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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During the last larval stage, corpora allata (CA) of Manduca sexta are inactivated by a factor from the brain. Apparently the same factor (allatinhibin, AI) is secreted by day 4 Vth instar brains kept overnight in Grace's medium. AI is rapidly inactivated by heat or acid but withstands exposure to alkali and can be recovered after freezing and lyophilization. Exposure to pronase, chymotrypsin, carboxypeptidases-A and -Y, as well as leucine aminopeptidase eliminated AI activity completely, whereas after exposure to trypsin and protease XVII-S, some residual activity remained. Inactivation by pyroglutamate aminopeptidase is interpreted as being due to prolinase activity of this enzyme. Incubation of CA with gentamicin, an aminoglycoside antibiotic, affects neither their ability to produce JH in vitro nor their viability in implantation assays. However, AI did not inactivate CA in the presence of low concentrations of gentamicin. This effect was used to guard against false positive assay results possibly produced by allatotoxic contamination. AI was purified by chromatography on Sephadex G-25. All activity recovered emerged from the columns in intermediate fractions with an apparent M(r) of 1,000-2,000.

Larvae of the tobacco hornworm moth Manduca sexta starved for the first 3 days of the last (fifth) stadium undergo a supernumerary moult. If they are provided with sucrose during the starvation period, they develop into normal pupae although pupation is delayed. The activities of the corpora allata (CA) from normal, starved, and sucrose fed larvae were followed through the fifth stadium with a radiochemical assay for Juvenile Hormone (JH) biosynthesis. An attempt was made to correlate CA‐activity with CA cell number, size, and protein content. In CA of normally fed larvae the rate of JH synthesis declined to undetectable levels by day 4 which was also the time of exposure of the dorsal vessel. In CA of starved larvae, the rate of JH synthesis at first decreased but began to increase on day 3 and reached a peak value by day7, at which time head capsule slippage occurred. In CA of sucrose fed larvae, the rate of biosynthesis declined as in normal larvae but the decline was extended over a longer period. Exposure of the dorsal vessel was delayed in the same manner and occurred on days 7–9. The major JH in all cases was JH‐II. The CA comprise c. 150 cells in the early fifth stadium, and this number remained constant during the fifth stadium in all three feeding regimens. In normal larvae, CA size and protein content increased several‐fold during the stadium whereas in starved and sucrose‐fed larvae they increased slowly and in agreement with the altered timing of developmental events. In none of the groups was the CA activity pattern correlated with morphometric changes of the CA. The rates of JH biosynthesis were not closely correlated with published JH titre curves. The in vivo mechanisms for regulation of JH production remain to be elucidated.