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

蜕皮或蜕皮是地球上最多样化和最丰富的动物在进化枝蜕皮动物（包括节肢动物，线虫，灵长类和盟友）中的一个特征。蜕皮对于这些动物的生长是必不可少的，因为它们的身体覆盖着无弹性的角质层或外骨骼，这些角质层或外骨骼必须定期脱落并更换为更大的覆盖物。据信蜕皮过程通常由蜕皮类固醇激素控制。虽然这一过程已经在昆虫和甲壳类动物中得到了很好的探索，但对其他节肢动物（如蜘蛛）以及最近的节肢动物亲戚：缓步动物（水熊）和ony-chophorans（天鹅绒蠕虫）的了解甚少。因此，我们将分析潜在的关键基因调控的生物合成的蜕皮甾类在缓步类Hypsibius exemplaris，onychophoran Euperi-patoides rowelli，和蜘蛛Parasteatoda tepidariorum。首先，将进行蜕皮诱导实验以阐明蜕皮类固醇激素原蜕皮激素及其活性形式20-羟基蜕皮激素是否参与蜕皮。第二，原位杂交和荧光原位杂交将进行定位涉及蜕皮甾类生物合成的组织。第三，将在不同蜕皮阶段和特定组织上完成实时定量逆转录PCR（qRT-PCR），以揭示这些基因的锡永水平随时间的变化并鉴定所涉及的另外的酶。第四，将进行功能分析（RNA干扰）以确认这些基因参与蜕皮。最后，我们的数据将被合并到一个大规模的，开放访问的数据库（MoultDB）和获得的，扩展的数据矩阵将用于系统发育分析。新的发现将有助于（i）澄清蜕皮激素，20-羟基蜕皮激素或ponasterone A是否在全节肢动物的最后一个共同祖先中起蜕皮诱导激素的作用;（ii）揭示在缓步动物，Onychophora和Chelice rata成员中合成蜕皮类固醇的组织;（iii）阐明这些组织在全节肢动物中的起源。以及（iv）提供对全节肢动物门和蜕皮动物门蜕皮起源和蜕皮激素途径进化变化的见解。

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.