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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 OHAMT) 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.

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 Msexta larvae.J H 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.Key words: juvenile hormone acid methyltransferase, juvenile hormone acid, juvenile hormone, imaginal disc, Manduca sexta Acknowledgments: We thank Drs. Gijnter Weirich and Judith Willis for reviewing the manuscript. We gratefully acknowledge the advice of Dr. Karl H. Dahm during the course of this work. This work was supported by Organized Research at Texas A&M University. This paper is a portion of the Master of Science thesis of Steven P. Sparagana.*Abbreviations: accessory sex glands = ASG; corpora allata = CA; hi h pressure liquid chromatography = HPLC; juvenile hormone = JH; juvenile hormone acicf methyltransferase = JHAMT; radioimmunoassay = RIA; S-adenosylmethionine = AdoMet; S-benzyl-O-ethyl phosphorarnidothiolate = BEPAT; thin-layer chromatography = TLC.

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Denaturingelectrophoresis of hemolymph from prepupaeof M. sexta showed trace amounts of polypeptides with mobilities correspondingto those of vitellogenin (Vg) apoproteins from adult females. Absence of the polypeptides in allatectomized insects suggested regulation by juvenile hormone (JH). Daily administration of 10 pg of the JH analog methoprene from day 4 of the fifth stage to day 0 of the pupal stage caused accumulation of these polypeptides. They were identified as apovitellogenins (apovgs) immunochemically with Vg antiserum. stimulation of Vg in response to methoprene varied with age. In all cases, day 0 female pupae were highly responsive. Vg synthesis was not stimulated when pupae were injected with 20-hydroxyecdysone (20-HE) in addition to methoprene. Methoprene-stimulated Vg synthesis was also abolished by inhibitors of mRNA or protein synthesis (a-amanitin, actinomycin, cycloheximide). This result indicated that methoprenestimulated Vg accumulation requires gene expression. A Vg cDNA (2.1 kb) obtained by imrnunoscreening of the Xgt 11 library, when used as a radiolabelled probe, hybridized with a 5.1 kb mRNA from total RNA of female fat body. It also hybridized with fat body RNA of normal prepupae and methoprene treated day 0 pupae but not with that of early fifth instars or solvent control pupae. The results indicate that the trace amounts of Vg found in prepupal stages are due to a weak expression of the Vg gene, which is stimulated by JH and repressed by 20-HE.01994 Wiley-Lis, Inc.