Fig. 3: Mineral precipitation on the cuticle in both in vitro cuticle synthetic studies and ant-rearing experiments.

a Scheme of in vitro mineralization experiment using Acromyrmex echinatior leaf-cutting ant cuticles as templates for biomineralization (Photo C.M.C.). b, c Pre- and post-incubation SEM images showing the original, uncoated cuticle (b) and the cuticle covered by a layer of precipitated carbonate (c) after incubation in Mg²⁺/Ca²⁺/Cl⁻/Na⁺/HCO₃⁻ solution for 7 days at 19 °C. d, XRD patterns of, from top to bottom, an uncoated ant cuticle, a cuticle after incubation in Mg²⁺/Ca²⁺/Cl⁻/Na⁺/HCO₃⁻ solution, a platinum-coated cuticle incubated in Mg²⁺/Ca²⁺/Cl⁻/Na⁺/HCO₃⁻ solution, and a cuticle after KOH protein hydrolysis incubated in Mg²⁺/Ca²⁺/Cl⁻/Na⁺/HCO₃⁻ solution. H: high-magnesium-calcite, A: aragonite, Pt: platinum. e XRD patterns of cuticles of ants representing different developmental stages, ranging from (from bottom to top), a newly formed pupa to an older worker, after incubation in Mg²⁺/Ca²⁺/Cl⁻/Na⁺/HCO₃⁻ solution. f Environmental scanning electron micrographs (eSEM) of ant epicuticles taken over a 10-day time series, from immediately after eclosion from pupa to adult (left), to 10 days post-eclosion (right), showing the formation of the biomineral layer over time (Photo H.L.). g Estimated magnesium concentration of the biomineral layer during 30 days of ant development based on the XRD d₍₁₀₄₎ value according to Graf and Goldsmith⁶⁴, showing the rapid integration of magnesium from days 6 to 8 and the continued presence of high-magnesium content for up to 30 days (n = 2 per treatment and the corresponding standard error are shown).