Structure of an amorphous calcium carbonate phase involved in the formation of Pinctada margaritifera shells.

Some mollusc shells are formed from an amorphous calcium carbonate (ACC) compound, which further transforms into a crystalline material. The transformation mechanism is not fully understood but is however crucial to develop bioinspired synthetic biomineralization strategies or accurate marine biomineral proxies for geoscience. The difficulty arises from the simultaneous presence of crystalline and amorphous compounds in the shell, which complicates the selective experimental characterization of the amorphous fraction. Here, we use nanobeam X-ray total scattering together with an approach to separate crystalline and amorphous scattering contributions to obtain the spatially resolved atomic pair distribution function (PDF). We resolve three distinct amorphous calcium carbonate compounds, present in the shell of Pinctada margaritifera and attributed to: interprismatic periostracum, young mineralizing units, and mature mineralizing units. From this, we extract accurate bond parameters by reverse Monte Carlo (RMC) modeling of the PDF. This shows that the three amorphous compounds differ mostly in their Ca-O nearest-neighbor atom pair distance. Further characterization with conventional spectroscopic techniques unveils the presence of Mg in the shell and shows Mg-calcite in the final, crystallized shell. In line with recent literature, we propose that the amorphous-to-crystal transition is mediated by the presence of Mg. The transition occurs through the decomposition of the initial Mg-rich precursor into a second Mg-poor ACC compound before forming a crystal.

Tracing key genes associated with the Pinctada margaritifera albino phenotype from juvenile to cultured pearl harvest stages using multiple whole transcriptome sequencing.

BACKGROUND: Albino mutations are commonly observed in the animal kingdom, including in bivalves. In the black-lipped pearl oyster Pinctada margaritifera, albino specimens are characterized by total or partial absence of colouration resulting in typical white shell phenotype expression. The relationship of shell colour with resulting cultured pearl colour is of great economic interest in P. margaritifera, on which a pearl industry is based. Hence, the albino phenotype provides a useful way to examine the molecular mechanisms underlying pigmentation. RESULTS: Whole transcriptome RNA-sequencing analysis comparing albino and black wild-type phenotypes at three stages over the culture cycle of P. margaritifera revealed a total of 1606, 798 and 187 differentially expressed genes in whole juvenile, adult mantle and pearl sac tissue, respectively. These genes were found to be involved in five main molecular pathways, tightly linked to known pigmentation pathways: melanogenesis, calcium signalling pathway, Notch signalling pathway, pigment transport and biomineralization. Additionally, significant phenotype-associated SNPs were selected (N = 159), including two located in the Pif biomineralization gene, which codes for nacre formation. Interestingly, significantly different transcript splicing was detected between juvenile (N = 1366) and adult mantle tissue (N = 313) in, e.g., the tyrosinase Tyr-1 gene, which showed more complex regulation in mantle, and the Notch1 encoding gene, which was upregulated in albino juveniles. CONCLUSION: This multiple RNA-seq approach provided new knowledge about genes associated with the P. margaritifera albino phenotype, highlighting: 1) new molecular pathways, such as the Notch signalling pathway in pigmentation, 2) associated SNP markers with biomineraliszation gene of interest like Pif for marker-assisted selection and prevention of inbreeding, and 3) alternative gene splicing for melanin biosynthesis implicating tyrosinase.

Toxic effects of leachates from plastic pearl-farming gear on embryo-larval development in the pearl oyster Pinctada margaritifera.

Pearl-farming leads to significant plastic pollution in French Polynesia (FP) as the end of life of most farming gear is currently poorly managed. Plastic debris released into the aquatic environment accumulates, with potentially detrimental effects on the lagoon ecosystem and pearl oyster Pinctada margaritifera, a species of ecological, commercial and social value. Here, we tested the effects of leachates from new (N) and aged (A) plastic pearl-farming gear (spat collector and synthetic rope) obtained after 24 h and 120 h incubation, on the embryo-larval development of the pearl oyster using an in-vitro assay. Embryos were exposed for 24 h and 48 h to a negative control (0) and the leachate from 0.1, 1, 10 and 100 g of plastic. L-1. After 24 h exposure to leachate at 100 g.L-1, effects were observed on embryo development (-38% to -60% of formed larvae) and mortality (+72% to +82%). Chemical analyses of plastic gear indicated the presence of 26 compounds, consisting of organic contaminants (PAHs) and additives (mainly phthalates). Screening of leachates demonstrated that these compounds leach into the surrounding seawater with an additional detection of pesticides. Higher levels of phthalates were measured in leachates obtained from new (6.7-9.1 µg.L-1) than from aged (0.4-0.5 µg.L-1) plastics, which could be part of the explanation of the clear difference in toxicity observed after 48 h exposure at lower concentrations (0.1-10 g.L-1), associated with mortality ranging from 26 to 86% and 17-28%, respectively. Overall, this study suggests that plastic gear used in the pearl-farming industry releases significant amounts of hazardous chemicals over their lifetime, which may affect pearl oyster development that call for in-situ exploration.

Strong genetic isolation of the black-lipped pearl oyster (Pinctada margaritifera) in the Marquesas archipelago (French Polynesia).

The French Polynesian islands are internationally known for their black pearls, produced by culture of the black lipped pearl oyster Pinctada margaritifera. The ongoing development of hatcheries for P. margaritifera in French Polynesia poses new challenges for the industry, particularly regarding the maintenance of genetic diversity in the hatchery stocks. This emphasizes the necessity to characterize the genetic diversity and differentiation within natural and exploited populations, to carefully select putative parental populations. The present study aimed at validating the phylogenetic status and investigating genetic attributes of populations from the only two non-exploited archipelagos of French Polynesia, the Marquesas archipelago, and the Australes archipelago, never analysed before. We found that individuals from both archipelagos belonged to P. margaritifera species. However, while the Australes population was genetically similar to non-exploited populations of the Tuamotu, the Marquesas populations were highly differentiated from the rest of the populations. This differentiation cannot not be only attributed to geographic distance and aquaculture status, but likely to hydrodynamic barriers allowing vicariant events to take place. Our results add up to other studies describing the Marquesas archipelago as a hotspot for biodiversity and differentiation, with some of the highest levels of endemism and vicariance found among marine species worldwide and provide precious information on available genetic resources for the implementation of P. margaritifera selective breeding and its genetic conservation in French Polynesia.

Relationship of the orange tissue morphotype with shell and pearl colouration in the mollusc Pinctada margaritifera.

Molluscs display a vast range of shell colours both between and within species. However, only a few species show colour variation in their soft tissues. In French Polynesia, the pearl oyster Pinctada margaritifera has three tissue morphotypes: the black wild-type and two rare mutations: white albino and orange mantle. Phenotypic transmission is known to occur from these phenotypes when they are used as graft donors for pearl production, leading to multicoloured and white pearls from black and albino mantle grafts, respectively. The present study furthers this knowledge by examining the phenotypic association between the orange mantle tissue morphotype and hard tissues: shells and cultured pearls. Based on a large experimental graft, shell colour quantification and pearl qualification showed that the orange morphotype is associated with light-coloured shells and pearls. Expression analysis of some candidate genes previously identified in the white mantle mutant, tested here on both graft and pearl sac tissues from orange mantle donors, confirmed the involvement of genes associated with shell matrix protein (shem4) and the melanin biosynthesis pathway (zinc). This study provides fundamental information on the mechanism behind mantle tissue colour in P. margaritifera and its association with biomineralisation and pigmentation processes that will be potentially valuable in future selection programs.

The Mendelian inheritance of rare flesh and shell colour variants in the black-lipped pearl oyster (Pinctada margaritifera).

Pinctada margaritifera is French Polynesia's most economically important aquaculture species. This pearl oyster has the specific ability to produce cultured pearls with a very wide range of colours, depending on the colour phenotypes of donor oysters used. Its aquaculture is still based on natural spat collection from wild stocks. We investigated three rare colour variants of P. margaritifera - orange flesh, and red and white shell colour phenotypes - in comparison with the wild-type black flesh and shell commonly found in this species. The study aimed to assess the geographic distribution and genetic basis of these colour variants. Colour frequencies were evaluated during transfer and graft processes of pearl oyster seed captured at collector stations. Among the collection locations studied, Mangareva Island showed the highest rate of the orange flesh phenotype, whereas Takaroa and Takume atolls had relatively high rates of red and white shell phenotypes respectively. Broodstocks were made of these rare colour variants, and crosses were performed to produce first- and second-generation progenies to investigate segregation. The results were consistent with Mendelian ratios and suggest a distinct model with no co-dominance: (i) a two-allele model for flesh trait, whereby the orange allele is recessive to the black fleshed type, and (ii) a three-allele model for shell trait, whereby the black wild-type allele is dominant to the red coloration, which is dominant to the white shell. Furthermore, the proposed model provides the basis for producing selected donor pearl oyster lines through hatchery propagation.