Modern morphological methods for tadpole studies. A comparison of micro-CT, and clearing and staining protocols modified for frog larvae

Abstract

Micro-computed tomography (micro-CT) scanning and three-dimensional reconstruction have revolutionized morphological studies. Whereas species descriptions and comparative studies formerly were based on external appearance and dissection, we now can visualize muscles, skeleton and viscera in intact animals. In most cases, visualization of internal structures depends on appropriate staining methods. Staining with iodine, phosphotungstic acid (PTA) and osmium tetroxide are established methods, but some problems remain. Agents like osmium tetroxide are toxic and the contrast of cartilage generally is unsatisfactory with osmium tetroxide, iodine or PTA. Furthermore, staining results vary for different animals and different developmental stages. We investigated critical point drying as an inexpensive, nontoxic and rapid alternative to staining for frog tadpoles. Critical point drying enables visualization of cartilage and its differentiation from muscle tissue. Shrinkage generally is acceptable. We also present a protocol for clearing and staining frog tadpoles.

Key words:

• Anura
• cartilage
• critical point drying
• frogs
• iodine
• larvae
• micro-computerized tomography
• osmium tetroxide
• phosphotungstic acid
• tadpoles

Acknowledgments

We thank Peter Rühr from the Zoologisches Forschungsmuseum Alexander Koenig and Joachim Feil from RJL Micro & Analytic for assisting with micro-CT scanning. We thank also Thomas Ziegler from the Cologne Zoo, Claudia Koch, Fabian Herder, Serkan Güse, Claudia Etzbauer and Ulla Bott from the Zoologisches Forschungsmuseum Alexander Koenig for practical, technical and organizational support and expertise. Special thanks to Susanne Pickhardt, who helped with the English. Special thanks to Thorsten Mahne for sharing his enormous experience in breeding frogs with us. We are grateful to the Alexander Koenig Society for financial support.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.