Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many aspects of the ...biomineralization process and its evolutionary origin remain unknown. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms that underlie its production of shells and pearls are not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins, chondroitin sulfotransferases, and regulatory elements. Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre suggests that elements of chitin- and collagen-based matrices have deep roots and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems.
The oyster species Pinctada maxima is cultured for the production of large pearls with high economic value. Pearl weight and thickness are related to the growth of P. maxima. The molecular mechanism ...underlying the growth of this species, however, remains poorly understood given the limited availability of the genetic and genomic information of this species. Here, the molecular mechanism of the asynchronous growth of P. maxima was investigated. The transcriptomes of large and small P. maxima individuals were sequenced using the Illumina HiSeq 2000 platform. A total of 145,877 unigenes were generated for the transcriptomes, and 1,921 differentially expressed genes (DEGs) were identified. Compared with the slow-growing group, the fast-growing group showed 879 and 1,042 significantly up- and down-regulated DEGs, respectively. The differential expression patterns of nine selected genes were obtained through real-time quantitative polymerase chain reaction analysis and showed consistency with those obtained through RNA-Seq analysis. The results of this study provide further insight on the complexity of the differential growth patterns of P. maxima individuals and will help guide the design of breeding programs for this economically important species.
•The basic knowledge on molecular mechanisms of unsynchronized growth of Pinctada maxima was studied.•Gene expression patterns of Pinctada maxima with different growth performances were different.•Many differentially expressed genes were linked to growth and immune responses.
Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of ...a CHST1 gene identified from the pearl oyster Pinctada fucata martensii (PmCHST1b) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5′ PSB motif and 3′ PB motif) which interacted with 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC (P<0.05), and sulfate polymer content in the extrapallial fluid significantly reduced (P<0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation.
Biomineralization‐controlled exo‐/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified ...“pearl” involved in invading defence. Notably, exo‐/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole‐genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization‐controlled growth and ectopic biomineralization. The insulin‐like growth factor (IGF) endocrine signal interacted with ancient single‐copy transcription factors to form the regulatory network. Moreover, the “cross‐phylum” regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo‐/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo‐/endoskeleton animals and ectopic biomineralization.
•We obtained the full length of TIMP gene in Pinctada martensii.•Pm-TIMP mRNA was highly expressed in the mantle and pearl sac.•RNA interference was used to elucidate the function of Pm-TIMP ...gene.•The obtained Pm-TIMP participated in nacre formation in Pinctada martensii.
Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901bp long, containing a 5′ untranslated region (UTR) of 51bp, a 3′ UTR of 169bp, and an open reading fragment (ORF) of 681bp encoding 226 amino acids with an estimated molecular mass of 23.37kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.
Increasing evidence demonstrated that microRNAs (miRNAs) play critical roles in innate immunity in vertebrates and invertebrates. MiR-146a/b is reported as a key regulator of the immune response ...through mediating Toll-like receptor and cytokine signalling. In this study, a novel miR-146a was identified and characterised from Pinctada martensii (designated as pm-miR-146a), and its roles in modulating the inflammatory response after LPS stimulation were also investigated. Pm-miR-146a ubiquitously expressed in all examined tissues, with the highest level in the mantle and lowest expression in the haemolymph. Pm-miR-146a increased at 24 h after lipoplysaccharide injection, in union with up-regulated NF-κB (P < 0.05). The over-expression of pm-miR-146a in vivo could significantly inhibit the expression of macrophage migration inhibitory factor (MIF), the potential target gene predicted by miRanda, while enforcing pm-miR-146a involved in the down-regulation of NF-κB. Thus, we propose that pm-miR-146a plays a role of negative feedback regulation to the NF-κB signal by repressing the expression of the pro-inflammatory cytokine MIF. These findings revealed that miR-146a represents a critical role in inflammatory response and offers new evidence for miRNAs in the innate immunity of molluscs.
•A novel miR-146a (pm-miR-146a) was identified from Pinctada martensii.•Pm-miR-146a increased after lipoplysaccharide injection.•The over-expression of pm-miR-146a could inhibit the expression of MIF.•Pm-miR-146a plays a potential role of negative feedback regulation to NF-κB signal.
MicroRNAs (miRNAs) are a class of non-coding RNA molecules with presumed post-transcriptional regulatory activity in various biological processes, such as development and biomineralization. Pinctada ...martensii is one of the main species cultured for marine pearl production in China and Japan. In our previous research, 258 pm-miRNAs had been identified by solexa deep sequencing in P. martensii, while it is far from the number of miRNAs found in other species. In this study, based on the transcriptome database of pearl sac, we identified 30 candidate pm-miRNAs by computational prediction. Among the obtained 30 pm-miRNAs, 13 pm-miRNAs were generated from the complementary strand of protein-coding mRNAs, and 17 pm-miRNAs could not be annotated using blastx and tblastn analysis. Notably, 10 of the 30 pm-miRNAs, such as pm-miR-1b, pm-miR-205b and pm-miR-375b, were homologous with the reported pm-miRNAs, respectively. To validate the existence of the identified pm-miRNAs, eight randomly selected pm-miRNAs were tested by stem loop quantitative RT-PCR analyses using 5.8S as the internal reference gene. Target prediction between the obtained pm-miRNAs and biomineralization-related genes by microTar, miRanda and RNA22 indicated pm-miR-2386 and pm-miR-13b may be the key factors in the regulation network by regulating the formation of organic matrix or the differentiation of mineralogenic cell during shell formation. Thus, this study enriched miRNA databases of pearl oyster and provided a new way to understand biomineralization.
Two calcium carbonate crystal polymorphs, aragonite and calcite, are the main inorganic components of mollusk shells. Some fossil evidences suggest that aragonite shell is more ancient than calcite ...shell for the Bivalvia. But, the molecular biology evidence for the above deduction is absent. In this study, we searched for homologs of bivalve aragonite-related and calcite-related shell proteins in the oyster genome, and found that no homologs of calcite-related shell protein but some homologs of aragonite-related shell proteins in the oyster genome. We explained the results as the new evidence to support that aragonite shells are more ancient than calcite shells in bivalves combined the published biogeological and seawater chemistry data.
Three wild populations of Meretrix meretrix sampled from Dongxing, Beihai, and Shankou along the coast of Guangxi, China, were investigated with morphometry and karyometry. Six morphological indices ...（shell length, shell height, shell width, hinge length, total wet weight and shell weight） were measured. Differences in all morphological indices except hinge length were significant among the three populations （P 〈 0.05）. The mean values of these indices （except for the hinge length） in the Dongxing population were larger than those in the Beihai and Shankou populations, although the latter had the largest hinge length. The karyotype of the Beihai, Shankou and Dongxing samples had ten metacentric, six submetacentric, and three subtelocentric chromosome pairs. No significant difference was shown in the centromeric index values of the chromosomes in the populations （P〉0.05）. However, the order of metacentric, submetacentric and subtelocentric chromosome pairs was variable among the three populations. The results indicate a high level of inter-population variation in morphology and karyotype.
In September 2011, two wild populations of pearl oysters were collected separately from Daya Bay, Shenzhen, and from Beibu Bay, Beihai, China. In April 2012, three cultured stocks were established by ...sampling breeders from the third-generation selected lines. The genetic variation within the five stocks was analyzed using eight SSR primer pairs. The effective population sizes of the five stocks were analyzed using the sibship reconstruction and multiple summary statistics methods. A total of 22–29 alleles were observed in the five stocks, with two to five alleles per locus and means ranging from 2.750 to 3.625 alleles per locus. The average expected and observed heterozygosities ranged from 0.3387 to 0.5029 and from 0.2917 to 0.4219, respectively. The effective population sizes of the five stocks ranged from 11.0 to 20.0 when estimated using sibship reconstruction and from 13.7 to 179.4 when estimated using multiple summary statistics. The effective population sizes estimated in the three cultured stocks were smaller than the number of breeders used to produce these stocks. Thus, it may be necessary to augment the number of breeders used to produce progeny stocks in selective breeding programs.
•We evaluated genetic variation and effective population sizes of Pinctada martensii.•The Beihai stock had higher genetic variation than the other cultured stocks.•Effective population sizes of the stocks were smaller than the numbers of breeders.•We suggested an augment in the number of breeders in selective breeding programs.
PDF
