Temperature-adaptive temporal hardening process in Tachypleus tridentatus: linking chitin metabolism to exoskeletal biomechanics

Horseshoe crabs possess a mineralized exoskeleton adapted to intertidal environments, yet how developmental temperature shapes chitin dynamics and their coupling to cuticle biomechanics remains unclear. We integrated microstructural, mechanical, and physiological analyses to track post‑ecdysis hardening in Tachypleus tridentatus under normal (25.8 ± 0.6°C) and high (30.2 ± 0.9°C) temperatures. Hardening followed a regulated sequence of biomineralization and Bouligand formation, with time-dependent stiffening; mechanical properties stabilized by day 14, and high temperature enhanced anisotropy. Chitin metabolism showed a dual regulation pattern: synthesis was mainly time-dependent, with trehalose and glucose depletion driving chitin accumulation via chitin synthase 1 (Chs1), whereas degradation was more temperature-sensitive via chitinase 6 (Cht6) and CHT activity. PCA revealed temporal separation along PC1 for both physiological and mechanical datasets, explaining 36.93% and 34.96% of the total variance, respectively. Multivariate analyses confirmed significant congruence (Procrustes: M² = 0.650, r = 0.592, P = 0.001; CoIA: Axis 1 = 74.6%, RV = 0.541, P < 0.001), with the CoIA axis 1 score increasing linearly with post ecdysis time (adjusted R² = 0.692, P < 0.001). This study provides an integrative framework linking environmental temperature, chitin dynamics, and exoskeletal mechanics during early cuticle development in this ancient arthropod.