33] Quantification of ecdysteroid biosynthesis during short-term organ culture

Abstract

Publisher Summary This chapter describes the methods for the quantification of ecdysteroid biosynthesis by ring glands of dipteran larvae during short-term organ culture. During the larval development of insects, molting and pupariation are triggered by ecdysteroids synthesized by the prothoracic gland in response to stimulation by the prothoracicotropic hormone (PTTH). Development of highly sensitive ecdysteroid radioimmunoassay (RIA) helps to measure in vitro ecdysteroid synthesis by single larval ring glands of small dipteran insects such as D . melanogaster . This has led to the identification of lethal mutations of Drosophila that cause ecdysteroid deficiency by directly impairing the ability of larval ring glands to synthesize ecdysteroids. These mutations are the insect equivalent of congenital abnormalities in adrenal and gonadal steroid synthesis in vertebrates. The chapter describes a sensitive radioimmunoassay for measuring femtomole quantities of ecdysteroids. Protocols are provided, for the in vitro culture of larval ring glands from two dipteran species, Drosophila melanogaster and the blowfly Sarcophaga bullata .