[1].A Rapid, Non-invasive Method for Anatomical Observations of Tadpole Vertebrae in Vivo[J].Asian Herpetological Research,2018,9(2):99-109.[doi:10.16373/j.cnki.ahr.180003]
 Guocheng SHU,Shan XIONG,Wenyan ZHANG,et al.A Rapid, Non-invasive Method for Anatomical Observations of Tadpole Vertebrae in Vivo[J].Asian Herpetological Research(AHR),2018,9(2):99-109.[doi:10.16373/j.cnki.ahr.180003]

A Rapid, Non-invasive Method for Anatomical Observations of Tadpole Vertebrae in Vivo()

Asian Herpetological Research[ISSN:2095-0357/CN:51-1735/Q]



A Rapid, Non-invasive Method for Anatomical Observations of Tadpole Vertebrae in Vivo
Guocheng SHU12 Shan XIONG12 Wenyan ZHANG12 Jianping JIANG1 Cheng LI1* and Feng XIE1*
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
micro-CT bone-cartilage double-stain tadpole vertebrae
The tadpole is a critical stage in the amphibian life cycle and plays an important role during the transition from the aquatic to the terrestrial stage. However, there is a large gap in tadpole research, which represents a vital component of our understanding of the diversity and complexity of the life history traits of amphibians, especially their developmental biology. Some aspects of this gap are due to limited research approaches. To date, X-ray microcomputed tomography (micro-CT) has been widely used to conduct osteology research in adult amphibians and reptiles, but little is known about whether this tool can be applied in tadpole studies. Thus, we compared the results of two methods (the bone-cartilage double-staining technique and micro-CT) to study vertebrae in tadpole specimens. The results revealed no significant difference between the two methods in determining the number of vertebrae, and micro-CT represents a rapid, non-invasive, reliable method of studying tadpole vertebrae. When scanning tadpoles, voltage is the most critical of the scanning parameters (voltage, current and scan time), and moderate scanning parameters are recommended. In addition, micro-CT performed better using specimens stored in 70% ethanol than those preserved in 10% formalin. Finally, we suggest that micro-CT should be more widely applied in herpetological research to increase specimen utilization.


Boistel R., Swoger J., Kr?i? U., Fernandez V., Gillet B., Reynaud E. G. 2011. The future of three-dimensional microscopic imaging in marine biology. Mar Biol, 32(4): 438–452
Broeckhoven C., Plessis A., Roux S. G., Mouton P. L. F. N., Hui C. 2017. Beauty is more than skin deep: A non-invasive protocol for in vivo anatomical study using micro-CT. Methods Ecol Evol, 8(3): 358–369
Campbell G. M., Sophocleous A. 2014. Quantitative analysis of bone and soft tissue by micro-computed tomography: Applications to ex vivo and in vivo studies. Bonekey Rep, 3: 564
Cannatella D. 1999. Architecture: Cranial and axial musculoskeleton. In McDiarmid R. W., Altig R. (Eds.), Tadpoles: the biology of anuran larvae. Chicago, USA: University of Chicago Press, 52–81
Chao W., Harteneck B. D., Liddle J. A., Anderson E. H., Attwood D. T. 2005. Soft X-ray microscopy at a spatial resolution better than 15 nm. Nature, 435(7046): 1210–1213
Chen Y., Lin G., Chen Y., Fok A., Slack J. M. 2012. Micro-computed tomography for visualizing limb skeletal regeneration in young Xenopus frogs. Anat Rec, 295(10): 1562–1565
Depew M. J. 2009. Analysis of skeletal ontogenesis through differential staining of bone and cartilage. In Westendorf (Eds.), Molecular Embryology: Methods and Protocols. Totowa, USA: Humana Press. 37–4
Descamps E., Buytaert J., De Kegel B., Dirckx J., Adriaens D. 2012. A qualitative comparison of 3D visualization in Xenopus laevis using a traditional method and a non-destructive method. Belg J Zool, 142(2): 99–111
Dingerkus G., Uhler L. D. 1977. Enzyme clearing of Alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technol, 52: 229–232
Dingerkus G. 1981. The use of various alcohols for Alcian blue in toto staining of cartilage. Stain Technol, 56: 128–129
Dodd M. H. I., Dodd J. M. 1976. The biology of metamorphosis. Physiol Amphibia, 3: 467–599
Du Plessis A., Broeckhoven C., Guelpa A., Le Roux S. G. 2017. Laboratory X-ray micro-computed tomography: A user guideline for biological samples. GigaScience, 6(6): 1–11
Faulwetter S., Vasileiadou A., Kouratoras M., Dailianis T., Arvanitidis C. 2013. Micro-computed tomography: Introducing new dimensions to taxonomy. ZooKeys, 263: 1
Fortuny J., Marcé-Nogué J., Heiss E., Sanchez M., Gil L., Galobart ?. 2015. 3D bite modeling and feeding mechanics of the largest living amphibian, the Chinese giant salamander Andrias davidianus (Amphibia: Urodela). PLoS One, 10(4): e0121885
Gignac P. M., Kley N. J., Clarke J. A., Colbert M. W., Morhardt A. C., Cerio D., Cost I. N., Cox P. G., Daza J. D., Early C. M., Echols M. S., Henkelman R. M., Herdina A. N., Holliday C. M., Li Z., Mahlow K., Merchant S., Müller J., Orsbon C. P., Paluh D. J., Thies M. L., Tsai H. P., Echols M. S. 2016. Diffusible iodine based contrast enhanced computed tomography (diceCT): An emerging tool for rapid, high resolution, 3D imaging of metazoan soft tissues. J Anat, 228(6): 889–909
Gosner K. L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16(3): 183–190
Handrigan G. R., Haas A., Wassersug R. J. 2007. Bony-tailed tadpoles: The development of supernumerary caudal vertebrae in larval megophryids (Anura). Evol Dev, 9(2): 190–202
Hanken J., Wassersug R. 1981. The visible skeleton. Funct Photo, 16(4): 22–26
Heyer R., Donnelly M. A., Foster M., Mcdiarmid R. 1994. Measuring and monitoring biological diversity: Standard methods for amphibians. Washington, USA: Smithsonian Institution Press. 289–297
Karhula S. S., Finnil? M. A., Lammi M. J., Yl?rinne J. H., Kauppinen S., Rieppo L., Pritzker K. P. H., Nieminen H. J., Saarakkala S. 2017. Effects of articular cartilage constituents on phosphotungstic acid enhanced micro-computed tomography. PLoS One, 12(1): e0171075
Kelly W. L., Bryden M. M. 1983. A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates. Stain Technol, 58:131–134
Kim E., Sung H., Lee D., Kim G., Nam D., Kim E. 2017. Nondestructive skeletal imaging of Hyla suweonensis using Micro-computed tomography. Asian Herpetol Res, 88(4): 235–243
Lauridsen H., Hansen K., Wang T., Agger P., Andersen J. L., Knudsen P. S., Maglia, A. M. 2003. Skeletal development of Pelobates cultripes and comparisons of the osteology of pelobatoid frogs. Sci Pap Univ Kansas Nat Hist Mus, 30: 1–13
McDiarmid R. W., Altig R. 1999. Tadpoles: the biology of anuran larvae. Chicago, USA: University of Chicago Press. 52–90
Metscher B. D. 2009a. MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions. Dev Dyn, 238(3): 632–640
Metscher B. D. 2009b. MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physio, 9(1): 11
Mizutani R., Suzuki Y. 2012. X-ray microtomography in biology. Micron, 43(2): 104–115
Parapar J., Candás M., Cunha-Veira X., Moreira J. 2017. Exploring annelid anatomy using micro-computed tomography: A taxonomic approach. Zool Anz, 270: 19–42
Porro L. B., Richards C. T. 2017, Digital dissection of the model organism Xenopus laevis using contrast-enhanced computed tomography. J Anat, 231: 169–191
Rasmussen A. S., Uhrenholt L., Pedersen M. 2011. Inside out: modern imaging techniques to reveal animal anatomy. PLoS One, 6(3): e17879
Redfern B. G., David W. L., Spence S. 2007. An alternative Alcian blue dye variant for the evaluation of fetal cartilage. Birth Defects Res B, 80(3): 171–176
Ritman E. L. 2004. Micro-computed tomography–current status and developments. Annu Rev Biomed Eng, 6: 185–208
Ritman E. L. 2011. Current status of developments and applications of micro-CT. Annu Rev Biomed Eng, 13: 531–552
Ro?ková H., Ro?ek Z. 2005. Development of the pelvis and posterior part of the vertebral column in the Anura. J Anat, 206(1): 17–35
R Development Core Team. 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: http://www.R-project.org (accessed 28 September 2017).
Scherrer R., Hurtado A., Machado E. G., Debiais-Thibaud M. 2017. MicroCT survey of larval skeletal mineralization in the Cuban gar Atractosteus tristoechus (Actinopterygii; Lepisosteiformes). MorphoMuseuM, 3(3):e3
Scherz M. D., Ruthensteiner B., Vences M., Glaw F. 2014. A new microhylid frog, genus Rhombophryne, from northeastern Madagascar, and a re-description of R. serratopalpebrosa using micro-computed tomography. Zootaxa, 3860(6): 547–560
Scherz M. D., Ruthensteiner B., Vieites D. R., Vences M., Glaw F. 2015. Two new microhylid frogs of the genus Rhombophryne with superciliary spines from the Tsaratanana Massif in northern Madagascar. Herpetologica, 71(4): 310–321
Sephenson M. T. 1960. The skeletal characters of Leiopelma harniltoni McCulloch, with particular reference to the effects of heterochrony on the genus. Trans Roy Soc, 88 (3): 473–488
Sephenson N. G. 1965. Heterochronous changes among Australian leptodactylid frogs. Proc Zod Soc, 144 (3): 339–350
Simons E. V., Van Horn J. R. 1971. A new procedure for whole-mount Alcian blue staining of the cartilaginous skeleton of chicken embryos, adapted to the clearing procedure in potassium hydroxide. Acta Morphol Neerl-Scand, 8: 281–292
Trueb L. 1973. Bones, frogs, and evolution. In Vial J. L. (Eds.) Evolutionary biology of anurans. Columbia: University of Missouri Press, 65–132
Vasquez S. X., Hansen M. S., Bahadur A. N., Hockin M. F., Kindlmann G. L., Nevell L., Isabel Q. Wu., David J. G., David M. W., Greg M. J., Christopher R. J., Johnl L. V., Mario R. C., Johnson C. R. 2008. Optimization of volumetric computed tomography for skeletal analysis of model genetic organisms. Anat Rec. 291(5): 475–487
Wassersug R. J. 1976. A procedure for differential staining of cartilage and bone in whole formalin-fixed vertebrates. Stain Technol, 51(2): 131–134
Williams T. W. 1941. Alizarin red S and toluidine blue for differentiating adult or embryonic bone and cartilage. Stain Technol, 16: 23–25
Yamada T. 1991. Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S. Teratology, 43(6): 615–619
Zhang M., Chen X., Chen X. 2016. Osteology of Quasipaa robertingeri (Anura: Dicroglossidae). Asian Herpetol Res, 7(4): 242–250

更新日期/Last Update: 2018-06-26