Transcriptome and exosome proteome analyses provide insights into the mantle exosome involved in nacre color formation of pearl oyster Pinctada fucata martensii.

Color polymorphisms in molluscan shells play an important economic in the aquaculture industry. Among bivalves, shell color diversity can reflect properties such as growth rate and tolerance. In pearl oysters, the nacre color of the donor is closely related to the pearl color. Numerous genes and proteins involved in nacre color formation have been identified within the exosomes of the mantle. In this study, we analyzed the carotenoids present in the mantle of gold- and silver-lipped pearl oysters, identifying capsanthin and xanthophyll as crucial pigments contributing to coloration. Transcriptome analysis of the mantle revealed several differentially expressed genes (DEGs) involved in color formation, including ferric-chelate reductase, mantle genes, and larval shell matrix proteins. We also isolated and identified exosomes from the mantles of both gold- and silver-lipped strains of the pearl oyster Pinctada fucata martensii, revealing the extracellular transition mechanism of coloration-related proteins. From these exosomes, we obtained a total of 1223 proteins, with 126 differentially expressed proteins (DEPs) identified. These proteins include those associated with carotenoid metabolism and Fe(III) metabolism, such as apolipoproteins, scavenger receptor proteins, ß,ß-carotene-15,15'-dioxygenase, ferritin, and ferritin heavy chains. This study may provide a new perspective on the nacre color formation process and the pathways involved in deposition within the pearl oyster P. f. martensii.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

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.

DNA Methylation Analyses Unveil a Regulatory Landscape in the Formation of Nacre Color in Pearl Oyster Pinctada fucata martensii.

Pearl color is regulated by genetics, biological pigments, and organic matrices and an important factor that influences the pearl economic value. The epigenetic regulation mechanism underlying pearl pigmentation remains poorly understood. In this study, we collected the mantle pallial (MP) and mantle central (MC) of the golden-lipped strain, and MP of the silver-lipped strain of pearl oyster Pinctada fucata martensii. The whole-genome bisulfite sequencing (WGBS) technology was employed to investigate the possible implication of epigenetic factors regulating nacre color variation. Our results revealed approximately 2.5% of the cytosines in the genome of the P. fucata martensii were methylated, with the CG methylation type was in most abundance. Overall, we identified 12, 621 differentially methylated regions (DMRs) corresponding to 3,471 DMR-associated genes (DMGs) between the two comparison groups. These DMGs were principally enriched into KEGG metabolic pathways including ABC transporters, Terpenoid backbone biosynthesis, and fatty acid degradation. In addition, integrating information about DMGs, DEGs, and function annotation indicated eight genes LDLR, NinaB, RDH, CYP, FADS, fn3, PU-1, KRMP as the candidate genes related to pigmentation of nacre color. A further study proved that the pigment in nacre is violaxanthin. The results of our study provide the support that there is an association between nacre color formation and DNA methylation profiles and will help to reveal the epigenetic regulation of nacre pigmentation formation in pearl oyster P. fucata martensii.

Transcriptome analysis reveals acclimation responses of pearl oysters to marine heatwaves.

Marine heatwaves (MHWs) are weather-timescale extreme events in the oceans and can have devastating effects on marine bivalves and ecosystems they support, with considerable socio-economic consequences. Yet, the extent to which marine bivalves have the capacity to acclimate and adapt to MHWs remains unknown. Understanding molecular responses to MHWs is imperative to develop strategies for conservation of ecologically and economically important marine organisms. Here, using RNA-Seq, we investigate how various MHWs scenarios elicit molecular changes in threatened and vulnerable pearl oysters, Pinctada maxima (Jameson). Acute exposure of MHWs - mimicked by rapid increases of seawater temperature from 24 °C to 28 °C and 32 °C, respectively - significantly affected the expression levels of metabolic and immune-related genes, with thermal stress-responsive genes especially like HSP20, HSP70 and HSP90 being remarkably up-regulated. Following repeat exposure to MHWs, encouragingly, pearl oysters exhibited evident acclimation responses, as best exemplified by significantly lowered expression levels of key stress-responsive genes involved in metabolism and immunity in comparison to those observed during acute exposure. Findings of the present study provide a better understanding of molecular processes underpinning the acclimation and adaptation of marine bivalves to MHWs in the context of climate change.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii.

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5'-UTR of 58bp and a 3'-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

Survival, retention rate and immunity of the black shell colored stocks of pearl oyster Pinctada fucata martensii after grafting operation.

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production.