Insights into unique anatomical structures of the ascidian Halocynthia papillosa obtained by multimodal imaging

Current understanding of anatomical structures of ascidians remains limited. This study presents multimodal imaging techniques, including Light, Thunder, and fluorescent confocal microscopy, to investigate neural structures and the tunic of Halocynthia papillosa , a common ascidian in the Mediterranean Sea. We demonstrate advanced 3D imaging methods, i.e., Magnetic Resonance Imaging, and High-Throughput Tomography (HiTT) at a synchrotron beamline. Imaging results show structural differences in the central nerve of H. papillosa compared to other ascidians and identify three distinct suborders of oral tentacles. We also document detailed autofluorescent patterns in ascidian cuticular sheds for the first time. HiTT imaging of the tunic reveals a spiralized structure emerging from cellulose layers. The state-of-the-art imaging techniques presented here encourage a broader use of HiTT to study functional anatomy in marine invertebrates. It establishes a strong foundation for future studies on solitary ascidians and highlights the need to expand research beyond model species.