Description. This proposal will readdress the roles of ecdysteroids and juvenile hormones (JH) in vitellogenesis in female Drosophila melanogaster in the light of novel information. Published studies with apterous⁴ (ap⁴) mutants have suggested that JH is essential for vitellogenesis. However, another allele, ap^56f, displays the same very low level of JH production as the ap⁴ homozygous animals, yet undergoes normal vitellogenesis, although the process is delayed by about 24h. The project will examine the regulation of ovarian ecdysteroid synthesis by JH, and the nature of the delay in vitellogenesis in ap^56f flies as it may relate to low levels of JH production. Wild-type females in diapause arrest development at a pre-vitellogenic stage, in association with a decreased level of JH and ecdysteroid production. Preliminary data suggest that diapause termination is preceded by an increase in ecdysteroid, but not JH production. Diapausing females will be used to test the hypothesis that ecdysteroids, and not JHs drive YP uptake by oocytes. 20-hydroxyecdysone will be injected into these flies to attempt to initiate YP-synthesis and uptake. JH and ecdysteroid binding activities will be examined in diapausing, and diapause-terminating animals. The production of ecdysteroids by ovaries from ap⁴ females (deficient in the production of both JHs and ecdysteroids) will be further examined and ecdysteroid injections will be used to attempt to rescue YP uptake. The possibility that ecdysteroids regulate YP uptake by regulating the production of receptor-mediated endocytosis-associated proteins, e.g. clathrin will be investigated by immunofluorescence microscopy. An amended model for the endocrine regulation of vitellogenesis is proposed: that JH can stimulate ovarian ecdysteroid production, and that this production is essential for YP uptake by the developing oocytes. The significance of these experiments lies with the unique position that D. melanogaster occupies in biological research as both a model insect and a model dipteran. No other insect is as well characterized in terms of its genetic make-up and while it is a small insect, it has proved possible to surgically dissect and manipulate the organs involved in the endocrine system. Given the importance of some cyclorrhaphous dipteran species as vectors of human disease, it is important that we understand the role of the endocrine system in the regulation of reproductive development.