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.     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

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.     

Titres of juvenile hormone I,II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Moulting and ecdysone release in response to electrical stimulation of protocerebral neurosecretory cells in Locusta migratoria

Locusta migratoria larvae were submitted to electrical stimulation of the protocerebral neurosecretory cells (median neurosecretory cells of the pars intercerebralis and lateral neurosecretory cells), during the last larval instar. The effects of the treatment were observed both on the duration of the stage and on the variations in haemolymph ecdysone levels. In untreated larvae, there was an initial ecdysone peak at the beginning of day 5, which was followed by 4 larger peaks between days 6 and 8. Stimulation of the median neurosecretory cells at the beginning of the instar resulted in the formation of one very large hormonal peak at the end of day 3: a day and a half earlier than in the control groups. Moulting was likewise accelerated. Stimulation also increased the size of the peaks, as compared with the controls. Stimulation of the lateral neurosecretory cells had a weaker ecdysiotropic effect; neither the number nor the size of the peaks were changed, though, like ecdysis, they occurred earlier. Stimulation of the deutocerebrum had no effect on either ecdysone titres or moulting. Electrical stimulation of the median neurosecretory cells at the end of day 5, that is after the occurrence of the first ecdysone peak, shortened the larval stage while having no significant effect on ecdysone levels in the haemolymph. The neuroendocrine control of ecdysis in Locusta is discussed.     

Hormonal regulation of pupation in the codling moth, Laspeyresia pomonella

ABSTRACT. According to the different reactions to the juvenoid Altosid®, the last larval instar (L₅) of Laspeyresia pomonella (L.) (Tortricidae) reared under 'long day' conditions (constant light) was subdivided into three sensitive phases: an additional larval instar, a larval–pupal intermediate, or a pupa. Under short day conditions, the prothoracotropic effect of juvenile hormone (JH) in L₅, which have a continuous high titre of JH during the whole instar, indicated that it is not a particular titre of JH but a rise in the titre that can induce the production of moulting hormone. Neck-ligation experiments showed that JH acts not directly on the prothoracic glands but via the head, probably via the neurosecretory system. The meaning of the JH-peak in mature L₅ reared under long days was determined either by injections with the anti-JH, precocene II, in combination with applications of Altosid, or by forcing precocene-treated larvae to a precocious moult by injecting them with ecdysterone. Precocene delayed, and JH accelerated pupation if administered 4.5 days after the L₅ -moult. JH was also found to stimulate the growth and differentiation of the imaginal discs. Moulting hormone in long-days reared insects was detected one day after the larvae had spun their cocoon, with a maximum on the second day after spinning. The hormone was also present in freshly moulted pupae. Neck-ligation of mature larvae indicated that the delay between activation of the prothoracic glands and the production of an effective amount of moulting hormone is less than one day.     

Temporal organization of endocrine events underlying larval-larval ecdysis in the silkworm, Bombyx mori

The timing of ecdysis in the penultimate instar of Bombyx mori was demonstrated to be under the control of a circadian clock. The temporal organization of secretion of prothoracicotropic hormone (PTTH), ecdysone and juvenile hormone was studied with particular attention to the circadian control of the timing of hormone release. PTTH release occurs, at least, in the second and third night. The latter is responsible for evoking the larval ecdysis. Prothoracic gland initiates ecdysone secretion abruptly with a very short span after the second PTTH release and secrete enough amount of ecdysone for larval moulting, which takes place 11 h later. Juvenile hormone titer is relatively high before the second PTTH release and corpus allatum becomes dispensable for ensuring the larval moulting in 1.5 h. Based on these findings, interpretations for the endocrine system underlying precocious pupation and formation of intermediates, which are produced by neck ligation, are presented.     

Induction of supernumerary larval moulting in the tobacco hornworm Manduca sexta: interaction of bisacylhydrazine ecdysteroid agonists with endogenous Juvenile Hormone

Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle.     

Ecdysone metabolism and endogenous moulting hormone titre during larval-pupal development in Choristoneura fumiferana

The titre and metabolism of ecdysone were studied in the last larval instar of the spruce budworm, Choristoneura fumiferana. Both in males and females a distinct ecdysone peak is present just before ecdysis. Injection of radioactive α-ecdysone into the insect when the endogenous level is low results in the transformation of most of the injected hormone into 3-dehydro-α-ecdysone and conjugates, with very little conversion to β-ecdysone. Whereas when the labelled material is administered when the endogenous level is high, the α-ecdysone is for the most part converted to β-ecdysone. The significance of the correlation between the endogenous titre of ecdysone and the metabolism of injected α-ecdysone is discussed.     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Effect of exogenous JHI on imaginal determination in Aeshna cyanea

Last instar larvae of Aeshna cyanea injected with 8 μg or more JHI before the end of the fourth ocular stage (day 7 or 8 of the instar) became perfect supernumerary larvae which indicates that adult determination does not occur before this time. When JHI is applied later (days 9–13) larval-adult intermediates are formed. It seems, therefore, that imaginal programming takes place at the end of the fourth ocular stage and might be correlated with the first ecdysone peak which occurs at the same time.     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

Larval diapause of the European corn borer, Ostrinia nubilalis: Further experiments examining its hormonal control

The possible involvement of juvenile hormone (JH) in controlling the mature larval diapause of the European corn borer, Ostrinia nubilalis, was examined using biological and chemical assays for JH titres, topical applications of JH mimic, and injections of 20-hydroxy-ecdysone. Bioassays of extracts of larval haemolymph showed that (1) 4th instar pre-diapausing larvae had a higher JH titre (ca. 1450 Galleria Units (GU)/ml) than equivalent non-diapausing larvae (ca. 340 GU/ml), and that (2) 5th instar pre-diapausing larvae contained a JH titre of ca. 320 GU/ml, which declined to ca. 90 GU/ml in newly-diapaused larvae. Chemical assasys carried out on extracts of whole larvae showed that early diapausing larvae contained an extremely low titre of JH. In addition, the application of JH mimic or 20-hydroxy-ecdysone or both agents to diapausing larvae failed to reveal the presence of a functional JH titre during diapause. The application of JH mimic to early 5th instar non-diapausing larvae produced moribund larval-pupal intermediates rather than supernumerary larvae. Our results, therefore, suggest that although JH may control some phases of diapause induction, it is not involved in maintaining diapause.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. VI. Inhibition by juvenile hormones.

In the salivary gland chromosomes of late-third instar larvae and in late (8- to 12-hr) prepupae of Drosophila melanogaster, there are ecdysone-induced sequences of puffing patterns which can be reproduced in vitro. These two sequences are separated by a period when the glands are thought to be exposed to a low titer of β-ecdysone and during which they acquire the competence to respond to ecdysone at the late prepupal puff sites. Attempts to modify either the late larval or the late prepupal responses to ecdysone in vitro by the simultaneous addition of juvenile hormone (JH) with ecdysone, to larval or prepupal glands, respectively, are unsuccessful. If, however, JH (ca. 10^?6M) is added to larval glands cultured 6 hr in ecdysone and then 3 hr in JH alone, the subsequent induction of prepupal ecdysone puffs is inhibited. Thus the role of JH appears to lie in modifying the acquisition of competence to respond to ecdysone rather than in a direct antagonism between the two hormones.     

Choristoneura fumiferana entomopoxvirus prevents metamorphosis and modulates juvenile hormone and ecdysteroid titers

Larvae of the spruce budworm, Choristoneura fumiferana, infected with C. fumiferana entomopoxvirus (CfEPV) continue to feed and grow without undergoing metamorphosis and die as moribund larvae. The lethal dose (LD(50)) and lethal time (LT(50)) values for fourth instar larvae are 2.4 spheroids and 25.2 days, respectively. One hundred percent of the control fourth instar larvae, which were fed water instead of virus, pupated by 18 days post feeding (PF). Only 30% of the larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose pupated by 18 days PF. Of the control larvae, 95% became adults by 24 days PF, whereas in the treated group only 2% of larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose became adults by 24 days PF. Some of the virus-treated larvae died as either larval/pupal or pupal/adult intermediates. These phenotypic effects were similar to the larval/pupal and pupal/adult intermediates, resulting from treating larvae with juvenile hormone (JH) or its analogs, which suggests that EPV may cause such abnormalities by modulating JH and/or ecdysteroid titers. In untreated sixth instar larvae the JH titer decreased to low levels by 24 h after ecdysis and remained low throughout larval life. EPV-fed sixth instar larvae had 2112 pg/ml on day 0, 477 pg/ml on day 1 and 875 pg/ml on day 8 of the sixth instar. Control larvae contained 860 ng of ecdysteroids per ml hemolymph on day 8 of the sixth instar, whereas EPV-treated larvae of the same age (30 days PF) had only 107 ng of ecdysteroids per ml of hemolymph. Thus, EPV infection results in increased JH titer and decreased ecdysteroid titer. Northern hybridization analysis was performed using RNA isolated from control and EPV-fed larvae and cDNA probes for (i) juvenile hormone esterase (JHE), which is JH inducible, (ii) Choristoneura hormone receptor 3 (CHR3), which is ecdysteroid inducible, and (iii) larval specific diapause associated protein 1 (DAP1), whose expression is larval specific. EPV-treated larvae showed higher levels of JHE and DAP1 mRNA and lower levels of CHR3 mRNA, indicating that they had higher levels of JH and lower levels of ecdysteroids. Thus, our data show that EPV prevents metamorphosis by modulating ecdysteroid and JH levels.     

The metabolism of 3H-moulting hormone in Calliphora erythrocephala during larval development

The activity in whole insects for converting ³H-α-ecdysone to ³H-β-ecdysone after injection is low (half-maximal) in young last instar larvae, maximal in mature larvae, and minimal (fourth-maximal) at the white puparial stage. Because moulting hormone titre is low throughout the last larval instar and increases at the formation of the puparium it appears that hydroxylation at C-20 is not a key step in regulating β-ecdysone biosynthesis during larval development.The activity for catabolizing ³H-β-ecdysone is maximal in second instar larvae, about thirdmaximal throughout most of the third instar, and minimal at pupariation (thirtieth-maximal). Thus inactivation may play a rôle in regulating moulting hormone titre during larval development.     

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.