Identification of key players of the ecdysteroid pathway and detection of ecdysteroid-producing tissues in tardigrades, onychophorans and chelicerates: Insights into the evolution of molting in Panarthropoda

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Molting or ecdysis is a characteristic feature of the most diverse and abundant animals on Earth united in the clade Ecdysozoa (including arthropods, nematodes, priapulids, and allies). Ecdysis is essential for growth in these animals, as their body is covered with an inelastic cuti¬cle or exo-skeleton, which has to be shed and replaced periodically by larger covering. The pro¬cess of molt¬ing is believed to be generally governed by the ecdysteroid hormones. While this process has been explored well in insects and crustaceans, only little is known about it in other arthro-pods, such as spiders, and the closest arthropod relatives: tardigrades (water bears) and ony-chophorans (velvet worms). We will therefore analyze the potential key genes regulating the bio-synthesis of ecdysteroids in the tardigrade Hypsibius exemplaris, the onychophoran Euperi-patoides rowelli, and the spider Parasteatoda tepidariorum. First, molt in¬duction experiments will be performed to clarify whether the ecdysteroid prohormone ecdysone and its active form 20-hydroxyecdysone are involved in molting. Second, in situ hybridization and immunohistochem-istry will be carried out to localize tissues involved in ecdysteroid biosynthesis. Third, Real-Time quan¬titative Reverse Transcription PCR (qRT-PCR) will be accomplished on different molt stages and specific tissues to reveal changes in the expres¬sion levels of these genes over time and to identify addi¬tional enzymes involved. Fourth, func¬tional analyses (RNA interference) will be performed to con¬firm the involvement of these genes in molting. Finally, our data will be incor-porated into a large-scale, open-access database (MoultDB) and the obtained, extended data matrix will be used for phylo¬genetic analyses. The new findings will (i) help to clarify whether ecdysone, 20-hydroxyecdysone or ponasterone A acted as molt-inducing hormones in the last common ances¬tor of Panarthro¬poda; (ii) reveal the tissues that synthesize ecdysteroids in members of Tardi¬grada, Onychophora and Chelice¬rata; (iii) shed light on the origin of these tissues across panarthropods; and (iv) pro¬vide insights into the origin of molting and evolutionary changes in the ecdys¬teroid pathway in Panarthropoda and Ecdysozoa.