Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans

Ecdysone-controlled pathways regulate ecdysozoan development through both a nuclear (nEcR) and a membrane receptor (mEcR). Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids. Nevertheless, there is still no clear consensus regarding the presence or functional role of ecdysone-like signaling in mollusks. This study aimed to advance our understanding of the evolution of ecdysone signaling by investigating the potential synthesis and physiological role of 20-hydroxyecdysone (20HE) in the great pond snail ( Lymnaea stagnalis ) by applying a complex experimental approach. Exposure to different concentrations (10 ng/L, 100 ng/L, and 1 µg/L) of 20HE had no effect on hatching, heart rate, locomotion (gliding), or feeding in Lymnaea embryos. Furthermore, the treatments did not alter the expression of Lymnaea homologs of Drosophila nEcR and mEcR. Using cluster analysis and phylogenetics to resolve the evolution of Halloween genes involved in ecdysone synthesis, we found no clear homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species, while Phantom and Shade appear to have a many to many homologues in lophotrochozoans. Although mass spectrometric analysis detected trace concentrations of 20HE in various tissues of adult Lymnaea specimens, we suggest that its presence is of dietary origin. Our findings clearly indicate that an endogenous ecdysone synthesis pathway is not present in Lymnaea and that 20HE has no effect on the physiology of Lymnaea embryos. Although further experiments are required on other molluscan models as well, we propose that molluscan sequences homologous to Drosophila nEcR and mEcR are unlikely to function as ecdysone receptors, and that ecdysone-like signaling is absent in lophotrochozoans.