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

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Abstract

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.

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Acknowledgements

We thank Prof. Ken’ichi Kanazawa and Prof. Susumu Izumi from Kanagawa University for fruitful advice and many discussions. We are thankful to Dr. Masato Owada (Kanagawa University) for technical advice on scanning electron microscopy. We also thank Prof. Hideho Uchiyama (Yokohama City University) for providing a protocol for Xenopus immunostaining.

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Correspondence to Ryuji Toyoizumi.

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Smovie 3: Ciliary flow around an external gill of a Pleurodeles larva. To visualize water flow around a gill, red polystyrene beads were suspended in artificial freshwater. Overall, ciliary flow occurs from an anterior-to-posterior direction, along the cranio-caudal axis. Additionally, adjacent to the frontal surface of the gill rachis, part of the water flow runs in a proximal-to-distal direction along the longitudinal axis of the rachis. Note also that, in the anterior front of the rachis, bloodstream flow is from the distal tip to the proximal stem, resulting in countercurrent flow between the ciliary water flow and the bloodstream. (MOV 2971 kb)

Smovie 5: Ciliary flow around the external gill of an Ambystoma mexicanum (Mexican salamander) larva. In Ambystoma larvae, in front of the gill rachis, beads flow from an anterior-proximal to a posterior-distal direction, along the surface of the rachis. This water flow pattern is essentially similar to that of the Pleurodeles larvae shown in Smovie 3. Note also that in Ambystoma larvae the bloodstream in the rachis runs countercurrent to the ciliary flow (see Fig. 5b). (MOV 1391 kb)

Smovie 6: Ciliary flow around the external gill of a Hynobius nebulosus (Kasumi salamander) larva. In H. nebulosus larvae, the ciliary flow in front of the frontal surface of the rachis runs from an anterior-proximal to a posterior-distal direction. This flow pattern resembles those of Pleurodeles and Ambystoma larvae. Also in H. nebulosus larvae, ciliary flow and bloodstream flow are countercurrent (see Fig. 6b). (MOV 1409 kb)

Smovie 7: Ciliary flow around the external gill of a Hynobius nigrescens (Black salamander) larva. In H. nigrescens salamander larvae, ciliary flow in front of the rachis runs from an anterior-proximal to a posterior-distal direction, as in Pleurodeles and Ambystoma larvae. Also in H. nigrescens larvae, the bloodstream and ciliary flow are countercurrent (see Fig. 7b). (MOV 1911 kb)

Smovie 8: Ciliary flow around the external gill of a Hynobius kimurae (Hida salamander) larva. H. kimurae larvae have large external gills relative to overall body size, and the morphology of the external gills is quite different from that of typical urodelan external gills. The anterior-to-posterior water flow pattern was not observed around the gills of H. kimurae larvae. However, water flow still occurred in a proximal-to-distal direction, along the gill branch. In addition, ciliary flow and bloodstream flow are countercurrent (see Fig. 8b). (MOV 1672 kb)

Smovie 9: Ciliary flow around the external gill of a Bufo japonicus formosus (Azuma toad) larva. In B. japonicus larvae, ciliary flow in front of the rachis runs from an anterior-proximal to a posterior-distal direction, as observed in Pleurodeles and other urodelan larvae. (MOV 1440 kb)

Smovie 10: Ciliary flow around the external gill of a Buergeria buergeri (Kajika frog) larva. Also in B. buergeri larvae, the ciliary flow pattern is essentially similar to that of Pleurodeles and other urodelan larvae. (MOV 1369 kb)

Smovie 1: Direction of the ciliary strokes of multi-ciliated cells on the frontal surface of a rachis in a Pleurodeles larva. Effective strokes of cilia beating from the proximal side (upper right) to the distal side (lower left) on the external gill. Gills vibrate gently due to the pulsation of the heartbeat. (MOV 5760 kb)

Smovie 2: Direction of the ciliary strokes of multi-ciliated cells on the fimbria of a Pleurodeles larva. Effective strokes of the cilia on the fimbria surface beat from the proximal side to the distal side, along the longitudinal axis, without exception. (MOV 5444 kb)

Smovie 3: Ciliary flow around an external gill of a Pleurodeles larva. To visualize water flow around a gill, red polystyrene beads were suspended in artificial freshwater. Overall, ciliary flow occurs from an anterior-to-posterior direction, along the cranio-caudal axis. Additionally, adjacent to the frontal surface of the gill rachis, part of the water flow runs in a proximal-to-distal direction along the longitudinal axis of the rachis. Note also that, in the anterior front of the rachis, bloodstream flow is from the distal tip to the proximal stem, resulting in countercurrent flow between the ciliary water flow and the bloodstream. (MOV 2971 kb)

Smovie 4: 3D-reconstruction of confocal laser scanning microscopy (CLSM) image of Pleurodeles external gill decorated with acetylated alpha-tubulin and AlexaFluor 488-conjucated secondary antibody. Circumferential ciliary alignment with similar spacing are confirmed by the 3D image corresponding to Fig. 4 k. (MOV 8076 kb)

Smovie 5: Ciliary flow around the external gill of an Ambystoma mexicanum (Mexican salamander) larva. In Ambystoma larvae, in front of the gill rachis, beads flow from an anterior-proximal to a posterior-distal direction, along the surface of the rachis. This water flow pattern is essentially similar to that of the Pleurodeles larvae shown in Smovie 3. Note also that in Ambystoma larvae the bloodstream in the rachis runs countercurrent to the ciliary flow (see Fig. 5b). (MOV 1391 kb)

Smovie 6: Ciliary flow around the external gill of a Hynobius nebulosus (Kasumi salamander) larva. In H. nebulosus larvae, the ciliary flow in front of the frontal surface of the rachis runs from an anterior-proximal to a posterior-distal direction. This flow pattern resembles those of Pleurodeles and Ambystoma larvae. Also in H. nebulosus larvae, ciliary flow and bloodstream flow are countercurrent (see Fig. 6b). (MOV 1409 kb)

Smovie 7: Ciliary flow around the external gill of a Hynobius nigrescens (Black salamander) larva. In H. nigrescens salamander larvae, ciliary flow in front of the rachis runs from an anterior-proximal to a posterior-distal direction, as in Pleurodeles and Ambystoma larvae. Also in H. nigrescens larvae, the bloodstream and ciliary flow are countercurrent (see Fig. 7b). (MOV 1911 kb)

Smovie 8: Ciliary flow around the external gill of a Hynobius kimurae (Hida salamander) larva. H. kimurae larvae have large external gills relative to overall body size, and the morphology of the external gills is quite different from that of typical urodelan external gills. The anterior-to-posterior water flow pattern was not observed around the gills of H. kimurae larvae. However, water flow still occurred in a proximal-to-distal direction, along the gill branch. In addition, ciliary flow and bloodstream flow are countercurrent (see Fig. 8b). (MOV 1672 kb)

Smovie 9: Ciliary flow around the external gill of a Bufo japonicus formosus (Azuma toad) larva. In B. japonicus larvae, ciliary flow in front of the rachis runs from an anterior-proximal to a posterior-distal direction, as observed in Pleurodeles and other urodelan larvae. (MOV 1440 kb)

Smovie 10: Ciliary flow around the external gill of a Buergeria buergeri (Kajika frog) larva. Also in B. buergeri larvae, the ciliary flow pattern is essentially similar to that of Pleurodeles and other urodelan larvae. (MOV 1369 kb)

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Ichikawa, R., Toyoizumi, R. Finely tuned ciliary alignment and coordinated beating generate continuous water flow across the external gills in Pleurodeles waltl larvae. Zoomorphology (2020). https://doi.org/10.1007/s00435-020-00479-0

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Keywords

  • Ciliated cells
  • Gill rachis
  • Gill fimbria
  • Ciliary beat
  • Ciliary flow
  • Urodela