Ecdysteroid-dependent molting in tardigrades

Although molting is a defining feature of the most species-rich animal taxa-the Ecdysozoa, including arthropods, tardigrades, nematodes, and others¹^,²-its evolutionary background remains enigmatic. In pancrustaceans, such as insects and decapods, molting is regulated by the ecdysteroid (Ecd) hormone and its downstream cascade (Figure 1A, see also the text).³^,⁴^,⁵ However, whether Ecd-dependent molting predates the emergence of the arthropods and represents an ancestral machinery in ecdysozoans remains unclear. For example, involvement of the Ecd hormone in molting regulation has been suggested only in some parasitic nematodes outside of arthropods,⁶^,⁷ and insect Ecd synthesis and receptor genes are lacking in some ecysozoan lineages (Figure S1A).⁸^,⁹^,¹⁰ In this study, 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, whereas 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 the Ecd hormone, supporting the ancestry of Ecd-dependent molting in panarthropods. Furthermore, we were able to identify the putative neural center of molting regulation in tardigrades. This region may be homologous to the neural center in the protocerebrum of pancrustaceans and represent an ancestral state of panarthropods. Together, our results suggest that Ecd-dependent molting evolved in the early-late Ediacaran, 22-76 million years earlier than previously suggested.¹¹.