Semi-automated differentiation of insect exo- and endocuticle in X-ray microtomography

One of the most versatile and complex biological composite materials, the insect exoskeleton shows a huge range in biomechanical properties. The cuticle exoskeleton can be differentiated into two main histologically different layers with distinct properties: the outer, more sclerotized exocuticle and inner, softer endocuticle. For most biomechanical research questions, it is of great importance to be able to selectively characterize geometrical features of these layers. However, most conventional preparation methods (cross-sections, histological staining, SEM) require complex and destructive sample preparation, which provides only two-dimensional information. Here, we present a novel, simple staining method using X-ray microtomography to distinguish between exo- and endocuticle in a 3D environment without sample destruction. We illustrate the power of our method using locust (Locusta migratoria) hindleg tibia, a well characterized biomechanical sample. Our method allows an easy and direct measurement of exo- and endocuticle and their respective geometric features. Applying our method will help to understand the biomechanical role of exo- and endocuticle within an insect exoskeleton and will allow us to understand its composition and morphological features in more detail.