Digital dissection of the pelvis and hindlimb of the red-legged running frog,Phlyctimantis maculatus, using Diffusible Iodine Contrast Enhanced computed microtomography (DICEμCT)

Abstract

Background The current study applies both traditional and Diffusible Iodine Contrast Enhanced computed microtomography (DICE µCT) techniques to reveal the musculoskeletal anatomy of Phlyctimantis maculatus. DICE µCT has emerged as a powerful tool to visualise intricate musculoskeletal anatomy. By generating 3D digital models, anatomical analyses can be conducted non-destructively, preserving the in situ 3D topography of the system, therefore eliminating some of the drawbacks associated with traditional methods. We aim to describe the musculature of the spine, pelvis, and hindlimb, compare the musculoskeletal anatomy and pelvic morphology of P. maculatus with functionally diverse frogs, and produce 3D digital anatomy reference data. Method An adult frog was stained using an aqueous Lugol’s solution and scanned in a SkyScan1176 in vivo µCT scanner. Scan images were reconstructed, resampled, and digitally segmented to produce a 3D model. A further adult female frog was dissected traditionally for visualisation of tendinous insertions. Results Our work revealed three main findings: (1) P. maculatus has similar gross muscular anatomy to Rana catesbeiana (bullfrog) but is distinct from those species that exhibit ancestral traits (leopelmids) and those that are highly specialised (pipids), (2) P. maculatus’s pelvic anatomy best fits the description of Emerson’s walking/hopping pelvic morphotype IIA, and (3) a split in the semimembranosus and gracilis major muscles is consistent with the reported myology in other anuran species. Discussion While DICE µCT methods were instrumental in characterising the 3D anatomy, traditional dissection was still required to visualise important structures such as the knee aponeurosis, tendinous insertions, and fasciae. Nonetheless, the anatomical data presented here marks the first detailed digital description of an arboreal and terrestrial frog. Further, our digital model presents P. maculatus as a good frog model system and as such has formed a crucial platform for further functional analysis within the anuran pelvis and hindlimb.

Kaiso regulates osteoblast differentiation and mineralization via the Itga10/PI3K/AKT signaling pathway

“…The implants were harvested and fixed in 4% paraformaldehyde (room temperature for 24 h) for µ -computed tomography ( µ CT) analysis. The bone mineral density (BMD) of implants was then generated by skyscan 1176 ( 22 ). The implants were decalcified in 10% EDTA and embedded in paraffin.…”

From fibre to function: are we accurately representing muscle architecture and performance?

“…This staining process enhances the contrast of soft tissue structures thus rendering individual muscles visible in these CT scans, from which various important anatomical variables (muscle volumes and fibre lengths) can be measured (Kupczik et al ., 2015). This method has been used to quantify the muscle architecture and musculoskeletal geometry of, among others, the jaw musculature of the crab‐eating macaque ( Macaca fascicularis ) (Dickinson, Stark & Kupczik, 2018), the pectoral muscles of the European starling ( Sturnus vulgaris ) (Sullivan et al ., 2019), various species of bat (Santana, 2018), as well as the hindlimbs of the mouse (Charles et al ., 2016a) and the red‐legged running frog ( Phlyctimantis maculatus ) (Collings & Richards, 2019). Some of these frameworks (Dickinson et al ., 2018; Sullivan et al ., 2019) were able to produce estimates of muscle fibre lengths from imaging alone through a form of digital fibre tracking, which generated a sample size of fibre lengths equivalent to that produced from DTI.…”

Hind foot drumming: Volumetric micro‐computed tomography investigation of the hind limb musculature of three African mole‐rat species (Bathyergidae)

“…Several studies have comparatively studied the masticatory musculature of African mole‐rats (Bathyergidae; Cox & Faulkes, 2014; Cox et al, 2020), bats (Santana, 2018) and anteaters (Ferreira‐Cardoso et al, 2020), however no statistical analysis comparing the species was performed in these studies as they compared only one individual per species. Locomotory studies on the limbs of crocodilians (Klinkhamer et al, 2017; Wiseman et al, 2021), frogs (Collings & Richards, 2019) and birds (Bishop et al, 2021; Sullivan et al, 2019) have also employed diceCT scanning.…”