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

Current understanding of the anatomical structures of ascidians remains limited, particularly in regionally confined but abundant species such as Halocynthia papillosa from the Mediterranean Sea. This study employed multimodal imaging techniques, including Light, Thunder, and fluorescent confocal microscopy, to investigate selected neural structures and the tunic of H. papillosa at various magnifications. We utilized advanced 3D imaging methods, such as Magnetic Resonance Imaging (MRI) and High-Throughput Tomography (HiTT) at a synchrotron beamline. Additionally, chemical analysis using Fourier-Transformation Infrared-Spectroscopy (FTIR) was conducted. Our 3D and isotropic high-resolution renderings revealed significant structural differences in this species compared to others, notably the absence of a cerebral thickening. HiTT imaging identified three distinct suborders of oral tentacles, each with its own innervation and blood supply. Using Thunder and fluorescent Confocal microscopy, we also documented autofluorescence in ascidian cuticular sheds for the first time in vivo and ex vivo in greater detail. Furthermore, HiTT X-ray imaging of the tunic revealed a spiralized structure emerging from the tunicin layers, which could be verified in this species for the first time using FTIR. The high-resolution, state-of-the-art imaging techniques presented in this study establish a strong foundation for future studies on H. papillosa and other solitary ascidians, and highlight the need to expand research beyond traditional model species.