Anatomical insights into fish terrestrial locomotion: a study of barred mudskipper (Periophthalmus argentilineatus) fins based on μCT 3D reconstructions

Mudskippers are a group of extant ray-finned fishes with an amphibious lifestyle and serve as exemplars for understanding the evolution of amphibious capabilities in teleosts. A comprehensive anatomical profile of both the soft and hard tissues within their propulsive fins is essential for advancing our understanding of terrestrial locomotor adaptations in fish. Despite the ecological significance of mudskippers, detailed data on their musculoskeletal anatomy remains limited. In the present research, we utilized contrast-enhanced high-resolution micro-computed tomography (μCT) imaging to investigate the barred mudskipper, Periophthalmus argentilineatus. This technique enabled detailed reconstruction and quantification of the morphological details of the pectoral, pelvic, and caudal fins of this terrestrial mudskipper, facilitating comparison with its aquatic relatives. Our findings reveal that P. argentilineatus has undergone complex musculoskeletal adaptations for terrestrial movement, including an increase in muscle complexity and muscle volume, as well as the development of specialized structures like aponeuroses for pectoral fin extension. Skeletal modifications are also evident, with features such as a reinforced shoulder-pelvic joint and thickened fin rays. These evolutionary modifications suggest biomechanically advanced fins capable of overcoming the gravitational challenges of terrestrial habitats, indicating a strong selective advantage for these features in land-based environments. The unique musculoskeletal modifications in the fins of mudskippers like P. argentilineatus, compared to their aquatic counterparts, mark a critical evolutionary shift toward terrestrial adaptations. This study not only sheds light on the specific anatomical changes facilitating this transition but also offers broader insights into the early evolutionary mechanisms of terrestrial locomotion, potentially mirroring the transformative journey from aquatic to terrestrial life in the lineage leading to tetrapods.

Methods
Specimens were collected from Okinawa Island, Japan, complemented by a zebrafish acquired from a local pet shop. Following humane euthanization with 2-phenoxyethanol, they were preserved first in 10% formalin and then stored in ethanol to maintain their integrity for scanning. Before micro-CT scanning, each specimen was stained with an iodine-based solution to enhance soft tissue contrast, thereby improving scan visibility. High-resolution micro-CT scans were then performed using a Zeiss Xradia Versa 510 at the Okinawa Institute of Science and Technology, ensuring detailed visualization of each specimen's anatomical structure.

To visualize the fins' skeletal and muscular structures and their locomotion functions, we developed an animated video. This entailed exporting segmented bone and muscle models in *.stl format from segmentation software into Blender for 3D animation. We programmed the animation sequence with Python in Blender's API, showcasing the muscle groups, movements, and transitions, complemented by manual camera animation to enhance feature visibility. Additionally, legends and labels were integrated into the animation using Python scripting and vector math for accurate placement. The animation was finalized and rendered clear with ffmpeg, providing a vivid representation of our findings. The final video was rendered and compiled using the ffmpeg program.