Protocol for quantifying the thickness and puncture resistance properties of solitary bee cocoons using Osmia lignaria as a model

This protocol presents a standardized method for quantifying cocoon thickness and puncture resistance in solitary bees, using Osmia lignaria as a model species. The approach addresses a key gap in the biomechanical characterization of silk-based protective structures by enabling reproducible preparation, mounting, and testing of cocoon segments. Thickness measurements are used to normalize puncture resistance metrics, allowing meaningful comparison across specimens and between sexes, which exhibit natural variability in cocoon morphology. The protocol combines custom 3D-printed fixtures with mechanical testing to generate quantitative parameters including thickness, maximum puncture load, stiffness, and work to puncture. By detailing specimen preparation, directional puncture testing, and data analysis workflows, the study provides a transferable framework for investigating puncture resistance in cocoons of other solitary bee species and layered biological membranes more broadly.