Element Sensitive Synchrotron-Based X-Ray Tomography as a Technique for Studying Transition Metals in the Cuticle of Insects

Several different lineages of invertebrates are known to augment their cuticle with the addition of inorganic elements. Perhaps the most well studied group of inorganic elements in the cuticle of insects are the transition metals, which increases cuticle hardness in specific structures that are exposed to high friction forces. A challenge in this field, however, is choosing the most suitable technique for identifying the specific locations of transition metals in structures and quantifying their concentrations. Here, we demonstrate the usefulness of element sensitive synchrotron-based X-ray tomography in locating and calculating concentrations of zinc (ranging from 9 to 27 mg/cm3) in the cuticle of mandibles from several different insect groups such as ants, caddisflies, and butterflies. This technique was combined with several other methodologies, including scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy, and histological studies with transmission electron microscopy and light microscopy to validate our findings with synchrotron imaging. The highly sclerotized areas of the cuticle (generally described as the exocuticle) of the incisor teeth and the molar area were the primary location for Zn deposition, where they mainly formed cap-like inlays. We found that element sensitive synchrotron-based X-ray imaging was useful for producing a three-dimensional image from the resulting tomographs and quantifying Zn concentrations. We found this technique to have several benefits over other techniques, including a simple sample preparation and ability to locate and map Zn deposition beyond the cuticle surface.