Finely tuned ciliary alignment and coordinated beating generate continuous water flow across the external gills in Pleurodeles waltl larvae

Urodelan amphibian larvae develop three pairs of branching external gills on both sides of the pharyngeal region, and this study focuses on motile cilia on the gill surface. High-speed camera was used to observe ciliary strokes on the surface of the external gills of Pleurodeles waltl larvae. We found that the directionality of ciliary beating is position-dependent on the gill surface, and this rule is applicable to all the samples examined. For visualizing water flow around the external gills, we used bead suspensions. We revealed continuous anterior-to-posterior water flow generated by coordinated ciliary beating. Around the frontal surface of the gill stem (gill rachis), water flows countercurrent to the bloodstream beneath the gill epidermis. These results suggest that ciliary beating in each ciliated cell is coordinated, which cooperatively generates continuous and directional ciliary flow. We next visualized the overall distribution of ciliated cells on the gill surface by immunostaining of acetylated alpha-tubulin. Our results showed that the fine branches of external gills (fimbriae) have a circumferential distribution of cilia aligned orthogonal to the longitudinal axes of fimbriae, which facilitates water flow from proximal to the distal part of the fimbriae through the gills. This ciliary distribution pattern and directionality of cilia-driven flow are shared among five urodelan and two anuran species. Taken together, our findings suggest that the distribution and motility of ciliated cells on the surface of external gills is finely controlled, and this might support efficient respiration by the gills in urodeles.