Definition of a juvenile hormone-sensitive period in Rhodnius prolixus

This paper is an attempt to establish the times of onset and termination of the juvenile hormone-sensitive period for metamorphosis in fifth-instar larvae of Rhodnius. Small regions of the abdominal integument were exposed to discrete pulses of the juvenile hormone analogue ZR-515 (methoprene) by applying small drops of a mixture in paraffin to the dorsum at various times after a bloodmeal and removing these drops after different time intervals. The diffusion coefficient of the analogue in the integument was estimated and used together with estimates of its metabolism to determine the lag times between application of the analogue and its rise to above threshold concentration in the epidermis, and between removal of the analogue source and its fall to below threshold concentration in the epidermis. These lag times were estimated to be 1.5 and 24h, respectively. Knowledge of the lag times makes it possible to establish the limits of the juvenile hormone-sensitive period or metamorphosis from the responses of larvae to variously-timed pulses of the analogue. The juvenile hormone-sensitive period has the following properties. For the population as a whole it lasts from about day 3 to about day 9 after a bloodmeal. Any individual in that population, however, only requires the presence of juvenile hormone during a 2 to 4-day period. The exact duration of an individual's sensitive period within these limits is a stochastic event. Surprisingly, for any individual, a pulse of juvenile hormone is equally effective when experienced early as when experienced late during its juvenile hormone-sensitive period.     

Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male).This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Juvenile hormone and photoperiodically controlled polymorphism in Aphis fabae: Postnatal effects on presumptive gynoparae

Long days (short nights) (LD 16:8) and high temperatures (> 15°C) have an apterizing effect on the short day (LD 12:12) induced, presumptive gynopara of Aphis fabae. Transfer of presumptive gynoparae to long days (15°C) or to 25°C (short days) at varying times during postnatal development demonstrate that the adult form is determined by the second day of the second instar, i.e. 5 days after birth at 15°C. Transfer on day 1 induces maximum apterization with the proportion of aphids affected decreasing with age at transfer.Apterization induced by long days immediately after birth can, to some extent, be cancelled by return to short days but only up to day 4. Thus long days are morphogenetically more potent than short days at the beginning of larval development. At temperatures above 15°C the proportion of aphids apterized increases almost linearly.Apterized insects can be distinguished from juvenilized insects in the fifth-instar. Topical application of juvenile hormone (JH) induces both apterization and juvenilization of presumptive gynoparae but at different times during larval development, JH treatment during the early-instars promotes apterization but induces little juvenilization, whereas maximum juvenilization, without apterization, is produced by middle-instar treatment. The apterizing effects of JH are, thus, not due to its neotenic action.The response profile of JH-induced apterization is similar to that observed with long days and 25°C. It is suggested that such conditions increase endogenous JH levels in A. fabae. The three naturally occurring JH's differ in activity in the order JH I > JH II > JH III. Both long-day and JH-apterized insects switch from the normal ovipara production of the adult gynopara to vivipara production.     

Juvenile hormone and photoperiodically controlled polymorphism in Aphis fabae: Prenatal effects on presumptive oviparae

All three naturally occurring juvenile hormones (JH's) were shown to have effects on the parthenogenetic/gamic polymorphism of Aphis fabae; they mimicked long day conditions by inducing parthenogenetic forms. When topically applied to fourth instar gynoparae, JH caused the appearance of oviparous/viviparous intermediate morphs in the progeny. JH induced both wing development and embryogenesis in embryonic, presumptive oviparae. Embryogenesis was induced by lower doses of JH. Adult, embryo-containing alatae produced by treatment with high JH doses were capable of flight, and whilst reluctant to reproduce, their few viable progeny were oviparae. They did, however, differ from normal gynoparae in size, occasional presence of scent plaques on the metathoracic tibiae, numbers of secondary rhinaria on the antennae and morphogenetic response to postnatal rearing in long day conditions. The presumptive, oviparous embryos most sensitive to JH treatment were shown to be ca 323 μm in length, close to the stage where their germaria differentiate as parthenogenetic or gamic. Similar effects were observed in the progeny of JH-treated, teneral adult gynoparae but there was no effect on the morph of progeny of long day, alate virginoparae. The JH's differed in potency in the order JH I > JH II > JH III. The treatment of fourth instar gynoparae also induced a terminal batch of apparently normal viviparous progeny in a number of aphids. This result was obtained even at JH doses below threshold for the appearance of oviparous/viviparous intermorphs.     

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.     

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.     

mRNA changes during imaginal determination of the epidermal cells in the pupa ofGalleria mellonella L

Summary Changes in the mRNA population of the mesonotal epidermal cells were investigated inGalleria mellonella during the first 48 h after pupation. Total RNA was extracted and assayed by in vitro translation. The translational products were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. The changing banding pattern of the in vitro synthesized proteins indicates changes in the cellular pattern of mRNAs, most of which occur between 6 h and 18 h after pupal ecdysis. These changes mostly consist in the decrease or disappearance of bands. The injection of juvenile hormone (JH) immediately after pupal ecdysis does not qualitatively influence mRNA changes, but does alter their time course, for they are postponed for 6–12 h. After the injection of 20-hydroxyecdysone (20HE) the same changes can again be seen, but they are greatly accelerated. A comparison of these results with known data on the time course of reprogramming and ecdysteroid titre leads to the conclusion that the mRNA changes in the epidermal cells are a prerequisite for the renewed expression of a developmental programme. This is independent of whether, in the absence of JH, a new programme is determined or whether, under the influence of JH, the previous programme is restored. 20HE does not have any effect on the change in the developmental programme. The change seems to occur as an active and autonomous process in the epidermal cells, in accordance with a genetically fixed developmental programme.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

Activity of disconnected corpora allata in Locusta migratoria: Juvenile hormone biosynthesis in vitro and physiological effects in vivo

Severance of nervi corporis allati I (NCA I) in day-1 adult female Locusta migratoria resulted in a significant decrease and a loss of the characteristic pattern of juvenile hormone biosynthesis by the corpora allata as determined by radiochemical assay. This decrease in the rate of juvenile hormone biosynthesis was not reflected in basal oöcyte growth. The lengths of the oöcytes were the same in NCA-transectioned and in the sham-operated females. The effect of severance of both NCA I and NCA II on juvenile hormone biosynthesis and ovarian maturation was similar to the effect of NCA I severance only.Rate of juvenile hormone biosynthesis by corpora allata of fourth-instar larvae exhibited a maximum of activity in the middle of the stadium. The severance of NCA I early in the stadium resulted in a very low rate of juvenile hormone biosynthesis and a disappearance of this peak. In NCA I-transectioned larvae, the duration of the stadium was significantly increased although larvae moulted into normal fifth instar.     

Identification of the juvenile hormone from the cricket, Teleogryllus commodus, and juvenile hormone titre changes

In the cricket, Teleogryllus commodus, eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Tissue-specific effects of the juvenile hormone analogue ZR 515 during metamorphosis in Drosophila cell cultures

The juvenile hormone analogue ZR 515 has specific effects on ecdysone-induced metamorphic differentation of Drosophila cells cultured in vitro. The number of vesicles containing imaginal cuticular structures is reduced to 10% of control levels. Similarly, the differentiation of adult fat body is partly inhibited by ZR 515. The differentiation of adult tubular and fibrillar muscles, however, is not affected. ZR 515 does not inhibit cuticle secretion by tracheal cells and larval epidermal cells.     

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis

Background

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.

Methods

Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.

Results

We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.

Conclusions

In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.

General significance

Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.     

The activity of juvenile hormone mimics for the eggs of Rhodnius prolixus

Twenty four juvenile hormone (JH) mimics of several different chemical classes were applied to freshly laid eggs of Rhodnius prolixus. Activity of the JH mimics was assessed in terms of their ability to inhibit eclosion. All chemical classes tested except one of the natural JHs and two very closely related long chain compounds contained members which inhibited eclosion; the most active chemicals were derivatives of geranyl para-aminobenzoic acid esters and aryl methylene dioxyphenyl ethers. In general, those chemicals of a particular class which had highest morphogenetic activity when applied to last instar larvae, were also most active in inhibiting eclosion. However, not all structure/activity relationships of JH mimics for last instar larvae were applicable to eggs; in particular the presence of a terminal epoxide group increased activity greatly for eggs but only slightly for last instar larvae. The dose per unit weight to inhibit eclosion is far greater than the equivalent dose to disrupt normal metamorphosis.     

Effects of a juvenile hormone analogue on the duration of the fifth instar in the milkweed bug Oncopeltus fasciatus

Topical application of JHA to fifth instar nymphs of Oncopeltus fasciatus, immediately following ecdysis from the fourth instar, decreases the duration of the fifth instar by approximately 36 hr in addition to inducing a supernumerary larval moult. JHA appears to accelerate the time of subsequent ecdysis in two ways: first, the onset of ecdysone secretion is accelerated, and is accompanied by a similarly premature initiation of mitotic activity in epidermal cells. This is the classical prothoracicotropic action of JH. Second, the period between the onset of mitotic activity and the time of ecdysis itself is shortened. That is, once cellular activities associated with the moulting cycle are triggered by ecdysone, such activities are completed more rapidly in the presence of JHA. It appears that the larval-larval moult induced by JHA requires intrinsically less time to accomplish than a normal metamorphic moult.