The role of the carapace in the accumulation of metals from seawater in the green crab (Carcinus maenas): Studies with radio-labeled calcium, zinc, and nickel.

The role of the carapace in the uptake and storage of newly accumulated metals was investigated in the green crab exposed to environmentally relevant concentrations of calcium ([Ca] = 389 mg L-1 or 9.7 mmol L-1), zinc ([Zn] = 82 µg L-1 or 1.25 µmol L-1), and nickel ([Ni] = 8.2 µg L-1 or 0.14 µmol L-1) in 12 °C seawater, using radio-tracers (45Ca, 65Zn, 63Ni). After 24-h exposure, carapace exhibited the highest concentration of newly accumulated Ca, whereas carapace and gills exhibited the highest concentrations of both newly accumulated Zn and Ni relative to other tissues. For all three metals, the carapace accounted for >85 % of the total body burden. Acute temperature changes (to 2 °C and 22 °C) revealed the highest overall temperature coefficient Q10 (2.15) for Ca uptake into the carapace, intermediate Q10 for Ni (1.87) and lowest Q10 (1.45) for Zn. New Ca uptake into the carapace continued linearly with time for 24 h, new Zn uptake gradually deviated from linearity, whereas Ni uptake reached a plateau by 6 h. Attachment of a rubber membrane to the dorsal carapace, thereby shielding about 20 % of the total crab surface area from the external water, eliminated both new Zn and Ni incorporation into the shielded carapace, whereas 36 % of new Ca incorporation persisted. When recently euthanized crabs were exposed, new Zn uptake into the carapace remained unchanged, whereas Ca and Ni uptake were reduced by 89 % and 71 %, respectively. We conclude that the carapace is a very important uptake and storage site for all three metals. All of the uptake of new Zn and new Ni, and most of the uptake of new Ca into this tissue comes directly from the external water. For Zn, the mechanism involves only physicochemical processes, whereas for Ca and Ni, life-dependent processes make the major contribution.

Modulation of host lipid metabolism by virus infection leads to exoskeleton damage in shrimp.

The arthropod exoskeleton provides protection and support and is vital for survival and adaption. The integrity and mechanical properties of the exoskeleton are often impaired after pathogenic infection; however, the detailed mechanism by which infection affects the exoskeleton remains largely unknown. Here, we report that the damage to the shrimp exoskeleton is caused by modulation of host lipid profiles after infection with white spot syndrome virus (WSSV). WSSV infection disrupts the mechanical performance of the exoskeleton by inducing the expression of a chitinase (Chi2) in the sub-cuticle epidermis and decreasing the cuticle chitin content. The induction of Chi2 expression is mediated by a nuclear receptor that can be activated by certain enriched long-chain saturated fatty acids after infection. The damage to the exoskeleton, an aftereffect of the induction of host lipogenesis by WSSV, significantly impairs the motor ability of shrimp. Blocking the WSSV-caused lipogenesis restored the mechanical performance of the cuticle and improved the motor ability of infected shrimp. Therefore, this study reveals a mechanism by which WSSV infection modulates shrimp internal metabolism resulting in phenotypic impairment, and provides new insights into the interactions between the arthropod host and virus.

Elastic constants of biogenic calcium carbonate.

Living organisms form complex mineralized composite architectures that perform a variety of essential functions. These materials are commonly utilized for load-bearing purposes such as structural stability and mechanical strength in combination with high toughness and deformability, which are well demonstrated in various highly mineralized molluscan shell ultrastructures. Here, the mineral components provide the general stiffness to the composites, and the organic interfaces play a key role in providing these biogenic architectures with mechanical superiority. Although numerous studies employed state-of-the-art methods to measure and/or model and/or simulate the mechanical behavior of molluscan shells, our understanding of their performance is limited. This is partially due to the lack of the most fundamental knowledge of their mechanical characteristics, particularly, the anisotropic elastic properties of the mineral components and of the tissues they form. In fact, elastic constants of biogenic calcium carbonate, one of the most common biominerals in nature, is unknown for any organism. In this work, we employ the ultrasonic pulse-echo method to report the elasticity tensor of two common ultrastructural motifs in molluscan shells: the prismatic and the nacreous architectures made of biogenic calcite and aragonite, respectively. The outcome of this research not only provides information necessary for fundamental understanding of biological materials formation and performance, but also yields textbook knowledge on biogenic calcium carbonate required for future structural/crystallographic, theoretical and computational studies.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis.

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: -0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

Interpreting life-history traits, seasonal cycles, and coastal climate from an intertidal mussel species: Insights from 9000 years of synthesized stable isotope data.

Understanding past coastal variability is valuable for contextualizing modern changes in coastal settings, yet existing Holocene paleoceanographic records for the North American Pacific Coast commonly originate from offshore marine sediments and may not represent the dynamic coastal environment. A potential archive of eastern Pacific Coast environmental variability is the intertidal mussel species Mytilus californianus. Archaeologists have collected copious stable isotopic (δ18O and δ13C) data from M. californianus shells to study human history at California's Channel Islands. When analyzed together, these isotopic data provide windows into 9000 years of Holocene isotopic variability and M. californianus life history. Here we synthesize over 6000 δ18O and δ13C data points from 13 published studies to investigate M. californianus shell isotopic variability across ontogenetic, geographic, seasonal, and millennial scales. Our analyses show that M. californianus may grow and record environmental information more irregularly than expected due to the competing influences of calcification, ontogeny, metabolism, and habitat. Stable isotope profiles with five or more subsamples per shell recorded environmental information ranging from seasonal to millennial scales, depending on the number of shells analyzed and the resolution of isotopic subsampling. Individual shell profiles contained seasonal cycles and an accurate inferred annual temperature range of ~ 5°C, although ontogenetic growth reduction obscured seasonal signals as organisms aged. Collectively, the mussel shell record reflected millennial-scale climate variability and an overall 0.52‰ depletion in δ18Oshell from 8800 BP to the present. The archive also revealed local-scale oceanographic variability in the form of a warmer coastal mainland δ18Oshell signal (-0.32‰) compared to a cooler offshore islands δ18Oshell signal (0.33‰). While M. californianus is a promising coastal archive, we emphasize the need for high-resolution subsampling from multiple individuals to disentangle impacts of calcification, metabolism, ontogeny, and habitat and more accurately infer environmental and biological patterns recorded by an intertidal species.

Hsp70 Knockdown in the Brine Shrimp Artemia franciscana: Implication on Reproduction, Immune Response and Embryonic Cuticular Structure.

The potential functional role(s) of heat shock protein 70 (Hsp70) in the brine shrimp, Artemia franciscana, a crucial crustacean species for aquaculture and stress response studies, was investigated in this study. Though we have previously reported that Hsp70 knockdown may have little or no impact on Artemia development, the gestational survival and number of offspring released by adult females were impaired by obscuring Hsp70 synthesis. Transcriptomic analysis revealed that several cuticle and chitin synthetic genes were downregulated, and carbohydrate metabolic genes were differentially expressed in Hsp70-knockdown individuals. A more comprehensive microscopic examination performed in this study revealed exoskeleton structural destruction and abnormal eye lenses featured in Hsp70-deficient adult females 48 h after Hsp70 dsRNA injection. Cysts produced by these Hsp70-deficient broods, instead, had a defective shell and were smaller in size, whereas nauplii had shorter first antennae and a rougher body epicuticle surface. Changes in carbohydrate metabolism caused by Hsp70 knockdown affected glycogen levels in adult Artemia females, as well as trehalose in cysts released from these broods, indicating that Hsp70 may play a role in energy storage preservation. Outcomes from this work provided novel insights into the roles of Hsp70 in Artemia reproduction performance, cyst formation, and exoskeleton structure preservation. The findings also support our previous observation that Hsp70 knockdown reduced Artemia nauplius tolerance to bacterial pathogens, which could be explained by the fact that loss of Hsp70 downregulated several Toll receptor genes (NT1 and Spaetzle) and reduced the integrity of the exoskeleton, allowing pathogens to enter and cause infection, ultimately resulting in mortality.

The lgi-miR-2d is Potentially Involved in Shell Melanin Synthesis by Targeting mitf in Manila Clam Ruditapes philippinarum.

Shell color as an important economic trait is also the crucial target trait for breeding and production. MicroRNA (miRNA) is an endogenous small non-coding RNA that can post-transcriptionally regulate the expression of target genes, it plays important roles in many life activities and physiological processes, such as shell color, stress response, and disease traits. In this study, we investigated the function of lgi-miR-2d in shell melanin formation and the expression patterns of lgi-miR-2d and target gene Rpmitf in Manila clam Ruditapes philippinarum. We further explored and verified the relationship between Rpmitf and lgi-miR-2d and identified the expression level of shell color-related gene changes by RNAi and injecting the antagomir of lgi-miR-2d, respectively. Our results indicated that lgi-miR-2d antagomir affected the expression of its target gene Rpmitf. In addition, the dual-luciferase reporter assay was conducted to confirm the direct interaction between lgi-miR-2d and Rpmitf. The results showed that the expression levels of melanin-related genes such as Rpmitf and tyr were significantly decreased in the positive treatment group compared with the blank control group after the Rpmitf dsRNA injection, indicating Rpmitf plays a crucial role in the melanin synthesis pathway. Taken together, we speculated that lgi-miR-2d might be negatively modulating Rpmitf, which might regulate other shell color-related genes, thereby affecting melanin synthesis in R. philippinarum.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

Transcriptomic and Physiological Analysis Reveal Melanin Synthesis-Related Genes and Pathways in Pacific Oysters (Crassostrea gigas).

Shell color is one of the shell traits of molluscs, which has been regarded as an economic trait in some bivalves. Pacific oysters (Crassostrea gigas) are important aquaculture shellfish worldwide. In the past decade, several shell color strains of C. gigas were developed through selective breeding, which provides valuable materials for research on the inheritance pattern and regulation mechanisms of shell color. The inheritance patterns of different shell colors in C. gigas have been identified in certain research; however, the regulation mechanism of oyster pigmentation and shell color formation remains unclear. In this study, we performed transcriptomic and physiological analyses using black and white shell oysters to investigate the molecular mechanism of melanin synthesis in C. gigas. Several pigmentation-related pathways, such as cytochrome P450, melanogenesis, tyrosine metabolism, and the cAMP signaling pathway were found. The majority of differentially expressed genes and some signaling molecules from these pathways exhibited a higher level in the black shell oysters than in the white, especially after L-tyrosine feeding, suggesting that those differences may cause a variation of tyrosine metabolism and melanin synthesis. In addition, the in vitro assay using primary cells from mantle tissue showed that L-tyrosine incubation increased cAMP level, gene and protein expression, and melanin content. This study reveals the difference in tyrosine metabolism and melanin synthesis in black and white shell oysters and provides evidence for the potential regulatory mechanism of shell color in oysters.

Outer fold is sole effective tissue among three mantle folds with regard to oyster shell colour.

Molluscs constitute the second largest phylum of animals in the world, and shell colour is one of their most important phenotypic characteristics. In this study, we found among three folds on the mantle edge of oyster, only the outer fold had the same colour as the shell. Transcriptome and mantle cutting experiment indicated that the outer fold may be mainly reflected in chitin framework formation and biomineralisation. There were obvious differences in SEM structure and protein composition between the black and white shell periostraca. The black shell periostraca had more proteins related to melanin biosynthesis and chitin binding. Additionally, we identified an uncharacterized protein gene (named as CgCBP) ultra-highly expressed only in the black outer fold and confirmed its function of chitin-binding and CaCO3 precipitation promoting. RNAi also indicated that CgCBP knockdown could change the structure of shell periostracum and reduce shell pigmentation. All these results suggest that the mantle outer fold plays multiple key roles in shell periostraca bioprocessing, and shell periostracum structure affected by chitin-binding protein is functionally correlated with shell pigmentation. The investigation of oyster shell periostracum structure and shell colour will provide a better understanding in pigmentation during biological mineralisation in molluscs.

Direct control of shell regeneration by the mantle tissue in the pearl oyster Pinctada fucata.

Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells.

A novel matrix protein PNU5 facilitates the transformation from amorphous calcium carbonate to calcite and aragonite.

For both nacre formation and biomineralization in mollusks, understanding the molecular mechanism is imperative. Biomineralization, especially shell formation, is dedicatedly regulated by multiple matrix proteins. However, ACC conversion to stable crystals still lacks positive factors. In this research, we found a novel matrix protein named PNU5 in Pinctada fucata that plays a regulatory role in both prismatic layer and nacreous layer formation. Functional studies in vivo and in vitro have shown that it might be involved in shell formation in a positive manner. RT-qPCR analysis showed that pnu5 was highly expressed in mantle pallial and participated in shell repairing and regeneration. RNAi-mediated repression of pnu5 could affect the normal structure of prismatic layer and nacreous layer. The recombinant protein rPNU5 significantly enhanced the precipitation rate of CaCO3 both in the calcite and aragonite crystallization systems, as well as altering the morphology of the crystals. Based on ACC transition experiments, the recombinant protein rPNU5 facilitated amorphous calcium carbonate (ACC) transformation into stable calcite or aragonite. This study could provide us with a better understanding of how positive regulatory mechanisms contribute to biomineralization.

Evolution of nacre- and prisms-related shell matrix proteins in the pen shell, Atrina pectinata.

The molluscan shell is a good model for understanding the mechanisms underlying biomineralization. It is composed of calcium carbonate crystals and many types of organic molecules, such as the matrix proteins, polysaccharides, and lipids. The pen shell Atrina pectinata (Pterioida, Pinnidae) has two shell microstructures: an outer prismatic layer and an inner nacreous layer. Similar microstructures are well known in pearl oysters (Pteriidae), such as Pinctada fucata, and many kinds of shell matrix proteins (SMPs) have been identified from their shells. However, the members of SMPs that consist of the nacreous and prismatic layers of Pinnidae bivalves remain unclear. In this study, we identified 114 SMPs in the nacreous and prismatic layers of A. pectinata, of which only seven were found in both microstructures. 54 of them were found to bind calcium carbonate. Comparative analysis of nine molluscan shell proteomes showed that 69 of 114 SMPs of A. pectinata were found to have sequential similarity with at least one or more SMPs of other molluscan species. For instance, nacrein, tyrosinase, Pif/BMSP-like, chitinase (CN), chitin-binding proteins, CD109, and Kunitz-type serine proteinase inhibitors are widely shared among bivalves and gastropods. Our results provide new insights for understanding the complex evolution of SMPs related to nacreous and prismatic layer formation in the pteriomorph bivalves.

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins.

The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva-larva settlement interactions. Other isolated Stains-all-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents.

Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

Expression of tyrosinase-like protein genes and their functional analysis in melanin synthesis of Pacific oyster (Crassostrea gigas).

Color polymorphism in Mollusca is of great interest for consumer preference. Although the heritability of shell color variation has been conducted by experimental crossing, little is known about molecular basis involved in these patterns. Tyrosinase-like proteins are important enzymes which are members of the type-3 copper protein superfamily. In this research, two tyrosinase-like protein genes including CgTyp-1 and CgTyp-3 were identified in the Pacific oyster Crassostrea gigas. Tissue expression analysis showed that CgTyp-1 and CgTyp-3 were dominantly expressed in the mantle. Particularly, they were expressed significantly higher in the edge mantle than that in the central mantle whether on the left or right mantles. Additionally, expressions of CgTyp-1 and CgTyp-3 were mainly found in the black shell color oysters, with relative lower levels in the white shell color oysters. In situ hybridization showed that positive signals for CgTyp-1 and CgTyp-3 were both detected within the outer epithelium of the outer fold either in the black or white shell color oysters. After interference, the expression levels of CgTyp-1 and CgTyp-3 mRNA were significantly attenuated, and the efficiency of RNAi reached 84.72% and 71.58%, respectively. Besides, knockdown CgTyp-1 or CgTyp-3, obviously decreased the tyrosinase activity of mantles. Furthermore, the number of the melanosomes within epithelium of the outer fold was sharply reduced by silencing of each Typ. These findings argue that CgTyp-1 and CgTyp-3 may be involved in the melanin synthesis, which lends insight into regulation mechanism of shell pigmentation in C. gigas.

An acidic protein, Hf15, from Haliotis fulgens involved in biomineralization.

Biomineralization leads to the hardening of mineralized materials, such as the shell of Mollusk, to fulfill a wide range of functions, such as (but not limited to) skeletal support, protection of the soft tissues, navigation, etc. The study of the proteins responsible for this process, shell matrix proteins (SMPs), allows addressing questions related to structure-function relationship and to the mechanism of mineral formation, which is limited in gastropod species. In this study, a low molecular weight protein was isolated from the insoluble fraction after decalcification with acetic acid of the shell of Haliotis fulgens and, named Hf15. The unglycosylated protein has a theoretical molecular weight of 15 kDa, it possesses calcium and chiting binding properties. Hf15 can precipitate calcium carbonate in vitro in presence of different salts. Analysis by LC-MS of the five peptide sequences of Hf15 generated by trypsinization revealed that two peptides displayed homology to an uncharacterized protein 3-like from Haliotis rufescens, Haliotis asinia and H. sorenseni. The results obtained indicated that Hf15 is a novel SMP involved in shell mineralization in Haliotis fulgens.

Shell Matrix Protein N38 of Pinctada fucata, Inducing Vaterite Formation, Extends the DING Protein to the Mollusca World.

In the animal kingdom, DING proteins were only found in Chordata and Aschelminthes. At present study, a potential DING protein, matrix protein N38, was isolated and purified from the shell of Pinctada fucata. Tandem mass spectrometry analysis revealed that 14 peptide segments matched between N38 and human phosphate-binding protein (HPBP). HPBP belongs to the DING protein family and has a "DINGGG-" sequence, which is considered a "signature" of HPBP. In this study, the mass spectrometry analysis results showed that N38 had a "DIDGGG-" sequence; this structure is a mutation from the "DINGGG-" structure, which is a distinctive feature of the DING protein family. The role of N38 during calcium carbonate formation was explored through the in vitro crystallization experiment. The results of scanning electron microscopy and Raman spectrum analysis indicated that N38 induced vaterite formation. These findings revealed that N38 might regulate and participate in the precise control of the crystal growth of the shell, providing new clues for biomineralization mechanisms in P. fucata and DING protein family studies. In addition, this study helped extend the research of DING protein to the Mollusca world.

Hic12, a novel acidic matrix protein promotes the transformation of calcite into vaterite in Hyriopsis cumingii.

Shell acidic matrix proteins are widely considered to be essential for shell formation given their low affinity and high loading for calcium ion. In the present study, a novel matrix protein, hic12, was isolated from the mantle of Hyriopsis cumingii. High expression in tissue and positive signals with in situ hybridization were detected in the mantle center and mantle pallium, indicating that hic12 mainly participated in the biomineralization of the shell nacreous layer. The expression pattern of hic12 in the pearl sac during early pearl formation indicated that it was involved in pearl biomineralization. Moreover, the recombinant protein, rGST-Hic12, was successfully expressed and purified. The addition of rGST-Hic12 could accelerate the calcium carbonate deposition rate, change the morphology of crystals, and promote the conversion of calcite to vaterite. These results may provide new insights into the molecular mechanisms of aragonite mollusk shell formation.

Optimized Sensory Units Integrated in the Chiton Shell.

The first step for animals to interact with external environment is to sense. Unlike vertebrate animals with flexibility, it is challenging for ancient animals that are less flexible especially for mollusca with heavy shells. Chiton, as an example, has eight overlapping shells covering almost the whole body, is known to incorporate sensory units called aesthetes inside the shell. We used micro-computed tomography combined with quantitative image analysis to reveal the optimized shell geometry to resist force and the aesthetes' global distribution at the whole animal levels to facilitate sense from diverse directions both in the seawater and air. Additionally, shell proteomics combined with transcriptome reveals shell matrix proteins responsible for shell construction and potentially sensory function, highlighting unique cadherin-related proteins among mollusca. Together, this multi-level evidence of sensory units in the chiton shell may shed light on the formation of chiton shells and inspire the design of hard armor with sensory function.