Identification of Key Players of the Ecdysteroid Pathway and Detection of Ecdysteroid Synthesizing Tissues in Chelicerates : Insights into the Evolution of Molting in Panarthropoda

Abstract

Ecdysteroids are crucial hormones that regulate molting and developmental progression in arthropods. While the biosynthesis pathway of these hormones has been extensively studied in insects, functional characterization of the involved components remains limited in chelicerates. This thesis focuses on the early-to-late ecdysteroid pathway genes <i>neverland</i> (<i>Pt-nvd</i>, Rieske-domain oxygenase), <i>shroud</i> (<i>Pt-sro</i>, short-chain dehydrogenase/reductase), the cytochrome P450 genes <i>spook</i> (<i>Pt-spo</i>), <i>disembodied</i> (<i>Pt-dib</i>), <i>shadow</i> (<i>Pt-sad</i>) and <i>shade</i> (<i>Pt-shd</i>) as well as the ecdysteroid-inactivating <i>CYP18A1</i> (<i>Pt-CYP18A1</i>) in the spider <i>Parasteatoda tepidariorum</i>. The aim is to evaluate functional conservation and potential evolutionary diversifications within these molting-related gene families beyond Pancrustacea. Single-cell RNA sequencing analyses combined with whole-mount <i>in situ</i> hybridizations reveal common expression domains in mid-embryonic hemocytes for <i>Pt-nvd</i>, <i>Pt-sro</i>, <i>Pt-spo</i>, <i>Pt-dib</i>, <i>Pt-shd</i> and <i>Pt-CYP18A1</i>, and a later shift to the cardiac vessel or its lumen for <i>Pt-nvd</i>, <i>Pt-sro</i>, <i>Pt-dib</i>, <i>Pt-sad</i>, and <i>Pt-CYP18A1</i> during late embryogenesis. <i>Pt-sad</i> additionally marks neuroectodermal precursors, implicating a function in early neurogenesis. Parental RNA interference against <i>Pt-sad</i> disrupts head lobe formation, significantly elevates embryonic lethality and interval until first ecdysis. This indicates essential roles of the gene in both endocrine and morphogenetic processes. Juvenile knockdowns of all studied genes drastically extend molt intervals (up to four-fold) and produce lethal molting defect phenotypes, confirming pathway conservation at the functional level. As in insects, postembryonic expressions of <i>Pt-nvd</i>, <i>Pt-sro</i>, <i>Pt-spo</i>, <i>Pt-dib</i> and <i>Pt-sad</i> are localized in a common tissue during postembryonic stages. However, instead of a centralized prothoracic gland, spiders appear to utilize hemocytes for ecdysteroidogenesis. The findings establish hemocytes as previously unrecognized ecdysteroid pathway domains in spiders and reveal spider-specific involvement of shadow in neurogenesis. Thereby, this work provides some of the first evidence for ecdysteroid pathway activity in chelicerates, reinforcing functional pathway conservation across panarthropods while exposing evolutionary flexibility of endocrine regulation.