Synchrotron based high resolution X-ray computed

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microtomography of freeze dried amphibians. T. Kleinteich ... that receive their nutrition by rasping, the hyobranchial musculature comprises less muscles and.
Synchrotron based high resolution X-ray computed microtomography of freeze dried amphibians T. Kleinteich and A. Haas Biozentrum Grindel und Zoologisches Museum Universität Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany

Evolutionary research in biology relies on the comparison of different individuals of different species in order to explore the history of todays biodiversity. The applications of synchrotron based high resolution X-ray computed microtomography (µCT) in the comparative field of evolutionary biology are virtually infinite (for examples see [1]). In this study, we use µCT imaging to investigate the evolution of a particular group of amphibians; caecilians. Caecilians (scientific name: Gymnophiona) are limbless, fossorial amphibians. The Gymnophiona comprise 171 species that are restricted to the tropics [2]. Caecilians show a variety of feeding modes during development (suction feeding, biting, rasping; [3], [4]). This project focuses on the anatomical and functional demands of different feeding modes on caecilian skulls and head muscles. It was necessary to have µCT datasets of different developmental stages and to collect µCT data from different species for comparison. µCT data of adult caecilians (4 individuals; 4 species) was collected within 72 h at beamline W2; developmental stages (6 specimens; 3 species) have been µCT scanned at beamline BW2 in a 48 h shift. The specimens had been decapitated and their heads were freeze dried prior to the µCT imaging procedure. Because the lengths of the samples exceeded the area that could be penetrated by the X-ray beam at once, several scans were performed for different regions of the specimens; the separate datasets were combined afterwards. The resulting datasets show high detail in hard and soft tissues. The resolution of the µCT data (voxel-sizes) ranges from 2 µm to 9 µm, depending on the size of the sample. Single muscle fibers, nerves, and connective tissues can be identified in the datasets. The richness in detail of our datasets is significantly higher, especially within soft tissues, than in any previously published vertebrate µCT scan (e.g. [5] and references therein). The µCT data was used for the comparison of caecilian anatomy in different species and developmental stages (Fig. 1A, B). The jaw closing musculature shows notable differences in the topographic relationships to other muscles and to the squamosal bone. The musculature that acts on the hyobranchium (i.e. gills and gill arches) in aquatic caecilians is well developed and covers the ventral and lateral side of the caudal head region; in caecilians that capture their prey by biting or that receive their nutrition by rasping, the hyobranchial musculature comprises less muscles and covers only the ventral side of the animal. A muscular tongue is absent in aquatic developmental stages but present in all other investigated specimens. Physical models of the skulls of 3 specimens have been created using a rapid prototyper with the µCT datasets as input data (Fig. 1C; cooperation with Dr. Adam Summers, UC Irvine). The models are up scaled in size and can easily be manipulated by hand. Interacting with the physical models gives new insights into the functional anatomy of caecilian heads. It is possible to interactively explore the degrees of freedom for movements of different bones in caecilian skulls. The high detail within soft tissues, especially musculature, of the µCT data made it possible to automatically measure the angles of muscle fibres in caecilians. The muscle fibre angles can be used in a lever arm model to estimate bite forces in the caecilian jaw closing mechanism. Results that were based on the µCT datasets have been presented in a talk at the International Conference on Vertebrate Morphology (ICVM) in Paris [6] and in a seminar talk at the Museum of Vertebrate Morphology at UC Berkeley (MVZ).

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Figure 1: µCT data in the study of caecilian evolution. A: Lateral view of a larval specimen of Ichthyophis kohtaoensis (specimen ZMH A08978; GKSS-ID: zim10); skin partially removed. B: Lateral view of an adult specimen of I. kohtaoensis (ZMH A08981; zim03); different planes of section for soft tissues and cranial bones. There are notable differences in the orientation and the presence of muscles between larva and adult within the same species. C: Physical model of an adult Typhlonectes natans (ZMH A08984; zim02) generated by rapid prototyping. The model is up scaled in size and can easily be manipulated by hand.

References [1] [2] [3] [4] [5] [6]

O. Betz, U. Wegst, D. Weide, et al., J. Microsc. 227, 51–71 (2007) D.R. Frost, Amphibian Species of the World: an online reference. Version 5.1 (2007) S.M. Deban, J.C. O'Reilly, K.C. Nishikawa, Am. Zool. 41, 1280–1298 (2001) A. Kupfer, H. Müller, M.M. Antoniazzi, et al., Nature 440, 926–929 (2006) Digital Morphology Group, University of Texas, http://www.digimorph.org (2007) T. Kleinteich, J. Morphol. 268, 1093 (2007)

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