Development of a New Reporter Gene Assay for Detecting Juvenile Hormone Agonists Using Yeast Expressing Methoprene-Tolerant of the Freshwater Cladoceran Daphnia magna

Abstract

Juvenile hormones (JHs) play crucial roles in regulating growth, metamorphosis, and reproduction in arthropods. Synthetic JH agonists (JHAs), categorized as insect growth regulators, have been widely employed as insecticides. Natural JHs and synthetic JHAs both exert their physiological effects by binding to the JH receptor methoprene-tolerant (Met), forming a functional heterodimer complex with steroid receptor coactivators (SRCs). These juvenoids induce male offspring production in various daphnids, including Daphnia magna, highlighting the significance of the Met-mediated signaling in environmental sex determination. As a representative invertebrate model for assessing aquatic endocrine-disrupting chemicals, D. magna is incorporated in the test guidelines of the Organization for Economic Corporation and Development. We herein introduced a newly developed yeast-based reporter gene assay (RGA) for easy and rapid screening of JH-like ligands for D. magna Met (Dapma-Met). Dapma-Met was expressed alongside the SRC of D. magna (Dapma-SRC) in yeast cells carrying the lacZ reporter plasmid with a JH-responsive element derived from the Bombyx mori Krüppel homolog 1 gene. The yeast RGA system for Dapma-Met revealed a dose-dependent response to various juvenoids. The rank order of the ligand potencies of natural JHs and synthetic JHAs examined in yeast RGA strongly correlated with those previously observed in RGAs for Daphnia Met proteins established in Chinese hamster ovary cells and positively correlated with the male neonate-inducing activity in vivo. Our novel yeast RGA offers a rapid, easy-to-handle, and cost-effective solution that will be valuable for discriminating Dapma-Met ligands among chemicals with male offspring-inducing activity.