<p>The hardness of the structural tools of insects can be increased through the integration of transition metals into the cuticle. However, questions remain regarding metal predominancies for insect cuticle (i.e., metal-prioritization hypothesis) and whether metals accumulate in the cuticle across life stages. To address these questions, we characterized structures in an insect-fungal-plant host system, including mandibles of invasive ambrosia beetles, their fungal diet, and the wood the fungus digests. Energy dispersive X-ray spectroscopy revealed low concentrations of transition metals in the wood, fungus, and neonate mandibles. High concentrations of zinc, however, were found in the distal regions of mandibles of third-instar larvae and adults, confirming diet as the source of transition metals and a lack of maternal inheritance. Confocal microscopy of mandibles showed sclerotization patterns that coincided with zinc distributions and nanoindentation confirmed increased hardness and elastic modulus in zinc-enriched locations. Patterns of zinc concentrations on the mandible surface suggest that metals do not accumulate in the cuticle through life stages. Instead, zinc-enriched cuticle is lost at each molt and replaced by zinc acquired from the previous instar’s diet, thus revealing a physiological mechanism by which specific transition metals are retained and accumulate within each instar for future cuticle deposition.</p>