Carbonate and phosphate biominerals, such as bones and shells, confer critical mechanical support and protection to various organisms [38], significantly contributing to the evolutionary success of invertebrates [31]. Proteins in biominerals, known as shell matrix proteins (SMPs), have been demonstrated to control the biomineralization of various biominerals [31]. Phylogenetics, genomics, and proteomics have provided comparative biological insights into calcium carbonate biomineralization: the biomineralization of calcium carbonate skeletons has independently originated and undergone convergent evolution across different phyla [19].

Over the past two decades, high-throughput methods such as RNA-seq and proteomics have greatly facilitated the discovery of SMPs, amassing proteomic data from the shells and skeletons of bivalves [3], gastropods [32], cephalopods [31], brachiopods [37], and corals [12]. These studies have highlighted the diversity of SMPs, confirming the presence of lineage-specific SMPs across multiple organisms [37,40]. Some conserved structural domains were shared among different organisms. For example, carbonic anhydrase (CA), chitin-binding domain 2, von Willebrand factor type A (VWA), and tyrosinase have been identified as common domains in the SMPs of bivalve shells [3]. Based on these findings, the classic “chitin-silk fibroin gel proteins-acidic macromolecules” model has been proposed to explain the process of calcium carbonate biomineralization [1].

However, the molecular mechanisms and biomineralization patterns underlying phosphate minerals in invertebrates remain unclear. Phosphate biominerals, in the form of crystalline or amorphous hydrogels, were widely present in the hard tissues of lophotrochozoans, including annelids, brachiopods, mollusks (Polyplacophora, Gastropoda and Bivalvia), arthropods (Cephalopoda) and holothurians [34]. Genomic and proteomic analyses of the brachiopod Lingula have revealed the independent origin of its calcium phosphate biomineralization, which was significantly different from the genetic mechanisms involved in the formation of calcium carbonate shells in mollusks [37].

The family Sternaspidae is an important group of phosphate-mineralized organisms within the phylum Annelida. They possess a bilaterally symmetrical shield on the ventro-caudal side of their body, composed of an amorphous iron phosphate hydrogel [34]. The study of composition of the matrix proteins in their shields is crucial for understanding the phosphate mineralization and evolution of lophotrochozoans. The shield was an important diagnostic feature for the family Sternaspidae, but recent molecular data have raised questions regarding the taxonomic efficacy of the shield. Wu et al. [56] described the widely distributed Sternaspis chinensis and Sternaspis liui in the China Sea, and the shield morphology was the key feature to distinguish them: the former has a harder shield with non-prominent main ribs and concentric lines, while the latter has a softer shield with prominent main ribs and concentric lines. Our previous study found that S. chinensis and S. liui syn represent two distinct shield morphology types of the same species, with elemental analysis showing significantly higher levels of iron, phosphorus, and calcium in the shields of S. chinensis [18]. Preliminary transcriptome analysis also revealed differential expression of biomineralization-related genes in tissues around the shield, suggesting that shield formation may be regulated by different molecular pathways for each morphology [18]. The SMPs are a key part of molecular regulation in the process of biomineralization, and their differential expression in the shell has been shown to lead to phenotypic plasticity of many mollusk shells [4]. However, the effect of shield matrix proteins (ShiMPs) on the morphological variation of shields is not clear.

This study conducted a quantitative analysis of the protein expression profiles of ShiMPs in the two distinct shield morphologies of S. chinensis (SC group, characterized by a harder shield with non-prominent main ribs and concentric lines, and higher iron phosphate and calcium content) and S. liui syn (SL group, characterized by a softer shield with prominent main ribs and concentric lines, and lower iron phosphate and calcium content). The goal was to identify key regulatory proteins responsible for phenotypic and chemical composition differences, and to elucidate the molecular basis of amorphous ferric phosphate hydrogel biomineralization in sternaspids. Then, we compared ShiMPs of sternaspids with SMPs of other marine invertebrates to enhance our understanding of phosphate biomineralization in annelids.