Shell Matrix Research Articles

Exploring the acid-insoluble shell organic matrix of freshwater mussels (Unionoida) as a proxy for palaeodietary and paleoenvironmental studies

ABSTRACT The shells of freshwater mussels, commonly found at archaeological sites associated with the Kodjadermen-Gumelnița-Karanovo VI human groups (5th millennium BCE, Romania and Bulgaria), were a significant dietary component for this population. The organic matrix of these ancient shells is often well-preserved and can be analysed using stable isotopes. This study establishes the empirical relationship between the isotopic composition of the acid-insoluble shell organic matrix (AIM) and the soft tissues of modern Unio tumidus. A quality criterion for AIM (C:N ratio 3.6 (3.3–<3.8)) was defined, and isotopic offsets (Δ15NAIM-defatted body = +0.95‰, Δ13CAIM-defatted body = +0.93‰ for soft tissues; Δ15NAIM-muscle = +1.7‰, Δ13CAIM-muscle = +0.3‰ for the adductor muscle) were determined. Our data suggest that the Gumelnița mussels were omnivorous, with a similar trophic level to carp and pond turtles, sourced from diverse water bodies (rivers, lakes …) based on δ13C values.

Study on corrosion mechanism of laser-clad Ni-WxC coating by in-situ electrochemical method under high temperature and pressure environment

Corrosion protection of oil and gas equipment by laser cladding technology has emerged as a crucial method to reduce costs and increase efficiency. The in-situ electrochemical method was employed to investigate the microstructure evolution and corrosion mechanisms of laser-clad Ni1725-WxC coatings under high temperature and pressure environment. The coating is composed of cellular γ-Ni (W, Cr, Fe), block W2C, WC/W2C eutectic structure with a shell of WC, and small amount of CrxCy (Cr7C3/Cr23C6) in Ni1725 matrix. The corrosion resistance of the coating is worsened with increasing temperature under in-situ electrochemical method owing to reduced reaction activation energy and increased transfer rate. Furthermore, the surface potentials of the coating follow the order: E1 (WC shell) > E2 (Ni1725 matrix (oxide)) > E3 (WC/W2C eutectic phase) > E4 (block W2C). Therefore, galvanic corrosion occurs between WC shell and Ni1725 matrix. W2C with lower potential corrodes preferentially as well at WC/W2C eutectic structure in WxC particle, which expands with increasing temperature to form a loose internal structure.

The adult shell matrix protein repertoire of the marine snail Crepidula is dominated by conserved genes that are also expressed in larvae

Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, ‘novel’ shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod species Crepidula fornicata and Crepidula atrasolea because they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development in C. atrasolea for the first time. Next, we leveraged previous findings in C. fornicata combined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% of C. fornicata’s SMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority of C. fornicata SMPs that were found conserved in C. atrasolea were expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development in C. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework in Crepidula that enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification.

Opal-Inspired SiO2-Mediated Carbon Dot Doping Enables the Synthesis of Monodisperse Multifunctional Afterglow Nanocomposites for Advanced Information Encryption.

Despite recent advancements in inorganic and organic phosphors, creating monodisperse afterglow nanocomposites (NCs) remains challenging due to the complexities of wet chemistry synthesis. Inspired by nanoinclusions in opal, we introduce a novel SiO2-mediated carbon dot (CD) doping method for fabricating monodisperse, multifunctional afterglow NCs. This method involves growing a SiO2 shell matrix on monodisperse nanoparticles (NPs) and doping CDs into the SiO2 shell under hydrothermal conditions. Our approach preserves the monodispersity of the parent NP@SiO2 NCs while activating a green afterglow in the doped CDs with an impressive lifetime of 1.26 s. Additionally, this method is highly versatile, allowing for various core and dopant combinations to finely tune the afterglow through core-to-CD or CD-to-dye energy transfer. Our findings significantly enhance the potential of SiO2 coatings, transforming them from merely enhancing the biocompatibility of NCs to serving as a versatile matrix for emitters, facilitating afterglow generation and paving the way for new applications.

Glucose-Responsive Microneedle Patch with High Insulin Loading Capacity for Prolonged Glycemic Control in Mice and Minipigs.

Transdermal microneedle-mediated glucose-responsive insulin delivery systems can modulate insulin release based on fluctuations in blood glucose levels, thus maintaining normoglycemia effectively in a continuous, convenient, and minimally invasive manner. However, conventional microneedles are limited by the low drug loading capacity, making it challenging to be applied on human skin at a reasonable size for a lasting glucose-controlling effect, thus hindering their clinical translation. Here, we design a microneedle patch with a solid insulin powder core to achieve a high loading capacity of insulin (>70 wt %) as well as a glucose-sensitive polymeric shell to realize glucose-responsive insulin release. Once exposed to hyperglycemia, the formation of negatively charged glucose-boronate complexes increases the charge density of the shell matrix, leading to swelling of the shell and accelerating insulin release from the core. We have demonstrated that this glucose-responsive microneedle patch could achieve long-term regulation of blood glucose levels in both type 1 diabetic mice and minipigs (up to 48 h with patches of ∼3.5 cm2 for minipigs >25 kg).

Reflectivity and Angular Anisotropy of Liquid Crystal Microcapsules with Different Particle Sizes by Complex Coalescence.

Cholesteric liquid crystal microcapsules (CLCMs) are used to improve the stability of liquid crystals while ensuring their stimulus response performance and versatility, with representative applications such as sensing, anticounterfeiting, and smart fabrics. However, the reflectivity and angular anisotropy decrease because of the anchoring effect of the polymer shell matrix, and the influence of particle size on this has not been thoroughly studied. In this study, the effect of synthesis technology on microcapsule particle size was investigated using a complex coalescence method, and the effect of particle size on the reflectivity and angular anisotropy of CLCMs was investigated in detail. A particle size of approximately 66 µm with polyvinyl alcohol (PVA, 1:1) exhibited a relative reflectivity of 16.6% and a bandwidth of 20 nm, as well as a narrow particle size distribution of 22 µm. The thermosetting of microcapsules coated with PVA was adjusted and systematically investigated by controlling the mass ratio. The optimized mass ratio of microcapsules (66 µm) to PVA was 2:1, increasing the relative reflectivity from 16.6% (1:1) to 32.0% (2:1) because of both the higher CLCM content and the matching between the birefringence of the gelatin-arabic shell system and PVA. Furthermore, color based on Bragg reflections was observed in the CLCM-coated ortho-axis and blue-shifted off-axis, and this change was correlated with the CLCM particle size. Such materials are promising for anticounterfeiting and color-based applications with bright colors and angular anisotropy in reflection.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein–protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Zero-waste approach to fluoride removal by contact precipitation using Hyriopsis cumingii shells with value-added products

We propose a zero-waste approach using Hyriopsis cumingii shells for fluoride (F) removal via contact precipitation with value-added products. H3PO4 was used as the precipitant for F removal. The F concentration could be reduced from 9.5 mg/L to 0.09–0.91 mg/L for over 900 BV with running-backwashing cycle. The precipitates formed during contact precipitation were easily stripped from the shell matrix by periodic backwashing, thus supporting the continuous dissolution of calcium (Ca) from the shell particles. X-ray diffraction (XRD)-Rietveld analysis showed that the backwashing residue was fluorapatite (FAP) with a small amount of hydroxy substitution. Therefore, FAP can be used as a raw material in phosphate fertilizer manufacturing. Economic analysis and sustainability evaluation showed that the contact precipitation process with H. cumingii shells as the substrate not only reduces chemical costs but also generates value-added products; thus, this process is environmentally friendly compared to traditional methods of F removal. The proposed approach for F elimination, waste shell utilization, and F and Ca resource recovery in the contact precipitation process represents a novel sustainable water treatment technology.

Nonlinear forced vibration of functionally graded hybrid three-phase nanocomposite toroidal shell segments reinforced by carbon nanotubes (CNTs) and graphene nanoplatelets (GPLs)

This study proposes a unique three-phase functionally graded (FG) hybrid nanocomposite material reinforcing the toroidal shell segments to investigate the nonlinear forced vibration using Reddy's higher-order shear deformation theory, von Karman geometrical nonlinearity and Stein-McElman's assumption along with the Hamilton principle. The examined toroidal shell's material consists of polymeric resin, carbon nanotubes (CNTs), and graphene platelets (GPLs) as nanocomposites. The material properties have been derived based on a modified Halpin-Tsai micromechanical model. Four distribution patterns have been studied: uniform (UD), FG-X, FG-O, and FG-V. The continuous distribution of GPLs and CNTs leads to inhomogeneous position-dependent properties throughout the shell thickness. The obtained differential equations of motion have been reduced to ordinary equations using Galerkin's technique. A multi-scales method (MSM) is used to estimate a closed-form solution representing the frequency-amplitude relation, and the state space representation is used along with numerical fourth-order Runge Kutta method (RK4) to obtain the nonlinear dynamic response of the toroidal shell. The accuracy of the current results obtained has been verified by comparing them with the relevant literature and numerical results using (RK4). In addition, the influence of both GPLs and CNTs weight fractions, nanofillers’ distribution types, longitudinal and circumferential wave numbers, elastic foundation parameters, static axial compression load, transverse excitation load, damping ratio, geometrical characteristics of the toroidal shell on the dimensionless natural frequency, nonlinear primary resonance (frequency-amplitude curve), and nonlinear dynamic response are carefully studied. The results found that combining (GPLs & CNTs) into the shell's matrix improves performance, especially in concave shells compared to convex ones. The FG-X pattern reduces peak resonance amplitude and improves natural frequency. A reduction of 12.3 % is observed for convex shells, and 21.5 % for concave shells compared to FG-O.

Transcriptome analysis of mantle and pearl sac of freshwater pearl mussel Lamellidens marginalis to identify biomineralisation-associated genes

ABSTRACT The freshwater pearl mussel, Lamellidens marginalis, is widely distributed in India and is cultured for freshwater pearl production. Understanding the biomineralisation process sheds light on the genes responsible for nacre secretion. However, information on the transcriptome of L. marginalis mantle tissue and pearl sac is lacking. This study generated transcriptome resources for L. marginalis, identifying genes involved in biomineralisation. Illumina paired-end sequencing yielded 11.13 million raw reads, assembled into 133,246 contigs and 26,373 unigenes. Of these, 21,033 unigenes exhibited homology with previously reported molluscan proteins. Genes related to biomineralisation, such as pif, perlucin, calreticulin, calmodulin, chitin synthase, chitin dehydrogenase, carbonic anhydrase, tyrosinase, shell matrix protein, dermatopontin isoform X1, chitinase 3, chitinase domain-containing protein 1, and putative chitinase-1were identified. This baseline information will help to compare the expression levels between the phenotypes of high-quality and low-quality pearls. Further investigations, incorporating large sample sizes and phenotype information, could assist in identifying type I genetic markers linked to pearl quality.

Scallop shells as biosorbents for water remediation from heavy metals: Contributions and mechanism of shell components in the adsorption of cadmium from aqueous matrix

To ascertain their potential for heavy metal pollution remedy, we studied the adsorption mechanism of cadmium onto scallop shells and the interactions between the heavy metal and the shell matrix. Intact shells were used to investigate the uptake and diffusion of the metal contaminant onto the shell carbonatic layers, as well as to evaluate the distribution of major and trace elements in the matrix. LA-ICPMS measurements demonstrate that Cd is adsorbed on a very thin layer on the inner and outer surfaces of the shell. Structural and thermal analyses showed the presence of 9 wt.-% of a CdCO3 phase indicating that the adsorption is mainly a superficial process which involves different processes, including ion exchange of Ca by Cd. In addition, organic components of the shell could contribute to adsorption as highlighted by different metal uptake observed for shells with different colours. In particular, darker shells appeared to adsorb more contaminant than the white ones. The contribution of the organic shell components on the adsorption of heavy metals was also highlighted by the element bulk content which showed higher concentrations of different metals in the darker specimen. Raman spectroscopy allowed to identify the pigments as carotenoids, confirmed by XRD measurements which highlighted the presence of astaxanthin phases. The results presented here provide new insights into the Cd adsorption mechanism highlighting the important contribution given by the organic components present in the biogenic carbonate matrix. Furthermore, the high efficiency of Cd removal from water by scallop shells, supported by adsorption kinetic and isotherm studies, has been demonstrated.

Diverting the Use of Hand-Operated Tablet Press Machines to Bioassays: A Novel Protocol to Test 'Waste' Insoluble Shell Matrices.

To mineralize their shells, molluscs secrete a complex cocktail of proteins-collectively defined as the calcifying shell matrix-that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease inhibitors offer the most tangible perspectives in applied sciences, health, and aquaculture. However, one technical obstacle in testing the activity of shell extracts lies in their high insolubility. In this paper, we present a protocol that circumvents this impediment. After an adapted shell protein extraction and the production of two organic fractions-one soluble, one insoluble-we employ a hand-operated tablet press machine to generate well-calibrated tablets composed of 100% insoluble shell matrix. FT-IR monitoring of the quality of the tablets shows that the pressure used in the press machine does not impair the molecular properties of the insoluble extracts. The produced tablets can be directly tested in different biological assays, such as the bactericidal inhibition zone assay in Petri dish, as illustrated here. Diverting the use of the hand-operated tablet press opens new perspectives in the analysis of insoluble shell matrices, for discovering novel bioactive components.

'Sorrento' and 'Tulare' Walnut Cultivars: Morphological Traits and Phytochemical Enhancement of Their Shell Waste.

Walnut processing generates considerable quantities of by-products that could be reprocessed into value-added products that have food and non-food applications. In this context, the aim of this study is to characterize the 'Sorrento' and 'Tulare' walnut cultivars using the UPOV guidelines and analyze the chemical composition and antioxidant activity of their shells. Insight into the chemical composition of the different granulometric fractions of walnut shell, obtained by sieving, was obtained following ultrasound-assisted extraction by Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS). The total phenolic, flavonoid, and tannin content and antiradical capacity, obtained by DPPH and ABTS assays, and the Fe(III) reducing power of the extracts were also evaluated. The UHPLC-HRMS analysis indicated the presence of thirty-two compounds ascribable to four major classes of specialized metabolites. Furthermore, the extraction efficiency of gallic acid, ellagic acid derivatives, as well as glansreginin A, increased with the decrease in shell matrix particle size in contrast to chlorogenic acids and flavonoid glycosides. This is the first study to highlight new knowledge on the chemical composition of walnut shells. The results obtained demonstrate the feasibility of recovering valuable bioactive components from agro-waste that may be further valorized.

Microcapsule Triggering Mechanics in Cementitious Materials: A Modelling and Machine Learning Approach.

Self-healing cementitious materials containing microcapsules filled with healing agents can autonomously seal cracks and restore structural integrity. However, optimising the microcapsule mechanical properties to survive concrete mixing whilst still rupturing at the cracked interface to release the healing agent remains challenging. This study develops an integrated numerical modelling and machine learning approach for tailoring acrylate-based microcapsules for triggering within cementitious matrices. Microfluidics is first utilised to produce microcapsules with systematically varied shell thickness, strength, and cement compatibility. The capsules are characterised and simulated using a continuum damage mechanics model that is able to simulate cracking. A parametric study investigates the key microcapsule and interfacial properties governing shell rupture versus matrix failure. The simulation results are used to train an artificial neural network to rapidly predict the triggering behaviour based on capsule properties. The machine learning model produces design curves relating the microcapsule strength, toughness, and interfacial bond to its propensity for fracture. By combining advanced simulations and data science, the framework connects tailored microcapsule properties to their intended performance in complex cementitious environments for more robust self-healing concrete systems.

Stable isotopes in the shell organic matrix for (paleo)environmental reconstructions

Stable isotope ratios of mollusc shell carbonates have long been used to reconstruct past environmental conditions. Although shells also contain organics, they are seldom used in (paleo)climatic studies. Here, we extract the acid-soluble and insoluble fractions of the organic matrix of modern Mytilus galloprovincialis shells from three sites along a coast–to-upper-estuary environmental gradient to measure their hydrogen (δ2H) and oxygen (δ18O) isotope compositions. Both organic fractions showed isotopic signatures significantly different from those of carbonate and water at each site, indicating the involvement of different fractionation mechanisms. The soluble fraction showed gradual differences in isotope values along the gradient, while the insoluble fraction showed δ2H-δ18O correlation regressions subparallel to the Global and Local Meteoric Water Lines. These results showed the great potential of the shell organic matrix stable isotopes as possible (paleo)environmental proxies, stimulating further research to better define the fractionation mechanisms involved.

Microencapsulation of fish oil with brewer’s spent grain proteins: Effect of citric acid and emulsion pH

The use of brewer’s spent grain (BSG) proteins as wall material for encapsulating fish oil was investigated for the first time. This study also demonstrated the effect of citric acid (CA)-crosslinking and emulsion pH on the encapsulating ability of BSG proteins. Four different BSG microcapsules were prepared, including non-crosslinked and crosslinked BSG microcapsules at pH either above or below the isoelectric point (pI) of BSG proteins, and their performance was compared with OSA-starch. Crosslinked BSG emulsions had larger droplet size than non-crosslinked ones. The largest droplet sizes were found in emulsions where pH < pI due to some degree of protein aggregation. Encapsulation efficiency of BSG microcapsules ranged from 81 to 93%. BSG microcapsules, regardless of crosslinking, showed considerably higher oxidative stability than OSA-starch microcapsules. CA-crosslinking further enhanced their oxidative stability. The highest and the lowest oxidative stabilities among BSG microcapsules belonged to the microcapsules prepared at pH < pI, where some degree of protein aggregation occurred in the emulsions. Crosslinked BSG microcapsule prepared at pH < pI exhibited the highest oxidative stability with peroxide and thiobarbituric acid reactive substances (TBARS) values remaining in the range allowed for fresh oils even after 15 days of storage at 40 °C. It was attributed to its denser shell matrix due to CA-crosslinking and multilayer formation resulting from protein aggregation. Non-crosslinked microcapsules prepared at pH < pI represented the lowest oxidation stability among BSG microcapsules due to larger pores. The results suggest a synergistic effect of CA-crosslinking and protein aggregation, which significantly reinforced shell matrix and effectively reduced oxygen permeability.

Biomineralization in a cold environment: Insights from shield compositions and transcriptomics of polar sternaspids (Sternaspidae, Polychaeta)

The survival and physiological functions of polar marine organisms are impacted by global climate changes. Investigation of the adaptation mechanisms underlying biomineralization in polar organisms at low temperatures is important for understanding mineralized organismal sensitivity to climate change. Here, we performed electron probe analysis on the shields of Antarctic polychaete Sternaspis sendalli and Arctic polychaete Sternaspis buzhinskajae (Sternaspidae), and sequenced the transcriptomes of the tissues surrounding shields to examine biomineral characteristics and adaptive mechanisms in persistently cold environments. Compared to the temperate relative species, the relative abundance of iron, phosphorus, calcium, magnesium, nitrogen, sulfur and silicon in two polar sternaspid shields was similar to Sternaspis chinensis. However, the diversity and expression levels of biomineralization-related shell matrix proteins differed between the polar and temperate species, suggesting distinct molecular mechanisms underlying shield formation in cold environments. Tubulin and cyclophilin were upregulated compared to the temperate species. Furthermore, 42 positively selected genes were identified in Antarctic S. sendalli, with functions in cytoskeletal structure, DNA repair, immunity, transcription, translation, protein synthesis, and lipid metabolism. Highly expressed genes in both polar species were associated with cytoskeleton, macromolecular complexes and cellular component biosynthesis. Overall, this study reveals conserved elemental composition yet distinct biomineralization processes in the shields of polar sternaspids. The unique expression of biomineralization related genes and other cold-adaptation related genes provide molecular insights into biomineralization in cold marine environments.

Verifiable (t, n) Secret Image Sharing Scheme Based on Slim Turtle Shell Matrix

As Internet technology advances rapidly, safeguarding confidential messages has become an increasingly pressing concern. Among the various schemes available, secret image sharing (SIS) has gained considerable attention. Particularly, the (t, n) secret sharing scheme offers robust protection for hidden secret messages. However, it remains challenging to expand the quality of t and n while maintaining acceptable image quality for the share images and enhancing security features. To address these challenges, we have developed a variant turtle shell known as the slim turtle shell, which we incorporate into our innovative (t, n)-SIS scheme. The slim turtle shell technique not only facilitates the creation of meaningful shares but also ensures that the resulting meaningful shares maintain a high level of visual quality, thanks to its impressive embedding capabilities. Additionally, we have devised two authentication mechanisms to validate the integrity of both shares and secret images. These mechanisms facilitate the detection of tampering during the secret message extraction and verification phase, should the integrity be compromised. Empirical results from our experiments demonstrate an impressive embedding capacity of 1,572,864 bits in our proposed scheme, coupled with an average PSNR (Peak Signal-to-Noise Ratio) of 45.80 dB. Furthermore, our scheme demonstrates superior authentication capabilities when compared to other existing schemes.

Proteomic analysis of shell matrix proteins from the chiton Acanthopleura loochooana

Most molluscs have mineralized shells to protect themselves. Although the remarkable mechanical properties of shells have been well-studied, the origin of shells is still elusive. Chitons are unique in molluscs because they are shelly Aculifera which diverged from Conchifera (comprising all the shell-bearing classes of molluscs) in the early pre-Cambrian. We developed a method to extract shell proteins from chiton shell plates (removing embedded soft tissues) and then compared the shell proteome to that of Conchifera groups. Sixteen shell matrix proteins from Acanthopleura loochooana were identified by proteomics, in which Nacrein-like, Pif-like proteins, and protocadherin were found. Additional evidences from shell proteome in another species Chiton densiliratus and comparative sequence alignment in five chitons supported a conserved biomineralization toolkit in chitons. Our findings shed light on the evolution of mineralization in chitons and pose a hypothesis that ancestral molluscs have already evolved biomineralization toolkits, which may facilitate the formation of mineralized shells.

EF-Hand-Binding Secreted Protein Hdh-SMP5 Regulates Shell Biomineralization and Responses to Stress in Pacific Abalone, Haliotis discus hannai.

The development of a shell is a complex calcium metabolic process involving shell matrix proteins (SMPs). In this study, we describe the isolation, characterization, and expression of SMP5 and investigate its potential regulatory role in the shell biomineralization of Pacific abalone Haliotis discus hannai. The full-length Hdh-SMP5 cDNA contains 685 bp and encodes a polypeptide of 134 amino acids. Structurally, the Hdh-SMP5 protein belongs to the EF-hand-binding superfamily, which possesses three EF-hand Ca2+-binding regions and is rich in aspartic acid. The distinct clustering patterns in the phylogenetic tree indicate that the amino acid composition and structure of this protein may vary among different SMPs. During early development, significantly higher expression was observed in the trochophore and veliger stages. Hdh-SMP5 was also upregulated during shell biomineralization in Pacific abalone. Long periods of starvation cause Hdh-SMP5 expression to decrease. Furthermore, Hdh-SMP5 expression was observed to be significantly higher under thermal stress at temperatures of 15, 30, and 25 °C for durations of 6 h, 12 h, and 48 h, respectively. Our study is the first to characterize Hdh-SMP5 comprehensively and analyze its expression to elucidate its dynamic roles in ontogenetic development, shell biomineralization, and the response to starvation and thermal stress.