Ecdysteroid-dependent molting in tardigrades

Molting is a defining feature of the most species-rich animal taxa, the Ecdysozoa, including arthropods, tardigrades, nematodes, and others. In pancrustaceans, such as insects and decapods, molting is regulated by the ecdysteroid (Ecd) hormone and its downstream cascade. However, whether the regulation of molting predates the emergence of the arthropods and represents an ancestral machinery of ecdysozoans remains unclear. Therefore, we investigated the role of Ecd in the molting process of the tardigrade Hypsibius exemplaris. We show that the endogenous Ecd level periodically increases during the molting cycle of H. exemplaris. The pulse treatment with exogenous Ecd induced molting while an antagonist of the Ecd receptor suppressed the molting. Our spatial and temporal gene expression analysis revealed the putative regulatory organs and Ecd downstream cascades. We demonstrate that tardigrade molting is regulated by Ecd hormone, supporting the ancestry of Ecd-dependent molting in panarthropods. Further, we were able to identify the putative neural center of molting regulation in tardigrades, which may represent an ancestral state of panarthropods homologous to the protocerebrum of pancrustaceans. Together, our results suggest that Ecd-dependent molting evolved 100 million years earlier than previously suggested.