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Biomineralization is a widespread strategy used by organisms to construct protective exoskeletons with enhanced structural and mechanical properties (H.A. Lowenstam, S. Weiner, On Biomineralization, Oxford University Press, New York, 1989, Weiner and Addadi, 2011). Calcium carbonate is the most abundant biomineral. In crustaceans, it serves as the primary mineral component of the cuticle, functioning as a protective barrier and a structural framework for muscle attachment. While most crustaceans inhabit marine environments, terrestrial isopods have successfully colonized land and exhibit distinct adaptations in their cuticle mineralization that allow survival under terrestrial constraints such as desiccation and predation pressure (Ziegler, 1996, Hornung, 2011).

Among terrestrial isopods, Armadillidium vulgare is a model species for studying the mineralized tergite cuticle (Becker et al., 2005, Hild et al., 2008, Seidl et al., 2012, Yamagata et al., 2022). Early transmission electron microscopy (TEM) studies showed that the tergite cuticle of terrestrial isopods is composed of four distinct layers: an outer epicuticle, followed by an exocuticle, an endocuticle, and an innermost membranous layer (Ziegler, 1996, Price and Holdich, 1980). The tergite cuticle comprises 59.8 wt% amorphous calcium carbonate (ACC), 13.0 wt% organic matrix, 12.0 wt% calcite, 10.7 wt% amorphous calcium phosphate (ACP), and small amounts of water (Becker et al., 2005). Scanning electron microscopy (SEM) and Raman spectroscopy have revealed that calcite and ACC are not randomly distributed but are segregated into distinct layers within the cuticle. Specifically, calcite is restricted to the outer layer, corresponding to the exocuticle, while ACC is predominantly found within the underlying endocuticle (Hild et al., 2008). Yokoo proposed two crystallization processes occurring in the exocuticle: epitaxial crystallization on pre-existing calcite plates and crystallization mediated by organic matrices (Yokoo, 2012). The fine calcite crystals, approximately 10 nm in size, have their c-axes oriented parallel to the surrounding organic fibers (Yokoo, 2012). Subsequently, Seidl et al. (Seidl et al., 2012) investigated the orientation distribution of calcite axes within the cuticle using electron back-scatter diffraction (EBSD). They demonstrated that the calcite is randomly oriented, leading to more isotropic mechanical properties in the cuticle. More recently, detailed investigations using nanoindentation and high-resolution spectroscopic analyses have revealed that the enhancement of mechanical properties from the interior to the epicuticle is attributed to an increasing degree of calcification, resulting in a highly graded protective cover that thwarts predation (Yamagata et al., 2022). These findings highlight that the tergite cuticle is a hierarchically organized bio-composite with structural and compositional gradients that underpin its remarkable mechanical performance.

The presence of ACC is particularly intriguing as it is over ten times more soluble than crystalline calcium carbonates (Brečević and Nielsen, 1989, Meiron et al., 2011). Therefore, ACC functions both as a transient precursor phase that transforms into crystalline phases and as a stable phase providing specific mechanical advantages (Yamagata et al., 2022). Specifically, the incorporation of ACC enhances mechanical compliance within the mineralized framework, enabling the formation of distinct gradients in stiffness and hardness that reduce brittleness while increasing overall toughness. Furthermore, ACC facilitates efficient stress redistribution and crack deflection at mineral–organic interfaces, thereby improving energy dissipation and damage tolerance under mechanical loading (Yamagata et al., 2022).

In this context, it is crucial to acknowledge that ACC exhibits “polyamorphism,” wherein the amorphous phase encompasses multiple structurally distinct forms. Unlike crystalline “polymorphism,” polyamorphism results from variations in short-range order, hydration state, and local coordination environments, yielding amorphous phases with varying thermodynamic stability and physicochemical attributes. Previous studies have identified proto-crystalline variants of ACC, such as proto-calcite ACC and proto-aragonite ACC, which possess short-range structural motifs similar to those of their respective crystalline polymorphs and are considered influential in subsequent crystallization pathways (Cartwright et al., 2012). These observations suggest that ACC comprises a heterogeneous array of polyamorphs, each possessing distinct structural features. Although ACC has been identified as a key component of the A. vulgare endocuticle, a systematic investigation of ACC phase diversity is lacking.

In terrestrial environments, A. vulgare acquires calcium primarily from substrates and dietary material in direct contact with the ground, where calcium carbonate typically occurs in crystalline polymorphs such as calcite and aragonite. Thus, the mineralogical form of ingested calcium constitutes the initial physicochemical input into the biomineralization pathway. Evaluating the effect of calcium carbonate polymorphs at the ingestion stage is essential for determining whether the organism passively converts all dietary calcium into a common amorphous precursor or selectively processes various polymorphs during mineral uptake and cuticle formation. An experimental ingestion-based approach offers an effective framework for investigating this issue, as analyses of only the intact cuticle cannot determine whether ACC characteristics arise from endogenous biological regulation or inherited mineral properties. By systematically supplying calcium carbonate in distinct polymorphic states, it becomes possible to evaluate whether the mineralogical form of dietary calcium influences the structural organization, phase distribution, and functional significance of ACC within the cuticle.

In the current study, we aimed to clarify the structural role and functional diversity of ACC in the tergite cuticle of A. vulgare. To this end, we examined how different external calcium carbonate sources influence cuticle mineralization and organization to determine whether A. vulgare discriminates among polymorphs during biomineral formation. This study provides new insights into the mechanisms and physiological regulation of biomineralization in terrestrial isopods.