Designing 3D Printed Braces for Imaging the Carolina Anole (Anolis carolinensis) Dewlap Musculature Using DiceCT

The form-function relationships of small and gracile features that contribute to multi-tissue phenotypes can be difficult to document due to their delicate nature. For our study we sought to analyze the structure and function of the anole dewlap, a cervical flap protruded via interaction of the hyoid bones, musculature, and integumentary tissues (supported by the second ceratobranchial bones). Previous research has implicated the Musculus branchiohyoideus and the M. ceratohyoideus in dewlap extension; however recent literature on this interaction is lacking in spite of novel 3D imaging techniques. We used diffusible iodine-based contrast-enhanced computed tomography (diceCT) to analyze the delicate muscles of the Carolina Anole (Anolis carolinensis) dewlap. We sought to observe the dewlap in both relaxed and extended positions to better understand the actions of different hyoid muscles in dewlap protrusion. We attempted multiple methods for diceCT staining the extended dewlaps: pulling the dewlap into the extended position with forceps, various store-bought holding clips, as well as designing de novo clips and support structures for in house 3D printing. We also manipulated the specimens by either maintaining or removing the integumentary fold of the dewlap, to observe which condition allowed for full extension of the second ceratobranchials. The ultimate design was a 3D printed brace belt that supported a strand of 3D printer filament, reinforced using surgical suture, with the skin of the dewlap removed to allow for maximum second ceratobranchial extension. The added support of the torso helped to keep the apparatus in place. This design enabled stabilization of the hyoid bones in the dewlap’s extended position, with the filament material composition allowing for the iodine staining and micro--‐CT scanning of the specimens in the desired, extended position.