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.     

A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Ecdysteroid levels during adult reproductive and embryonic developmental stages of Sarcophaga bullata (Sarcophagidae: Diptera)

Ecdysteroid levels were determined during the period of the adult reproductive cycle in the ovovivparous fleshfly Sarcophaga bullata. Low levels were found in males during the 10 days following eclosion while the entire adult female showed a significant peak of ecdysteroid activity at 190 h post eclosion (i.e. during embryogenesis). When the female reproductive tract was analyzed it was observed that the ovaries became vitellogenic a short time after a protein meal was offered at 96 h post eclosion: at 140 h, ecdysteroid activity was recorded in the developing oöcytes. The major peak of ecdysteroids during the reproductive cycle was found in developing embryos at 190 h. The significance of these releases of ecdysteroids is discussed in relation to major embryonic developmental events.     

Developmental Arrest and Ecdysteroid Deficiency Resulting from Mutations at the Dre4 Locus of Drosophila

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.     

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant,an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

Larval moulting hormone of trichophoran Hemiptera-Heteroptera: Makisterone A,not 20-hydroxyecdysone

The haemolymph ecdysteroids were examined in fifth-stage larvae of Nezara viridula, Podisus maculiventris and Dysdercus cingulatus (Hemiptera-Heteroptera) using high-pressure liquid chromatography to separate the ecdysteroids and a radioimmunoassay to detect the fractionated ecdysteroids. The length of the fifth stage ranged from 5 to 8 days, and a peak in ecdysteroid titre (1700–2650 ng/ml) occurred 2–3 days prior to ecdysis to the adult. An ecdysteroid matching the retention time of makisterone A (24-methyl-20-hydroxyecdysone) was clearly present in haemolymph taken at the time of peak titre in all 3 of these true bugs, whereas little, if any, ecdysone or 20-hydroxyecdysone was detected. These data, along with previously reported data for the milkweed bug Oncopeltus fasciatus, are persuasive evidence that makisterone A is the larval moulting hormone of a group of closely related Heteroptera called the Trichophora (Lygaeoida, Pentatomoidea, Pyrrhocoroidea and Coreoidea).     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

Hormonal regulation of dopa decarboxylase during a larval molt

Cuticular sclerotization in insects requires dopamine derivatives and thus the presence of dopa decarboxylase (DDC), the enzyme which converts dopa to dopamine. During the last half of the larval molt of the tobacco hornworm, Manduca sexta, beginning at 16 hr after head capsule slippage, the epidermal DDC activity increased fourfold. By contrast, allatectomized larvae which were destined to produce a melanized cuticle showed a sevenfold increase. This increase in DDC activity was prevented by infusion of 20-hydroxyecdysone (20HE) into the larva, indicating that the fall of the ecdysteroid titer is necessary for the increase. In vitro 20HE also prevented the increase in a dose-dependent manner when the epidermis was explanted at 16 hr after head capsule slippage but had less effect on epidermis explanted 3 hr later. Both 5 micrograms/ml alpha-amanitin and 100 micrograms/ml cycloheximide also prevented the increase. Application of juvenile hormone I showed that the critical period for determination of the level of the later increase in DDC activity was about 4 hr after head capsule slippage at the peak of the ecdysteroid titer. Apparently then the rise and fall of ecdysteroid regulate different aspects of DDC synthesis, the rise determining its later appearance and the fall timing this appearance.     

Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster

The relative DNA content of Drosophila melanogaster imaginal leg disc nuclei during larval growth and pupal and adult differentiation was measured by microspectrophotometry. During the larval proliferative phase there were twice as many nuclei in the 4C class as nuclei in the 2C class. At the end of the third larval instar, the proportion of nuclei with a 4C DNA value increased. By 3 hr after pupariation, during pupal cuticle secretion, 90% of the nuclei were in this class. After pupal apolysis which occurs at 12 hr after pupariation, the 4C to 2C ratio was reversed. The increase in the proportion of nuclei with a 2C value was observed until 24 hr after pupariation when 90% of the nuclei were in this class. We propose that most cells divide at least once between pupal and adult differentiation. All of these changes in the cell cycle were correlated temporally with changes in the ecdysteroid titers that occur during these periods.     

Simultaneous determination of molting and juvenile hormone titers of the greater wax moth

A simple and rapid extraction procedure was developed to determine simultaneously the molting hormone (MH) and juvenile hormone (JH) activity in a single insect tissue sample. From the onset of the last larval stage to adult eclosion of the greater wax moth, Galleria mellonella, three JH peaks were noted: at the time of the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and at the time of adult eclosion. Three MH peaks were recorded for the male: at 1 day before the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and 2 days after pupation. In the female, a fourth peak was shown at the time of adult eclosion. This fourth peak exhibits the highest molting hormone activity of all samples, 1600 Musca units/g of fresh tissue or an equivalent of 5.6 μg/g of ecdysterone. Eighty per cent of this MH accumulated in the ovary. The significance of MH and JH titers as related to the endocrine regulation of development is discussed in the light of this finding.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Growth and respiratory enzyme activity in the prothoracic glands of Galleria

In the penultimate-larval instar, the total volume of the prothoracic gland and the activities of some oxidative mitochondrial enzymes (cytochrome oxidase, NADH: cytochrome c oxidoreductase, succinate: cytochrome c oxidoreductase) undergo cyclic variations associated with larval growth. These specifically larval-larval growth cycles are absent in the prothoracic glands of normal last-instar larvae. Here the cycles can be induced artificially by implantation of brain or corpora cardiaca-allata complexes or, by exogenous application of juvenile hormone. The smallest size of the prothoracic gland in relation to the size of the body, as well as the minimal activity of all the three mitochondrial enzymes in the gland, have been found exactly at the moment of the pre-pupal peak of ecdysteroid in the body. The possibility that the prothoracic glands alone can synthetize ecdysteroid during the peak is questioned.     

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.     

Juvenile hormone esterase activity in the pupating and diapausing larvae of Sesamia nonagrioides

The development of the Mediterranean corn borer, Sesamia nonagrioides, under long-day (LD) photoperiod is associated with juvenile hormone (JH) decline and pupation in the 5th or 6th larval instar. The larvae grown under short-day (SD) conditions maintain a moderate JH titer and enter diapause during which they undergo several extra larval molts. Both types of larvae exhibit similar levels of juvenile hormone esterase (JHE) activity that increases in each instar during the period of low ecdysteroid titer and drops when the titer rises to a molt-inducing peak. A suppression of JHE activity within 24h after application of an ecdysteroid agonist suggests that the drop of activity is a rapid and possibly direct response to ecdysteroids or their agonist. Esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) suppressed more than 98% of the JHE activity without affecting pupation timing and adult development. The data indicate that JHE is not crucial for the switch between larval development, diapause, and metamorphosis in S. nonagrioides.