The Halloween gene shadow is involved in embryonic development and postembryonic molting in the spider Parasteatoda tepidariorum

Ecdysteroids are key hormones that regulate molting and developmental processes in arthropods, including insect metamorphosis. Understanding the molecular roles of the genes involved in ecdysteroid biosynthesis could provide crucial insights into the evolutionary conservation of regulation and regulatory modifications in arthropods. While extensively studied in insects, the functions of the so-called Halloween genes, encoding enzymes responsible for the conversion of cholesterol to ecdysone and 20-hydroxyecdysone, remain unclear in arachnids. Therefore, this study aims to elucidate the functions of the Halloween gene shadow (Pt-sad) in the spider Parasteatoda tepidariorum in both embryonic and postembryonic stages and to assess its potential role in molting. In situ hybridization was employed to characterize the expression patterns of Pt-sad, and RNAi-mediated knockdown (RNA interference) was conducted to explore its effects on development and molting. Knockdown of Pt-sad resulted in increased embryonic mortality and developmental delays, while it disrupted molt cycles in postembryonic stages, supporting a role in ecdysteroidogenesis. Expression analysis revealed Pt-sad activity in specific regions of embryos and juveniles, including the central nervous system, appendages, and circulatory system. These findings highlight the multifunctionality of shadow in the spider. While the involvement of this gene in the ecdysteroid pathway is most likely conserved in arthropods, this role may have diversified in spiders to include additional, unique functions that differ from their insect counterparts.