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.     

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.     

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.     

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.     

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。     

Sexual differentiation in the CNS of the moth,Manduca sexta. II. Target dependence for the survival of the imaginal midline neurons

While majority of the neurons in the adult nervous system of the moth Manduca sexta are produced postembryonically, little is known about how these cells interact with their targets during development. Few of these cells are motor neurons; most of Manduca's adult motor neurons are respecified larval motor neurons do develop postembryonically, including a large class of mixed neurosecretory and motor neurons called the imaginal midline neurons (IMNs). A subset of these cells show an unusual pattern of sex-specific development and survival (Thorn and Truman, 1994, J. Neurobiol. in press), which led us to suspect that factors extrinsic to the cells were controlling their fates. We analyzed one such potential factor by altering the contacts between a subset of these developing IMNs and their adult-specific target, the male sperm duct. When we transected the nerve that innervated the sperm duct in the pupa, we observed a loss of many sperm duct IMNs. In contrast, a transection of the same nerve in larvae showed no neuron loss. Immunocytochemistry showed that the pupal nerve transections were accompanied by a loss of axon endings on the sperm duct, while the larval nerve transections showed no such loss. Using local hormone application to slow the development of the sperm duct while leaving the nerve intact still resulted in a loss of IMNs. These results suggest that these IMNs need contact with a robust developing target in the pupa to survive metamorphosis. 1994 John Wiley & Sons, Inc.     

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.     

Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ-endotoxin of sodium- or potassium gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

Rapid filtration assays were used to determine the effects of barium, calcium and an insecticidal δ-endotoxin from Bacillus thuringiensis on sodium and potassium ion gradient dependent phenylalanine accumulation by brush border membrane vescles from the larval midgut of the tobacco hornworm (Manduca sexta). Neither barium nor calcium had a significant effect on sodium ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent phenylalanine accumulation by the membrane vesicles. B. thuringiensis δ-endotoxin inhibited both sodium and potassium ion gradient dependent phenylalanine accumulation by the vesicles. Inhibition of both sodium and potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing δ-endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml^?1 on day 1 to 0.27 U ml^?1 on day 3. When larvae were treated with (7S)-hydroprene (a JH analog), the titer remained elevated (2.09 U ml^?1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml^?1 on day 5 to 1.62 U ml^?1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head-ligated day 5 larvae were treated with either (7S)-hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml^?1 and 2.05 U ml^?1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml^?1). The combined results indicate the titer of the hemolymph factor is regulated by JH.