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Shell matrix proteins have important roles in the biomineralization of shells. In this study, we isolated and identified a novel shell matrix protein gene, hic7, from the mussel Hyriopsis cumingii. The cDNA of hic7 was 459 bp long, including a 240-bp open reading frame. It encoded a 79 amino acid-long protein, with amino acids 1-19 constituting the signal peptide. The resulting hic7 is rich in cysteine (16.5%). After removing the signal peptide, the molecular weight was 8.85 kDa and the theoretical isoelectric point was 6.34, indicating that hic7 is a weakly acidic shell matrix protein. Hic7 is mainly expressed in the mantle tissue of H. cumingii. In situ hybridization showed hic7 signals at the edge and dorsal region of the mantle outer fold, indicating that it is related to the formation of the prismatic and nacreous layer of the shell. RNA interference indicated that when hic7 was inhibited by 80%, the crystal morphology of the prism and nacre layers of the shell were irregular and disordered. In addition, the expression of hic7 during the early development of the pearl sac indicated that it has an important role in the transformation of calcium carbonate crystals from a disordered to an orderly deposition pattern. These results suggest that matrix protein hic7 take part in constructing the framework of crystal nucleation and regulating the calcium carbonate crystal morphology of the nacreous and prismatic layers of shells and pearls., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with post-transcriptional regulatory activity in various biological processes. Pearl oyster Pinctada fucata martensii is one of the main species cultured for marine pearl production in China and Japan. In this study, we constructed two small RNA libraries of mantle central (MC) and mantle edge (ME) from P. f. martensii and obtained 24,175,537 and 21,593,898 clean reads, respectively. A total of 258 miRNAs of P. f. martensii (Pm-miRNA) were identified, and 93 differentially expressed miRNAs (DEMs) including 49 known Pm-miRNAs and 44 novel Pm-miRNAs were obtained from the MC and ME. The target transcripts of these DEMs were obviously enriched in neuroactive ligand-receptor interaction pathway, and others. After over-expression of Pm-miR-124 and Pm-miR-9a-5p in the MC by mimic injection into the muscle of P. f. martensii, nacre exhibited a disorderly growth as detected by scanning electron microscopy. Pm-nicotinic acetylcholine receptor alpha subunit, Pm-neuropeptide Y and Pm-chitin synthase were investigated as the targets of Pm-miR-124; and Pm-tumor necrosis factor receptor associated factor 2 and Pm-chitin synthase were investigated as the targets of Pm-miR-9a-5p. These predicted target transcripts were down-regulated after the over-expression of Pm-miR-124 and Pm-miR-9a-5p in MC. This study comprehensively analyzed the miRNAs in mantle tissues to enhance our understanding of the regulatory mechanism underlying shell formation., (Copyright © 2020. Published by Elsevier Inc.)

The formation of the primary shell is a vital process in marine bivalves. Ocean acidification largely influences shell formation. It has been reported that enzymes involved in phenol oxidation, such as tyrosinase and phenoloxidases, participate in the formation of the periostracum. In the present study, we cloned a tyrosinase gene from Crassostrea angulata named Ca-tyrA1, and its potential function in early larval shell biogenesis was investigated. The Ca-tyrA1 gene has a full-length cDNA of 2430bp in size, with an open reading frame of 1896bp in size, which encodes a 631-amino acid protein that includes a 24-amino acid putative signal peptide. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that Ca-tyrA1 transcription mainly occurs at the trochophore stage, and the Ca-tyrA1 mRNA levels in the 3000ppm treatment group were significantly upregulated in the early D-veliger larvae. WMISH and electron scanning microscopy analyses showed that the expression of Ca-tyrA1 occurs at the gastrula stage, thereby sustaining the early D-veliger larvae, and the shape of its signal is saddle-like, similar to that observed under an electron scanning microscope. Furthermore, the RNA interference has shown that the treatment group has a higher deformity rate than that of the control, thereby indicating that Ca-tyrA1 participates in the biogenesis of the primary shell. In conclusion, and our results indicate that Ca-tyrA1 plays a vital role in the formation of the larval shell and participates in the response to larval shell damages in Crassostrea angulata that were induced by ocean acidification., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chitinase is an enzyme that plays an important role in the chitin metabolism of a wide range of organisms. However, the function of chitinase in the pearl oyster Pinctada fucata is yet to be determined. In this study, a chitinase gene (named PfChi1) was cloned from P. fucata and its expression profiles were investigated. The full-length cDNA of PfChi1 was 2743bp and consisted of a 2187-bp open reading frame encoding 728 amino acid residues, a 47-bp 5'-untranslated region (UTR), and a 509-bp 3'-UTR. Similar to other known chitinases, the PfChi1 protein is composed of an N-terminal leading signal peptide, a catalytic domain, a linker region, and a C-terminal chitin-binding domain. The results of qRT-PCR showed that PfChi1 was expressed in a wide range of tissues with relatively high levels in the mantle, muscle, gill, and gonad, and relatively low levels in hemocytes, the intestine, and the digestive gland (P<0.05). In situ hybridization showed that PfChi1 was mainly expressed in the mantle edge, particularly in the outer epithelial cells of the inner fold, whereas few hybridization signals were detected in the inner epithelial cells of the middle fold. A shell damage experiment indicated that PfChi1 transcript levels were up-regulated significantly (P<0.05) at 24h after shell damage and decreased gradually thereafter, followed by shell regeneration, indicating that PfChi1 is involved in shell formation. In addition, PfChi1 expression was higher in trochophore larvae than in other developmental stages (P<0.05), indicating a possible association with the formation of prodissoconch shells. To the best of our knowledge, this study is the first to report the potential biomineralization function of a chitinase in P. fucata., (Copyright © 2016 Elsevier Inc. All rights reserved.)