of Calpodes ethlius was determined by radioimmunoassay. During the last larval stadium, four significant ecdysteroid peaks are present, two of which have been reported for other Lepidoptera. The first peak occurs 12 hr after ecdysis and correlates temporally with nucleolar activity, RNA synthesis and organelle formation in the fat body and epidermis. It correlates also with fat body DNA synthesis, polyploidy and the initiation of a low rate of lipid synthesis. Another peak, at 78 hr, starts its increase when the prothoracic glands no longer require the influence of the brain to produce ecdysone for pupation, and marks the first critical period. It correlates with the initiation of epidermal DNA synthesis and mitosis, and with the progressive determination of pupal characteristics (change in commitment, reprogramming). This ecdysteroid peak may also be involved in the massive intermoult syntheses in the epidermis (lamellate cuticle, wax) and the fat body (lipid, protein). The largest ecdysteroid peak is seen at 162 hr, 6 hr after the tissues no longer require the prothoracic glands for pupation (second critical period). It correlates temporally with the cessation of massive synthetic activity in both epidermis and fat body and initiates preparation for pupal synthesis in both tissues. At this time the ratio of ecdysone: 20-hydroxyecdysone is ～ 1 : 6.6.In common with other Lepidoptera, a single large ecdysteroid peak occurs during the first half of the pupal stadium. Comparisons between these events and the ecdysteroid titre are made between Calpodes and other insects.     

Qualitative and quantitative analyses of juvenile hormone and ecdysteroids from the egg to the pupal molt in Trichoplusia ni

A method was developed to determine in the same extract juvenile hormone and various types of ecdysteroids in precisely staged eggs and larvae of Trichoplusia ni. Ecdysteroids were tentatively identified on the basis of their retention time in ion suppression reversed-phase HPLC and their cross-reactivity with two relatively non-specific, complimentary antibodies, whereas juvenile hormone was identified using reversed-phase HPLC combined with Galleria bioassay. Freshly laid eggs contained low levels of immunoreactive ecdysteroids. Mid-polar ecdysteroids increased in the phase of segmentation (14-18 h) and 1st larval cuticle formation (36-44 h), when 20-hydroxyecdysone and 20,26-dihydroxyecdysone were found to be predominant. Only traces of ecdysone and 26-hydroxyecdysone were seen. Toward hatching ecdysteroids decreased and represented mainly compounds more polar than 20,26-dihydroxyecdysone. In larval development ecdysteroids were low at the beginning of the feeding phases, increased toward cessation of feeding, and reached highest levels 12-15 h before ecdysis. In feeding stages ecdysone and 20-hydroxyecdysone were predominant, whereas in molting stages they were seen together with 20,26-dihydroxyecdysone and 20-hydroxyecdysonoic acid. The juvenile hormone titer was very low in freshly laid eggs and was high (approximately 25 ng/g) in embryos at the stage of 1st larval cuticle formation and eye pigmentation. In eggs we tentatively identified juvenile hormones I and II, whereas in larval stages juvenile hormone II appeared to be the predominant or exclusive juvenile hormone. Its titer fluctuated rapidly and was high in early 1st-instar larvae and again before the molts into the 3rd, 4th, and 5th instar. Highest titers were reached concomitant with the peak in 20-hydroxyecdysone 12-15 h before ecdysis.     

Reduced titres of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica

Changes in ecdysone titre of the larvae of the German cockroach, Blattella germanica, exposed continuously to the juvenile hormone (JH), or to the insect growth regulator (IGR) with JH activity, can be correlated with the nature of the substance applied, its dose, and the time of application. The younger larvae exposed to the high dose of the IGR die in the next ecdysis, whereas the same treatment induces a diapause-like stage of developmental arrest in the last larval stage. The affected larvae have very little or no ecdysone, the synthesis of which takes place in the second part of the instar. The same treatment after this period has a lesser effect. The extent of the effect is correlated to the amount of ecdysone synthesized before the application of IGR. Last instar larvae exposed to the lower dose of the IGR or JH lack the peak of ecdysone normally found in the controls at the end of the second third of the instar when metamorphosis takes place. In these insects the first rise of the ecdysone titre begins towards the end of the instar, and ecdysis into the supernumerary larval stage is initiated when the ecdysone titre reached a level permitting ecdysis.A direct or indirect antagonism between these hormones, both fundamental to insect development, can explain the morphogenetic, inhibitory, and lethal effects observed in insects treated with JH or IGR with JH activity.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro.

This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro. Beta-ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined. Beta-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by cultured P.interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated.timulated both evatination and cuticle deposition of wing discs of Plodia interpunctella(Hubner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hour pulse of beta-ecdysone (0.5-5.0 mug/ml); (b) continuous treatment with 0.2 mug/ml beta-ecdysone; or (c) continuous treatment with 0.5 to 50.0 mug/ml beta-ecdysone in medium conditioned with larval fat body...     

Chitin synthesis in larval and pupal epidermis of the Indian meal moth,Plodia interpunctella (Hübner), and the greater wax moth,Galleria mellonella (L.)

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.     

Changes in the Activity and Pattern of Hemolymph Esterases in the Larvae of Greater Wax Moth Galleria mellonellaL. (Lepidoptera, Pyralidae) during Mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonellalarvae infected by the fungi Metarhizium anisopliae, Beauveria bassianaor Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.     

Epithelial polarity and cell separation in the neoplastic l(1)dlg-1 mutant of Drosophila.

Lethal (1) discs-large-1 [l(1)dlg-1] is a non-epithelial overgrowth or neoplastic mutant of Drosophila, which results in tumor-like imaginal discs and enlarged larvae that never pupariate. In an ultrastructural analysis we found that the wing discs develop convoluted monolayers of epithelial cells characterized by well-defined apical-basal polarity and that these layered cells secrete large amounts of basement membrane material. Immuno-EM indicates that Drosophila laminin and collagen are components of this matrix. Late in development clusters or 'rosettes' of separated cells lacking cell-cell junctions and apical-basal polarity form. In in vitro culture experiments l(1)dlg-1 wing discs did not respond to a pulse of exogenous ecdysone by secreting cuticle or losing basement membrane as normal discs do. Our observations are consistent with the hypothesis that cell-cell interaction and communication is required for termination of disc cell proliferation, which must occur prior to a cellular response to ecdysone.     

"EXTRANUMERARY" CUTICLE DEPOSITION IN PIECES OF LARVAL INTEGUMENT TRANSPLANTED TO A PUPA IN THE SILKWORM BOMBY X MORI

The author (K ato , 1973b) reported that in Bombyx mori , pieces of larval integument, if transplanted to a pupa, produced two layers of cuticle during the metamorphosis of the host pupa to an adult. In the present transplantation experiments, deposition of the two layers of cuticle was temporally examined to determine the timing of the two depositions, compared with the host's cuticle deposition, using 0-day-old fifth-instar or 1-day-old fourth-instar larvae as donors. In the transplant the first deposition occurred within 4 days of transplantation, with the second deposition at 6 to 8 days, while the host began cuticle deposition 6 days after receiving the transplant. These results show a failure of simultaneous deposition between the transplant and the host. These moulting events in the transplant, e.g. , the fairly early appearance of the first cuticle and temporally-separated ecdyses, are discussed in view of the change of ecdysone titer in the host. Furthermore, the temporal pattern of cuticle deposition in the transplant appeared to have a definite relationship with the type of cuticle deposited.     

Action in vivo d'ecdysones sur la morphogénèse imaginale d'Aeshna cyanea (Odonata)

Single amounts of α or β ecdysone were injected during the last larval instar of Aeshna cyanea at various times after ecdysis. In these experimental conditions, α and β ecdysone had similar effects. Very large amounts of brown or black cuticle appeared on the tarsal claws soon after hormone injection, so that the cuticular synthesis of the larvae which were injected at the beginning of the last stage appears about two or three times more quickly than in controls. Nearly all the larval characters were exhibited by animals injected on the day of or the day after the last larval ecdysis. If the hormonal injection was further delayed, only adultoid forms were obtained. No perfect adults appeared. The effects evoked by α or β ecdysone may be different from one organ to another.On the other hand, some results were different according to the type of ecdysone. Darkening of the tarsal claws (and perhaps sclerotization) appears sooner when β ecdysone is supplied. The morphology of the external organs which degenerate during metamorphosis is not always the same after injection of equal amounts of α or β ecdysone at the same time. The regression of the larval organs seems to be more explicit and appears sooner when β ecdysone was administrated. The morphogenesis of the organs which grow during metamorphosis was either weaker or non-existent with β ecdysone.These results are discussed with regard to previous work.     

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.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

Synthesis of giant HnRNA in the epidermal cells of Calliphora and the role of the ring gland.

The appearance of giant HnRNA in the epidermal cells of Calliphora in the transition period from larva to pupa is ecdysone-dependent. In this specific stage of development, the initial increase of this RNA species occurs shortly after the appearance of a small peak of ecdysone. Another main peak of the hormone is a prerequisite for pupation. The removal of the ring gland, the source of ecdysone, prior to the appearance of the second peak of the hormone, was found to have no pronounced effect on HnRNA synthesis for at least 48 h after the operation. It is concluded that the larval pupal differentiation is a two-step process. Initially a gen activation occurs dependent on a low ecdysone titer. This is followed by a second final step of pupal cuticle synthesis and pupation, dependent on the presence of a high concentration of the hormone.