Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Juvenile hormone and ovarian growth in Manduca sexta

In Manduca sexta the major size increase of ovarian follicles is accomplished by two processes: (1) vitellogenesis in which follicular volume and dry weight increase simultaneously, and (2) hydration in which absorption of water by the oocyte accounts for an 80% increase in volume prior to chorion formation. Vitellogenic growth occurs in both a slow and rapid phase. Rapid vitellogenic growth is initiated only by follicles of a threshold size (1 mm) and is a juvenile hormone (JH)-dependent event. In the absence of JH follicles grow to 1 mm and then degenerate.     

Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta

The mechanisms of degradation of juvenile hormone esterase (JHE) were investigated in larvae of the tobacco hornworm, Manduca sexta. JHE is removed from the hemolymph by the pericardial cells by receptor-mediated endocytosis and is ultimately degraded in the lysosomes. Immunoprecipitation experiments and native PAGE followed by Western blotting showed that JHE associates with a putative heat shock cognate protein (Hsp). Approximately 25% of the active JHE in the pericardial cell complex is associated with the putative Hsp 1 h postinjection of affinity purified JHE. Electron microscope analysis revealed that the putative Hsp is located in the trans-Golgi network of pericardial cells, where it is hypothesized to be involved in sorting of proteins destined for the lysosomes, from those destined for the cell membrane. Data acquired from immunoprecipitation and Western blotting experiments argue against the involvement of ubiquitin in the degradation of JHE. Injection of radiolabeled JHE into larvae of M. sexta followed by SDS-PAGE of pericardial cell homogenates revealed covalent binding of an unidentified protein to JHE in the pericardial cell complex. Arch. Insect Biochem. Physiol. 34:275–286, 1997. © 1997 Wiley-Liss, Inc.     

重组保幼激素酯酶的纯化和生物学效应

培养昆虫细胞生产重组昆虫保幼激素酯酶时细胞培养液的蛋白质浓度为153.2～188.0 μg/mL。批量处理纯化重组保幼激素酯酶时酶蛋白活力回收率33%,效果与梯度分离方法相当,但简便快速,可作为大量分离纯化的第一步。重组保幼激素酯酶对烟草天蛾Manduca sexta幼虫的生物学活性测定结果验证了重组保幼激素酯酶对烟草天蛾幼虫和自身天然酶有相似的生物学活性。     

Effects of endogenous esterases and an allatostatin on the products of Manduca sexta larval corpora allata in vitro

Abstract A rapid and simple method has been developed for the simultaneous measurement of juvenile hormone (JH) and JH acid synthesized in vitro by larval corpora allata (CA) of the tobacco hornworm, Manduca sexta. An organic solvent partition of incubation medium efficiently separates JH acid from JH, and a radioimmunoassay which recognizes the two moieties equivalently is then employed to quantify each. The change in the biosynthetic product of the CA from JH to JH acid appears to begin slowly at the time of ecdysis to the last (fifth) larval stadium and is not complete until just prior to wandering (day 4). The inclusion of the JH esterase inhibitor S-benzoyl-O-ethyl phosphoramidothiolate in incubations of corpora allata revealed that the activity of JH esterases from the gland parallels gland activity and that significant hydrolysis of newly synthesized JH by these esterases occurs in incubations of glands taken at the beginnings of the fourth and fifth larval stadia. An allatostatin, which is proposed to inhibit the corpus allatum during the time of the change in its product, inhibits both JH I and JH I acid synthesis.     

The molting hormone activity in Sarcophaga buttata in relation to metamorphosis and reproduction

Ecdysteroid levels were determined by radioimmunoassay, using an antiserum with higher affinity for ecdysterone than for ecdysone. In both sexes, the ecdysteroid level remains low until day 3 after eclosion. When fed liver on day 3, the ecdysteroid titre declines within 4 h in both sexes, stays low in males, but increases again after 12 h in females. The content in non-liver-fed animals, specially females, was significantly higher than if given liver. The level in liver-fed whole animals increased during oogenesis from the onset of follicular growth until the 4B—4C stage, and decreased thereafter.

During embryogenesis, a new high level was found in females, due to the developing embryos which contained a high titre. In isolated ovaries, the ecdysteroid content increased during follicular growth, with a steep increase during the last phase (4C—M and from M to uterine eggs). The ecdysteroid content in hemolymph of 7-day-old females, fed liver or not, was around 10 ng/ml, but in hemolymph of males of the same age no activity was found.

Ovariectomized females contained 12.4 ng/g ecdysteroid. The level during metamorphosis was also determined, mainly to be able to compare our radioimmunoassay results with known bioassay data. Our results support the statement that the ovary cannot be the only site of synthesis of molting hormone in adult S. buttata.     

昆虫的变态发育研究

昆虫变态发育使得昆虫成为地球陆地上种类最多、数量最大、分布最广、生活环境最多样化的一群生物。变态使昆虫在其生命周期中的不同发育时期表现出完全不同的形态、结构、功能和生活习性的变化,有利于昆虫迁飞转移,扩大其求偶交配、生活和生存环境空间。昆虫变态发育的变化是长期自然环境适应、协同进化的结果,受激素、营养和基因的精确调控。本文简要介绍了昆虫变态的类型、激素调控、营养调控和基因调控方面的研究进展,以及研究昆虫变态发育的科学和应用意义。     

Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis

Changes in activity of the corpora allata (CA) during larval-pupal-adult development of the tobacco hornworm Manduca sexta were studied by transplantation assays, measurements of in vitro juvenile hormone (JH) and JH acid synthesis, and determination of JH acid methyltransferase (JHAMT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities. The data from these assays demonstrate that the CA cease to secrete JH by day 4 of the last larval instar (wandering stage). With regard to JH synthesis, they remain inactive throughout the prepupal, pupal, and most of the pharate adult periods. CA of females, but not of males, resume JH synthesis shortly before eclosion. The biochemical basis of the inactivation process is the loss of JHAMT activity. However, prepupal CA produce JH acids, as shown by enzyme and in vitro assays. Pupal and pharate adult CA do not synthesize JH acids although levels of HMG-CoA reductase activity seem to remain relatively high. Radiolabeled JH was recovered from hemolymph of allatectomized prepupae that had been injected with radiolabeled JH acid. These results provide further evidence that certain peripheral tissues (eg, imaginal discs) convert JH acid secreted by the prepupal CA to JH and, thus, that JH acid is a prohormone in the prepupal period. The CA change from hormone secretion to prohormone secretion during larval-prepupal transformation, a unique functional alteration in an endocrine gland.     

Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin-immunoreactive peptide in Periplaneta americana

Antisera to Manduca sexta allatoropin were raised in rabbits and were used to develop a competitive enzyme immunoassay for this neuropeptide. The detection limit of the assay is less than 2 fmol/well. A useful quantification can be obtained from 2 to 30 fmol/well. No cross-reactivity was observed with several insect peptides, but the enzyme-linked immunosorbent assay does recognize [Ala⁶, Leu⁷, Ser⁸]-allatotropin, a myotropin recently isolated from Locusta migratoria. The assay was used to study the distribution of allatotropin within the nervous system of Manduca sexta. The peptide is present in the retrocerebral complex, the brain, and the ventral nerve cord of this species, in quantities of respectively 0.01, 1.2, and 1.7 pmol per insect. An allatotropin-immunoreactive peptide was found in the nervous system of Periplaneta americana. It is present in the ventral nerve cord (3.3 pmol/insect), brain (1.9 pmol/insect), and retrocerebral complex (0.09 pmol/insect). These data suggest that peptides of this family are generally present in insects. © 1993 Wiley-Liss, Inc.     

昆虫蜕皮激素信号转导途径研究进展

蜕皮与变态是全变态昆虫典型的发育特征。调控昆虫蜕皮与变态的激素主要有蜕皮激素和保幼激素。目前已经阐明了蜕皮激素的核受体EcR及部分核信号转导途径,但蜕皮激素是否存在膜受体及膜信号转导途径研究很少。研究证明,蜕皮激素存在细胞质中的信号转导分子和途径,蜕皮激素通过NTF2和Ran调控EcR入核启动基因转录。蜕皮激素使细胞质中的热休克蛋白Hsc70部分入核与USP结合启动基因转录。蜕皮激素通过蛋白激酶PKC使伴侣蛋白calponin磷酸化,参与蜕皮激素信号途径的基因转录。这些研究结果说明蜕皮激素除了有核受体和核受体信号转导途径外,还存在细胞膜受体和细胞膜信号转导途径。     

Induction and regulation of juvenile hormone esterases during the last larval instar of the cabbage looper, Trichoplusia ni

Juvenile hormone esterase titres were monitored in gate I and gate II last instar larvae of Trichoplusia ni using JH III as substrate. Two peaks of activity were observed for both gate I and gate II larvae, although the first and second juvenile hormone esterase peaks for the gate II larvae are extended and delayed one day, respectively. Head or thoracic ligations before the prepupal stage lower or block the appearance of both esterase peaks. Juvenile hormone I and II, as well as homo and dihomo juvenoids can induce the second juvenile hormone esterase peak in both normal and ligated larvae, and increase the esterase titre during the first peak in nonligated larvae. Induction of the juvenile hormone esterases is possible in non-ligated larvae as soon as the moult to the last instar has occurred and in ligated larvae as soon as the first esterase peak has started to decline. Distinct mechanisms of regulation are present for the first and second juvenile hormone esterase peaks. Juvenile hormone does not appear to be involved in regulating its own metabolism by directly inducing the first esterase peak; however, evidence is consistent with a brief burst of juvenile hormone which occurs prior to pupation inducing the production of the second peak of juvenile hormone esterase activity.     

Steroid control of muscle remodeling during metamorphosis in Manduca sexta

During metamorphosis in the tobacco hornworm, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. The degeneration of muscle DEO1 involves the dismantling of its contractile apparatus followed by the degeneration of muscle nuclei. As some nuclei are degenerating, others begin to incorporate 5-bromodeoxyuridine (BrdU), indicating the onset of nuclear proliferation. This proliferation is initially most evident at the site where the motoneuron contacts the muscle remnant. The developmental events involved in muscle remodeling are under the control of the steroid hormones, the ecdysteroids. The loss of the contractile elements of the larval muscle requires the rise and fall of the prepupal peak of ecdysteroids, whereas the subsequent loss of muscle nuclei is influenced by the slight rise in ecdysteroids seen after pupal ecdysis. Incorporation of BrdU by muscle nuclei depends on both the adult peak of the ecdysteroids and contact with the motoneuron. Unilateral axotomy blocks proliferation within the rudiment, but it does not block its subsequent differentiation into a very thin muscle in the adult. © 1996 John Wiley & Sons, Inc.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Preliminary characterization of glycogen phosphorylase activating hormone and adipokinetic hormone from Manduca sexta corpora cardiaca

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G-25 and from Sephadex LH-20 columns. In the separation experiments with glass beads and G-25 the activities eluted as a single peak, but using LH-20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × V_t, which is very similar to locust AKH, while the smaller second peak eluted at O.74 × V _t. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Comparison of structurally analogous allatotoxins on the molting and morphogenesis of Rhodnius prolixus,and the reversal of ecdysial stasis by ecdysone

The allatotoxic effect of 3-ethoxy-4-methoxy-6-iso-pentenylphenol on nymphal molting and metamorphosis of Rhodnius prolixus was examined. Continuous contact treatment with IPP induced the formation of precocious adults and retarded molting or initiated a permanent ecdysial stasis. Insects treated with 7-ethoxy-6-methoxy-2,2-dimethylchrornene were similarly affected. Ecdysone given orally counteracted the ecdysial stasis and also reduced the duration of the molting delay caused by IPP.     

Sexual differentiation in the CNS of the moth,Manduca sexta. I. Sex and segment-specificity in production,differentiation, and survival of the imaginal midline neurons

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineage of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation. 1994 John Wiley & Sons, Inc.     

Segment‐specific muscle degeneration is triggered directly by a steroid hormone during insect metamorphosis

During metamorphosis of the hawkmoth, Manduca sexta, some larval muscles degenerate while others are respecified for new functions. In larvae, accessory planta retractor muscles (APRMs) are present in abdominal segments 1 to 6 (A1 to A6). APRMs serve as proleg retractors in A3 to A6 and body wall muscles in A1 and A2. At pupation, all APRMs degenerate except those in A2 and A3, which are respecified to circulate hemolymph in pupae. The motoneurons that innervate APRMs, the APRs, likewise undergo segment‐specific programmed cell death (PCD), as a direct, cell‐autonomous response to the prepupal peak of ecdysteroids. The segment‐specific patterns of APR and APRM death differ. The present study tested the hypothesis that APRM death is a direct, cell‐autonomous response to the prepupal peak of ecdysteroids. Prevention of the prepupal peak prevented APRM degeneration, and replacement of the peak by infusion of 20‐hydroxyecdysone restored the correct segment‐specific pattern of APRM degeneration. Surgical denervation of APRMs did not perturb their segment‐specific degeneration at pupation, indicating that signals from APRs are not required for the muscles' segment‐specific responses to ecdysteroids. The possibility that instructive signals originate from APRMs' epidermal attachment points was tested by treating the epidermis with a juvenile hormone analog to prevent pupal development. This manipulation likewise did not alter APRM fate. We conclude that both the muscles and motoneurons in this motor system respond directly and cell‐autonomously to prepupal ecdysteroids to produce a segment‐specific pattern of PCD that is matched to the functional requirements of the pupal body. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005