Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

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.     

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

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

Role of juvenile hormone in regulating tyrosine glucoside synthesis for pupal tanning in Manduca sexta (L.)

Tyrosine glucoside (α-d-glucopyranosyl-O-l-tyrosine) serves as a reservoir of tyrosine and glucose for pupal and adult cuticle formation, tanning, and pigmentation in several Lepidopteran insects. In the tobacco hornworm, Manduca sexta (L.), detectable quantities appear in the haemolymph 1–2 days after ecdysis of the fifth instar and very high concentrations accumulate between the fourth and eighth days of the stadium. If juvenile hormone II or a mimic (methoprene) is injected into fifth-instar larvae at 24-h intervals after ecdysis, tyrosine glucoside synthesis is almost completely suppressed. Temporary starvation of newly ecdysed larvae that results in the maintenance of a high endogenous juvenile hormone titre, also suppresses and delays the onset of tyrosine glucoside synthesis. The decrease and eventual disappearance of juvenile hormone after ecdysis of the last-larval instar appears to be a necessary prerequisite for the synthesis or activation of tyrosine glucoside synthetase along with the initiation of other metamorphic events.     

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

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

Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (approximately day 3.5). At about days 6--7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of edcysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.     

Caste-specific differences in ecdysteroid titers in early larval stages of the bumblebee Bombus terrestris

Mounting evidence implicates ecdysteroids in queen-worker differentiation during the last larval instars of highly social insects. In the present study, we analyzed ecdysteroid titers in queen and worker larvae of the bumblebee Bombus terrestris from the second to the early fourth instar. B. terrestris is of particular interest because caste is already determined in the second instar, presumably by a pheromonal signal emitted by the egg-laying queen. Caste differences in the adults, however, are only expressed at the physiological and not at the morphological level, except for the distinctly larger size of the queen. In the second and third instar, ecdysteroid titers in queen larvae were generally higher than those of workers. These early caste-specific differences, however, were abolished in the fourth instar. In the early fourth instar we could detect two small ecdysteroid peaks, with the one preceding the cocoon-spinning phase presenting the characteristics of a pupal commitment peak. The synchrony of caste differences in ecdysteroid and juvenile hormone titers suggests a synergistic action of these hormones in caste determination.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Effects of juvenile hormone on some phase characteristics in the common cutworm, Spodoptera litura

Phase characters of the common cutworm, Spodoptera litura, were influenced by different rearing densities from the 4th-larval instar. Primarily the final feeding period of isolated larvae was 1 day longer than that of crowded larvae causing an increase in pupal weight. Applications of juvenile hormone I, II, or methoprene to crowded larvae caused an increased feeding period similar to that of isolated larvae when the juvenile hormones were applied within 1 day after the last-larval ecdysis. Allatectomy of isolated Spodoptera during the moult to the final-larval instar decreased the duration of the final feeding period to that of intact crowded larvae. These results suggested that one of the characters of phase variation, pupal weight, is influenced by the differences in the regulation and activity of the corpora allata during the last-larval instar. Other characteristics of phase variation such as behaviour (feigned death) and colour were not affected by alteration in juvenile hormone levels after the last larva ecdysis.     

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

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

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.     

Epoxide hydrase activity and its relationship to development in the southern armyworm, Prodenia eridania

Enzymatic epoxide hydration of the cyclodiene insecticide HEOM by the southern armyworm was investigated throughout the late larval, pupal and adult stages of development. Epoxide hydrase activity reaches a maximum in the period between ecdysis of the last instar and the larval-pupal ecdysis, decreases during pupal life and becomes essentially zero following adult emergence. Comparison was made with variations occurring in juvenile hormone hydrase activity, and the significance of these age-dependent changes in relation to regulation of insect development are discussed.     

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.     

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.     

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ? adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.