Proposed is a continuing study of the hormonal regulation of growth, molting and metamorphosis in Drosophila melanogaster, concentrating on the key role of the endocrine gland (prothoracic gland) that produces the steroid molting hormone ecdysone in size assessment and thus the timing of the onset of metamorphosis. Juvenile hormone (JH) plays an important role in allowing larval growth and molting by preventing metamorphosis. The presence of JH at the onset of the molt prevents the ecdysone-induced switch from a larval program to the metamorphic program during the molt by regulation of the ecdysone-induced transcription factor Broad. The loss of JH after the attainment of the critical size for metamorphosis then determines the final size of the larva and subsequent adult. Growth itself is dependent on the nutrient-dependent insulin signaling system. Therefore, size control depends on an interaction between the classical endocrine system and this insulin signaling system. Our specific aims in the next 4 years are: 1) To complete the study of the regulation of second pupal cuticle formation by JH, concentrating on the regulation of the Broad and Kruppel-homolog transcription factors. 2) To determine how the prothoracic gland interacts with the insulin signaling pathway and other growth factors to assess the size of the animal and signal the onset of metamorphosis. 3) To determine how JH regulates the final size at pupariation. 4) To determine what are the critical tissues for size assessment. 5) To determine how insulin signaling affects growth differentially before and after the critical weight stage. 6) To determine the role of E75A in the prothoracic gland and/or target tissues to ensure sufficient ecdysteroid for larval molting. These studies should lead to a better understanding of the ways in which JH prevents the ecdysone- induced gene switching that is necessary for metamorphosis and how the nutrition-dependent insulin signaling pathway interacts with the JH regulatory pathway so that metamorphosis occurs at a time optimal for reproduction. The control of growth and the tinning of its cessation leading to reproductive maturation is a problem that all animals including humans face. These studies in insects will provide models for the types of cellular and molecular mechanisms underlying the cessation of growth at puberty in humans. These studies also should provide new insights into how developmental switches common to all developing systems are controlled by endocrine signals. Finally, the studies may provide the basis for novel strategies of insect control that will provide for new strategies for control of the vectors of human disease such as mosquitoes and biting flies. [unreadable] [unreadable] [unreadable]