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Ocean warming challenges marine organisms' resilience, especially for species experiencing temperatures close to their upper thermal limits. A potential increase in thermal tolerance might significantly reduce the risk of population decline, which is intrinsically linked to variability in local habitat temperatures. Our goal was to assess the plastic and genetic potential of response to elevated temperatures in a tropical bivalve model, Pinctada margaritifera. We benefit from two ecotypes for which local environmental conditions are characterized by either large diurnal variations in the tide pools (Marquesas archipelago) or low mean temperature with stable to moderate seasonal variations (Gambier archipelago). We explored the physiological basis of individual responses to elevated temperature, genetic divergence as well as plasticity and acclimation by combining lipidomic and transcriptomic approaches. We show that P. margaritifera has certain capacities to adjust to long-term elevated temperatures that was thus far largely underestimated. Genetic variation across populations overlaps with gene expression and involves the mitochondrial respiration machinery, a central physiological process that contributes to species thermal sensitivity and their distribution ranges. Our results present evidence for acclimation potential in P. margaritifera and urge for longer term studies to assess populations resilience in the face of climate change.Le réchauffement des océans remet en question la résilience des organismes marins, en particulier pour les espèces connaissant des températures proches de leurs limites thermiques supérieures. Une augmentation potentielle de la tolérance thermique pourrait ainsi réduire considérablement le risque de déclin de la population. L'objectif de cette étude était d'évaluer le potentiel plastique et génétique de la réponse à l'exposition courte et chronique à températures élevées chez une espèce de bivalve tropical, Pinctada margaritifera. Ce modèle bénéficie de l'existence de deux écotypes pour lesquels les conditions environnementales locales sont caractérisées soit par de fortes variations diurnes associées aux marées (archipel des Marquises) soit par une température moyenne plus basse et des variations saisonnières prononcées (archipel des Gambier). Nous avons exploré les bases physiologiques des réponses individuelles ainsi que la divergence génétique et quantifié la plasticité en combinant des approches lipidomique et transcriptomique. Nous montrons que P. margaritifera possède des capacités d'acclimatation à des températures élevées sur le long terme jusqu'à présent largement sous-estimées. La divergence génétique entre populations est par ailleurs associée à des différences d'expression des gènes et implique la machinerie respiratoire mitochondriale, un processus physiologique central qui contribue à la sensibilité thermique des espèces et à leurs répartitions. Nos résultats présentent les bases des potentiels d'acclimatation chez P. margaritifera et soulignent l'importance d'études à plus long terme pour évaluer la résilience des populations face au changement climatique.

International audience; Aquaculture of nacreous gems, such as cultured pearls, mabé or keshi, is done mostly using different mollusc species grown in countries of the Indo-Pacific region. To date, no single species has been exploited for the simultaneous generation of more than one of these bioproducts, but all require animals with colourful shells. Historically, Pinctada species have mainly been used for nucleated pearl production, selecting the rarer colourful individuals to be used as graft donors. By contrast, colourful Pteria species have mostly been used for mabé production, as the grafting operation for pearl production is associated with low yield. In this study, we report the potential for cumulating cultured pearl and mabé (MP), or keshi and mabé (MK) production, using a colourful hatchery-produced G2 family of P. margaritifera. For these trials, MP and MK combinations were compared with the operations to produce pearls (P), mabé (M) or keshi (K) alone in an experimental design using groups of small and large recipients from the G2 family. Results showed no significant impact of combining operation types on subsequent pearl weight, keshi weight, or mabé thickness within recipient oyster size group. By contrast, significant differences were observed between the large and small recipients. The small group produced the thickest mabé, while the large group produced the heaviest pearls and keshi. These contrasting results revealed: 1) the relative independence between the two tissues capable of biomineralisation activities, the mantle (shell and mabé growth) and the pearl sac (pearl or keshi growth); 2) the potential compensatory growth of the small recipient oyster group, which had the highest shell growth performance; and 3) the regulation capacity of the larger oyster group of pearl sac activity. With the same growing area and number of cultured oysters, it would be possible for the P. margaritifera pearl industry to benefit from hatchery propagation of selected colourful shell and produce valuable keshi and mabé together with cultured pearls.

Ocean warming is a particularly challenging threat for tropical marine bivalves’ species because many live already near their upper thermal limits. The thermal sensitivity of organisms is a strong contributor to the biogeographic boundaries of populations and species. The potential of thermal plastic response (range of thermal breadth) is typically reduced for marine populations living in stable thermal environments, e.g., in extreme latitudes organisms as traduced in the climatic variability hypothesis. However, regional-scale heterogeneity among tropical environments, such as archipelagos in French Polynesia, might also serve in modulating this plastic potential. The questions remain now, how tropical organisms are able to cope with abnormally elevated temperature on long-term (several weeks) and how environmental-variability might drive the potential of resilience? To answer these questions, we benefit from two ecologically divergent populations of a marine tropical mollusc species,Pinctada margaritifera, that usually experience either large diurnal variations (tide-pools, Marquesas archipelago) or lower temperature with stable to moderate variations (Gambier archipelago). Individuals were maintained in common garden experiment at several controlled temperature conditions (23°C, 28°C, 32°C and 34°C) over a 48 days period. We explored genetic divergence as well as thermal plastic responses by combining lipidomic and transcriptomic approaches. We show thatP. margaritiferahave capacities to adjust to long-term elevated temperatures that was thus far largely underestimated. Furthermore, we identified genetic variation between populations that overlapped with genes expression variations, including genes involved in the respiration machinery, a central process delimiting critical temperatures in marine invertebrates. This study is the first of a series looking at the global adaptation and acclimation mechanisms in response to climate change inPinctadaspecies.

The shell color of the Mollusca has attracted naturalists and collectors for hundreds of years, while the molecular pathways regulating pigment production and the pigments themselves remain poorly described. In this study, our aim was to identify the main pigments and their molecular pathways in the pearl oyster Pinctada margaritifera—the species displaying the broadest range of colors. Three inner shell colors were investigated—red, yellow, and green. To maximize phenotypic homogeneity, a controlled population approach combined with common garden conditioning was used. Comparative analysis of transcriptomes (RNA-seq) of P. margaritifera with different shell colors revealed the central role of the heme pathway, which is involved in the production of red (uroporphyrin and derivates), yellow (bilirubin), and green (biliverdin and cobalamin forms) pigments. In addition, the Raper–Mason, and purine metabolism pathways were shown to produce yellow pigments (pheomelanin and xanthine) and the black pigment eumelanin. The presence of these pigments in pigmented shell was validated by Raman spectroscopy. This method also highlighted that all the identified pathways and pigments are expressed ubiquitously and that the dominant color of the shell is due to the preferential expression of one pathway compared with another. These pathways could likely be extrapolated to many other organisms presenting broad chromatic variation.

International audience; The shell color of the Mollusca has attracted naturalists and collectors for hundreds of years, while the molecular pathways regulating pigment production and the pigments themselves remain poorly described. In this study, our aim was to identify the main pigments and their molecular pathways in the pearl oyster Pinctada margaritifera—the species displaying the broadest range of colors. Three inner shell colors were investigated—red, yellow, and green. To maximize phenotypic homogeneity, a controlled population approach combined with common garden conditioning was used. Comparative analysis of transcriptomes (RNA-seq) of P. margaritifera with different shell colors revealed the central role of the heme pathway, which is involved in the production of red (uroporphyrin and derivates), yellow (bilirubin), and green (biliverdin and cobalamin forms) pigments. In addition, the Raper–Mason, and purine metabolism pathways were shown to produce yellow pigments (pheomelanin and xanthine) and the black pigment eumelanin. The presence of these pigments in pigmented shell was validated by Raman spectroscopy. This method also highlighted that all the identified pathways and pigments are expressed ubiquitously and that the dominant color of the shell is due to the preferential expression of one pathway compared with another. These pathways could likely be extrapolated to many other organisms presenting broad chromatic variation

Today, it is common knowledge that environmental factors can change the color of many animals. Studies have shown that the molecular mechanisms underlying such modifications could involve epigenetic factors. Since 2013, the pearl oyster Pinctada margaritifera var. cumingii has become a biological model for questions on color expression and variation in Mollusca. A previous study reported color plasticity in response to water depth variation, specifically a general darkening of the nacre color at greater depth. However, the molecular mechanisms behind this plasticity are still unknown. In this paper, we investigate the possible implication of epigenetic factors controlling shell color variation through a depth variation experiment associated with a DNA methylation study performed at the whole genome level with a constant genetic background. Our results revealed six genes presenting differentially methylated CpGs in response to the environmental change, among which four are linked to pigmentation processes or regulations (GART, ABCC1, MAPKAP1, and GRL101), especially those leading to darker phenotypes. Interestingly, the genes perlucin and MGAT1, both involved in the biomineralization process (deposition of aragonite and calcite crystals), also showed differential methylation, suggesting that a possible difference in the physical/spatial organization of the crystals could cause darkening (iridescence or transparency modification of the biomineral). These findings are of great interest for the pearl production industry, since wholly black pearls and their opposite, the palest pearls, command a higher value on several markets. They also open the route of epigenetic improvement as a new means for pearl production improvement.

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.

Cultured pearl production from Pinctada margaritifera uses the biomineralization capacities of the mantle graft, the saibo, which is usually obtained from only the middle mantle section of the donor oyster. To evaluate the potential for using other parts of the mantle, this study explores and describes the cultured pearl quality traits, pearl size, shape, surface defects and colour parameters obtained with saibo from the entire length of the mantle, comprising the four following sections: 1) posterior, 2) connection with the gills, 3) middle, (the section usually used commercially), and 4) anterior. Rates of nucleus retention and oyster mortality were also recorded and compared between sections. For this, two experimental grafts were designed and conducted in two contrasting culture sites, using 10 selected wild donor oysters in each to perform a total of 1536 grafts. Mantle section comparison revealed that the anterior section was different from the three other sections, showing: 1) the lowest nacre deposition rate in terms of weight and thickness, 2) the palest pearls, with lowest rate of the attractive overtone colour and the 3) a lower rate of pearls with lustre. For pearl circles and shape, no difference was recorded among the different mantle sections. Posterior, connection and middle sections showed similar pearl quality traits, revealing how the number of high quality saibo obtainable from the same batch of donors can easily be increased, thus benefitting the P. margaritifera pearl industry. This finding could provide significant benefits to pearl farmers and the further development of current pearl grafting practices.

The pearl oyster is one of the rare animal models that support two distinct genomes, through the surgical graft process operated for culture pearl production. This grafted organism is assimilated to a chimera whose physiological functioning remains poorly known. The question of the energy expenditure comparison between chimera and non-chimera animals arises. To answer this question, grafted and non-grafted pearl oysters were evaluated for their energetic needs by the indirect calorimetry method. This method made it possible to measure the energy expenditure based on the respiration rate (RR) measurement, reflecting the basal metabolism. The results showed that the RR values for grafted and non-grafted pearl oysters were not significantly different (p 3 and proteins synthesis was 0.237 ± 0.064 J h−1, representing 0.64% of the total energy expenditure of grafted pearl oysters. This study made it possible, for the first time, to see the energy cost of cultured pearl formation in P. margaritifera and the little impact in the energetic metabolism of the chimera organism.

Beaded cultured pearl farming is a lengthy aquaculture process, particularly when the pearl oysters are produced through a hatchery propagation system, and includes the key steps of artificial breeding, larval and spat rearing before graft operations can take place. Within its genus, Pinctada margaritifera has the ability to produce the widest range of pearl colours, thanks to the donor colour polymorphism of the inner shell, which is mainly responsible for colour transmission. As hatchery spat production in P. margaritifera leads to several colour phenotypes (at 3 months old), the aim of this study was to determine whether a relation exists between the colour of the donors as spat and the final pearl colour. In the experiment, which took place over a four-year period, earlier spat colour selection was applied to two hatchery-produced P. margaritifera families. The spat were traced and then used as donors at the adult stage. A total of 1100 experimental grafts were made, using originally grey, green, red and yellow spat phenotypes as donors. The results showed that all spat colour phenotypes mainly produced pearls in the moderately dark (78.4%) and grey colour (56.7%) classes. Differences in darkness level were produced by red and yellow spat, whose pearls were about twice as pale as those from the grey and green phenotypes. Concerning the pearl colour categories, the results showed that the attractive green/blue pearls were obtained twice as often when using grey and green spat phenotypes and that aubergine/peacock pearls were obtained four times more often by using the red and yellow spat phenotypes. This preliminary study suggests that earlier phenotypic colour selection could be applied in P. margaritifera spat as a useful indicator in both pearl production cycles and family selection for donor oyster lines of specific colour propagation.

The bivalve Pinctada margaritifera has the capacity to produce the most varied and colourful pearls in the world. Colour expression in the inner shell is under combined genetic and environmental control and is correlated with the colour of pearls produced when the same individual is used as a graft donor. One major limitation when studying colour phenotypes is grader subjectivity, which leads to inconsistent colour qualification and quantification. Through the use of HSV (Hue Saturation Value) colour space, we created an R package named ‘ImaginR’ to characterise inner shell colour variations in P. margaritifera. Using a machine-learning protocol with a training dataset, ImaginR was able to reassign individual oysters and pearls to predefined human-based phenotype categories. We then tested the package on samples obtained in an experiment testing the effects of donor conditioning depth on the colour of the donor inner shell and colour of the pearls harvested from recipients following grafting and 20 months of culture in situ. These analyses successfully detected donor shell colour modifications due to depth-related plasticity and the maintenance of these modifications through to the harvested pearls. Besides its potential interest for standardization in the pearl industry, this new method is relevant to other research projects using biological models.

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.

The pearl oyster Pinctada margaritifera has the specific ability to produce pearls with the widest range of colours among all pearl oyster species. This pearl colour diversity originates from the mantle biomineralising tissue (graft) of the donor oyster, which is originally responsible for the variety of colours of the inner shell surface. This study aimed to: 1) assess the geographic distribution and establish a first stocklist of the colourful oyster phenotypes used as donors in French Polynesia, and 2) investigate the phenotypic relation between inner shell colouration and the corresponding colour of harvested pearls. With the support of a pearl farmers' network, we investigated the different donor phenotype frequencies among five collection sites (Ahe, Apataki, Takaroa, TakumeandMangareva). This donor evaluation was made during grafting of pearl oysters (N=49,938) obtained from collector stations. Results showed that pearl production is mainly based on six common colourful donor phenotypes classified as monochromatic and polychromatic profiles, which shown different frequencies among the collection sites. Experimental grafts (N=4640)were then realised and subsequent culture conducted at a single site in order to avoid pearl colour variation due to environmental influences. Traceability between donors (N=232) and pearls (N=2776), revealed that: 1) yellow (gold) and aubergine (reddish) pearls could be mostly obtained by using the monochromatic yellowand red donor phenotypes, respectively, and 2) one third to one quarter of grey pearlswas inevitably harvested, whatever the polychromatic phenotype chosen as the donor, which leaves at least half the harvest composed of the attractive green and peacock colours. This preliminary stocklist of colour range together with analysis of the colour phenotype transmission between inner shell and pearl provide the basis for producing multiple pearl oyster “colour lines” through hatchery propagation and would be helpful for future selective breeding programs. Statement of relevance: Donor shell colour selection predict colours of pearls

The black-lipped pearl oyster, Pinctada margaritifera , produces the largest and most valuable coloured pearls in the world. Cultured pearl size remains one of the most important quality traits. Despite the great geographical area covered by pearl farms in the atolls of French Polynesia, little is known about the influence of grow-out site effects on pearl size attained. To explore the genetic and environmental impact on the size of pearls as well as the genetic × environment interaction, a uniform experimental graft was designed on two contrasting macro-geographical lagoons. Five biparental families of donor oysters were grafted and then reared at both sites. After 18 months of culture, phenotypic parameters corresponding to pearl size, i.e. nacre weight and thickness, were recorded among the harvested pearls. The expression of Pif-177 gene, a biomarker encoding protein in the aragonite nacreous layer, was analysed in the corresponding pearl sac. The results show a family effect for nacre weight and thickness on both sites, with family F058 producing the heaviest and thickest nacre, and F805 the lightest and thinnest. By contrast, inter-site comparison revealed no significant site effects for these two parameters. In addition, grow-out location did not modify the relative gene expression of Pif-177 in the pearl sac between donor families in either culture site. Both nacre weight and thickness were positively correlated with the level gene expression of Pif-177. These results suggest that pearl size parameters were not affected by the environment in the present study and this is supported by the relative gene expression of Pif-177 observed. This knowledge constitutes an initial step in the study of pearl size trait inheritance, which will be helpful in the near future for the diffusion of genetically selected donor oyster lines produced by hatchery systems throughout production sites.

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.

In French Polynesia, the P. margaritifera pearl aquaculture industry is spread over a vast area, as large as Europe. All the oysters for this the highly economically important activity are supplied from just a few collection lagoons, but they are grown in numerous sites across three archipelagos (Gambier, Society and Tuamotu). Many oyster transfers thus indirectly bring about grafting combinations mixing different geographic origins and production sites. This study aims to examine the impact of such graft combinations on cultured pearl quality traits. For this, six homogeneous and standardised experimental graft combinations (N = 6197) were conducted at commercial scale in the two growing locations the most frequently used in French Polynesia: Arutua atoll (Tuamotu) and Mangareva island (Gambier), using oysters supplied from by the top three collection sites: Ahe, Takapoto and Mangareva lagoons. At harvest, four main pearl quality traits: nacre weight deposition speed, pearl colour components (darkness level and green overtone), grade and shape categories were recorded by a professional sorter from the Tahiti auction and compared. Results revealed effects of the combinations of oyster origin and grow-out location, with: 1) significant origin × site interaction for nacre weight deposition speed; 2) colour variation at intra- and inter-site scales, with Ahe origin producing the most dark pearls and Gambier highest rate of the attractive green coloured pearls; and 3) higher grade categories for the Gambier origin and rearing location. These oyster-site combination effects highlight the benefit for the Polynesian pearl industry of switching from a mono-site/ company production system to a new multi-site production strategy to maximize overall cultured pearl quality expression. Keywords: Pearl oyster, Pinctada margaritifera, Graft combinations, Pearl quality traits, Environment

BackgroundCultured pearls are unique gems produced by living organisms, mainly molluscs of the Pinctada genus, through the biomineralization properties of pearl sac tissue. Improvement of P. margaritifera pearl quality is one of the biggest challenges that Polynesian research has faced to date. To achieve this goal, a better understanding of the complex mechanisms related to nacre and pearl formation is essential and can now be approached through the use of massive parallel sequencing technologies. The aim of this study was to use RNA-seq to compare whole transcriptome expression of pearl sacs that had producing pearls with high and low quality. For this purpose, a comprehensive reference transcriptome of P. margaritifera was built based on multi-tissue sampling (mantle, gonad, whole animal), including different living stages (juvenile, adults) and phenotypes (colour morphotypes, sex).ResultsStrikingly, few genes were found to be up-regulated for high quality pearls (n = 16) compared to the up-regulated genes in low quality pearls (n = 246). Biomineralization genes up-regulated in low quality pearls were specific to prismatic and prism-nacre layers. Alternative splicing was further identified in several key biomineralization genes based on a recent P. margaritifera draft genome.ConclusionThis study lifts the veil on the multi-level regulation of biomineralization genes associated with pearl quality determination.

Nucleated pearls are produced by molluscs of the Pinctada genus through the biomineralisation activity of the pearl sac tissue within the recipient oyster. The pearl sac originates from graft tissue taken from the donor oyster mantle and its functioning is crucial in determining key factors that impact pearl quality surface characteristics. The specific role of related gene regulation during gem biogenesis was unknown, so we analysed the expression profiles of eight genes encoding nacreous (PIF, MSI60, PERL1) or prismatic (SHEM5, PRISM, ASP, SHEM9) shell matrix proteins or both (CALC1) in the pearl sac (N = 211) of Pinctada margaritifera during pearl biogenesis. The pearls and pearl sacs analysed were from a uniform experimental graft with sequential harvests at 3, 6 and 9 months post-grafting. Quality traits of the corresponding pearls were recorded: surface defects, surface deposits and overall quality grade. Results showed that (1) the first 3 months of culture seem crucial for pearl quality surface determination and (2) all the genes (SHEM5, PRISM, ASP, SHEM9) encoding proteins related to calcite layer formation were over-expressed in the pearl sacs that produced low pearl surface quality. Multivariate regression tree building clearly identified three genes implicated in pearl surface quality, SHEM9, ASP and PIF. SHEM9 and ASP were clearly implicated in low pearl quality, whereas PIF was implicated in high quality. Results could be used as biomarkers for genetic improvement of P. margaritifera pearl quality and constitute a novel perspective to understanding the molecular mechanism of pearl formation.

Grafting mantle tissue of a donor pearl oyster into the gonad of a recipient oyster results in the formation of a chimera, the pearl sac. The phenotypic variations of this chimera are hypothesized to be the result of interactions between the donor and recipient genomes. In this study, the heritability of phenotypic variation and its association with gene expression were investigated for the first time during Pinctada margaritifera pearl production. Genetic variance was evaluated at different levels, 1) before the graft operation (expression in graft tissue), 2) after grafting (pearl sac tissue expression in chimera), and 3) on the product of the graft (pearl phenotype traits) based on controlled biparental crosses and the F1 generation. Donor-related genetic parameter estimates clearly demonstrate heritability for nacre weight and thickness, darkness and color, and surface defects and grade, which signifies a genetic basis in the donor oyster. In graft relative gene expression, the value of heritability was superior to 0.20 in for almost all genes; whereas in pearl sac, heritability estimates were low (h2 < 0.10; except for CALC1 and Aspein). Pearl sac expression seems to be more influenced by residual variance than the graft, which can be explained by environmental effects that influence pearls sac gene expression and act as a recipient additive genetic component. The interactions between donor and recipient are very complex, and further research is required to understand the role of the recipient oysters on pearl phenotypic and gene expression variances.

The objective of this study was to observe the impact of temperature on pearl formation using an integrative approach describing the rotation of the pearls, the rate of nacre deposition, the thickness of the aragonite tablets and the biomineralizing potential of the pearl sac tissue though the expression level of some key genes. Fifty pearl oysters were grafted with magnetized nuclei to allow the rotation of the pearls to be described. Four months later, 32 of these pearl oysters were exposed to four temperatures (22, 26, 30 and 34°C) for 2 weeks. Results showed that the rotation speed differed according to the movement direction: pearls with axial movement had a significantly higher rotation speed than those with random movement. Pearl growth rate was influenced by temperature, with a maximum between 26 and 30°C but almost no growth at 34°C. Lastly, among the nine genes implicated in the biomineralization process, only

Understanding the respective roles played by donor and recipient pearl oysters in pearl quality determination in relation to the environment is a challenge for the pearl industry. In most Pinctada species, pearl size is mainly related to recipient oyster growth performance, but also relies to some extent on the biomineralisation activity of the pearl sac, a tissue that originates from the donor oyster mantle. We examined donor effect on pearl size in response to culture in the lagoons on Arutua and Apataki atolls. Overall nacre weight and thickness were greater in Arutua than in Apataki, but sensitivity to the environment differed between donors. Some donors were associated with significantly heavier and thicker nacre in Arutua (I group), while others had similar results at the two sites (NI group). On average, up to 20% of the pearl size could be attributed to the donor but, in group I, donor effect was responsible for up to 36% of nacre weight determination. Additionally, a real-time PCR expression study of eight matrix protein genes related to biomineralisation in the pearl sac showed that MSI60, pearlin and pif177 were significantly and positively correlated with nacre weight and thickness, with the latter two genes explaining the larger pearl size observed in Arutua. Donor oysters in P. margaritifera therefore play a key role in pearl size improvement, related to the role of the shell matrix protein genes. Understanding such contributions could help in the design of genetic selection plans for specific and adapted donor oyster lines.

Pinctada margaritifera is an economically important marine bivalve species for cultured pearl production in French Polynesian aquaculture. In order to evaluate the influence of donor oyster age on pearl quality traits, experiments were conducted over 6 years using both grafts and surgreffe operations. At harvest, 6 pearl quality traits were recorded and compared: surface defects, luster, grade, darkness level, and visual color. Analyzing the quality traits of pearls harvested in the initial graft process and those of pearls obtained from surgreffe experiments allowed a comparison of the influence of pearl sac cells originating from the initial mantle graft, which aged together with their recipient oysters. The results demonstrated a significant decrease between these successive grafts in luster, grade (A-B-C,) darkness level, and green color – traits that are of major importance in the pearl market. The duplicated graft experiment allowed the comparison of donor oyster families at 2 and 5 years old, where a mantle graft was inserted into recipient oysters aged 2.5 years old. The results showed the same tendencies to a lesser extent, with 1) an improved pearl grade, predominantly through a most important rate of 0 surface defect category, and 2) a green / grey ratio in favor of the younger donor. A comparison between the graft-surgreffe and the duplicated graft experiments also highlighted: 1) the indirect role played by the younger recipient oysters, which must be optimized for optimal pearl quality realization, and 2) the complex interplay between donor and recipient oysters.

The black-lipped pearl oyster Pinctada margaritifera is of high economic importance in French Polynesia. Arecent decline in pearl value led to the initiation of several genetic breeding programs aiming to improve production traits, including oyster shell growth, which dictates the time of grafting, size of the implanted nuclei, and biomineralization capacities. We assessed shell diameters on hatchery-produced spat to analyze juvenile growth performance of four half-sib families derived from polyandry (one dam crossed with two sires) and polygyny (two dams crossed with one sire) using gametes obtained by thermal shock or gonad scarification. Spat growth was monitored over 3 mo, with shell diameter measured weekly. Results revealed that the spawning method had no significant effect on juvenile growth; however, the half-sib families produced with the polygyny mating design showed significant differences in average shell diameter between dams throughout the experiment, whereas none were observed between sires with the polyandry mating design. Precocious larval size selection within each family was performed by separation into batches of small, medium, and large size, and their maintenance through juvenile stages, providing the possibility for early growth selection. These findings are important for genetic breeding programs (1) as breeding of sires and dams exhibiting the most colorful inner shell phenotypes would be possible with the gonad scarification spawning procedure without affecting overall growth performance and (2) because P. margaritifera is a protandrous hermaphrodite species, genetic selection strategies in the pearl industry must take into account the differential influence of polygyny and polyandry mating designs.

Cultured pearls produced with Pinctada margaritifera, using the surgreffe method (implantation of a second nucleus following pearl harvest) were studied for the first time to: (1) examine family effect on nacre thickness, nacre weight and nacre deposition speed and (2) compare variation in these three traits with that obtained from the cultured pearls previously harvested after the corresponding initial grafts. A surgreffe experiment using 783 recipient oysters was realized in Rangiroa atoll (French Polynesia). After 24 months of culture, 389 cultured pearls were harvested. Significant donor family effect was found for the harvested pearl rate from surgreffe (P = 0.046). Highly significant donor family effect was recorded for nacre thickness (P = 0.004). Very highly significant donor family effects were recorded for nacre weight and nacre deposition speed (P

The bivalve Pinctada margaritifera exhibits three main transplant phenotypes derived from the donor (from which a mantle graft tissue, the saibo, is excised), the recipient (into which the saibo is implanted with a nucleus, leading to the formation of a pearl sac “chimera”) and the cultured pearls themselves. This first phenome study on the species derived from a large experimental graft. Transplant phenotype was assessed at three scales: 1) macro, pearl size, colour, grade, 2) micro, pearl surface microstructure, and 3) molecular, biomineralisation gene expression level in saibo and pearl sac tissues. From donor to pearl, the phenome revealed fine variations of quality traits dependent on the position on the mantle where the saibo was cut, whose variation could overlap with inter-individual donor phenotype differences. A single donor phenotype could therefore produce multiple pearl phenotypes at the scale of the saibo position, mirroring its original activity at the mantle position level and the colour and shape of the shell. This phenome study provides essential information on phenotypic trait architecture enabling us to explore and explain the main biological functions and pave the way for a phenomic project on P. margaritifera that could benefit the pearl industry.

La perle est une production conchylicole originale qui, en Polynesie francaise, se place au 2e rang economique. Cette industrie s’appuie fortement sur la recherche pour introduire du progres dans les pratiques. Cet article a pour ambition de presenter les bases techniques de sa production en detaillant les travaux menes en lien avec les enjeux de developpement durable de la perliculture. Les recherches sont principalement axees sur l’amelioration de la qualite des perles via la selection genetique. Cela necessite de comprendre les processus physiologiques et moleculaires en jeu. Nous concluons sur les mises au point, les developpements scientifiques et techniques en cours ou restant a mener.

International audience; The mollusc Pinctada margaritifera is the top economic aquaculture species in French Polynesia (export value) and forms the basis of the black pearl industry. Mass production of this unique gem, produced by a living organism, relies on a surgical operation requiring tissue from a donor pearl oyster to be grafted, together with a nucleus made of shell, into the gonad of a receiving pearl oyster. This technique is performed by expert grafters, whose work constitutes the first step influencing pearl farm production yield. This study makes the first report of effects mediated by individual grafters and by the season of grafting on three rates scored at 45 days post operation: nucleus retention, nucleus rejection and pearl oyster mortality. These results were obtained in a very large scale grafting experiment, designed and conducted in a single culture site in the atoll of Arutua (Tuamotu Archipelago) and involving a total of 52,910 grafts performed by ten professional grafters, during two contrasting seasons: autumn (four grafting experiments) and spring (three grafting experiments). Statistical analysis using linear mixed-effect model for both univariate and multivariate analysis was performed. The results show a very highly significant season effect (p < 0.001), with a higher rate of nucleus retention in autumn than spring: 91.1 versus 79.4 %, respectively. A very highly significant season effect was also recorded for nucleus rejection rate (p < 0.001), which was greater in spring than in autumn: 12.6 versus 7.7 %. Oyster mortality rate was up to six times higher in spring than in autumn (p < 0.001). Within the two seasons, there was no significant difference between the grafting experiments for any of the three variables, adding robustness to the inter-season results. Taking into account this seasonal variation effect, grafter skill had a significant impact on the three variables (p < 0.001), making it possible to rank grafters from most to least efficient. Aside from skill differences between grafters, grafting in autumn may significantly increase graft success. Water temperature and its indirect consequences could be one of the most important environmental factors implicated in overall efficiency. These findings may help the potential development of an annual grafting schedule according to season/ lagoon temperature as a way to maximize production yield for the black-lipped pearl oyster industry in French Polynesia.

Cultured pearl nacre deposition rate in Pinctada margaritifera oysters was studied to examine its influence on Tahitian classification grade and colour and to further our understanding of using selected donor oysters produced in a hatchery system for the improvement of these traits. A large-scale grafting experiment using 150 farmed donor oysters was realised in Rangiroa atoll (French Polynesia). After 18 months of culture, 874 pearls were harvested and 7 pearls quality traits were recorded: (1) nacre deposition rate assessed by nacre thickness and weight, (2) classification grade, lustre and surface defects, and (3) colour assessed by pearl colour darkness level and visual colour categories. Nacre deposition rate significantly influence pearl grade, surface defects, darkness level and colour categories, but not the lustre. Grade A pearls had the thickest and heaviest nacre on average, compared with grade D pearls and rejects. In addition, pearls without any surface defect had thicker and heavier nacre. Pearls with low darkness were the thinnest and the lightest. Pearls of aubergine–peacock secondary colours were the thickest and the heaviest. These results highlight the importance of rapid nacre deposition rate for increasing the production of grade A pearls, with less surface defects and dominance of a black tone colour. Fast nacre deposition may, however, have a negative impact on other pearl quality traits, such as pearl lustre. This study demonstrates the importance of examining relationships between pearl quality traits in a way that provides solid information that can be directly used for breeding and genetic selection programmes.

Dans le résumé [B] a été remplacé par (B) afin de ne pas perturber l'export de la notice; The black-lipped pearl oyster, Pinctada margaritifera, is the most important farmed mollusc species in French Polynesia. Donor oyster selection among wild P. margaritifera individuals, chosen according to their inner shell colour, makes it possible to obtain the broadest range of cultured pearl colours of any species. This study demonstrates the relative influence of using black or red [R] outer shell phenotypes, combined with green [G] or yellow [Y] inner shell phenotypes, on pearl darkness level, colour categories and lustre. A large scale grafting experiment was designed and carried out over five grow-out locations, covering three archipelagos: Tuamotus, Society and Gambier. Results revealed that the [B+G] phenotypes may be used as donors to produce dark green pearl, which suit the demands of the Asian market; whereas, phenotypes incorporating [R] and/or [Y] phenotypes may be used to obtain multicolour pearls of medium/light darkness, which suit the demands of the European market. From an environmental point of view, the 1) phenotype showed no significant variation for light and other pearl colour production, and 2) [Y] phenotype produced both the same rate of pearl darkness level and green colour pearls whatever the grow-out location. A classification tree model was built to predict, according to shell phenotype and culture location, the colour and darkness level of harvested pearls. Lustre was shown to be more influenced by the environment than by phenotype. These results should be taken into account in pearl farm production management and in selective breeding programmes.

International audience; n French Polynesia, the aquaculture of Pinctada margaritifera is carried out in numerous grow-out sites, located over three archipelagos (Gambier, Society and Tuamotu). To evaluate the impact of macro-geographical effects of these growing sites on pearl quality traits, five hatcheries produced families were used as homogeneous donor oysters in an experimental graft. The molluscs were then reared in two commercial locations: Tahaa Island (Society) and Rangiroa atoll (Tuamotu). At harvest, eight pearl quality traits were recorded and compared: surface defects, lustre, grade, circles, shape categories, darkness level, body and secondary colour and visual colour categories. Overall inter-site comparison revealed that: (1) all traits were affected by grow-out location except for lustre and round shape, and (2) a higher mean rate of valuable pearls was produced in Rangiroa. Indeed, for pearl grade, Rangiroa showed twice as many A-B and less reject samples than Tahaa. This was related to the number of surface defects (grade component): in Rangiroa, twice as many pearls had no defects and less pearls had up to 10 defects. Concerning pearl shape, more circled and baroque pearls were found in Tahaa (+10%). For colour variation, 10% more pearls have an attractive green overtone in Rangiroa than in Tahaa, where more grey body colour were harvested. Lustre does not seem to be affected by these two culture site (except at a family scale). This is the first time P. margaritifera donor family have been shown to vary in the quality of pearls they produce depending on their grow-out location.

Size is the most important and valuable quality trait of cultured pearls produced by the black-lipped pearl oyster, Pinctada margaritifera. In French Polynesia, several breeding programmes have been started that aim to improve this size trait, which is highly related to shell growth rate in both recipient and donor oysters. Shell growth rate dictates the time of grafting, size of implanted nuclei and bio-mineralisation potential of the mantle and pearl sac. We assessed shell growth rate through routine digital shell biometric analysis on 22 hatchery families produced between 2005 and 2008. These included full-sib families and half-sib families derived from polyandry (one dam crossed with two or more sires). Results showed that: 1) a significant family effect was recorded for growth performance, analysed according to the Von Bertalanffy model, 2) a significant male effect was observed for some of the half-sib families and 3) a relationship was found between the shell growth performances of five families randomly selected and used as graft donors in a grafting experiment and the final weight of the cultured pearls produced. These results have important implications for the breeding of pearl oysters with high growth capacities: it may be possible to select oyster lines for the potential to produce large pearls using shell equivalent diameter estimated by the digital method as a selection criterion.

In French Polynesia, the aquaculture of Pinctada margaritifera (Linnaeus, 1758) covers a large maritime exploitation area, spread over nearly 20 degrees latitude and longitude, with numerous pearl farms located in three archipelagos (Gambier, Society, and Tuamotu). As these archipelagos have specific seasonal temperature patterns each year, pearl oysters are subject to disparate and contrasting environmental regimes. This study aimed to examine the specificity of commercial pearl quality traits (n ¼ 2,236 samples) at the archipelago scale, in such a way as to provide preliminary data to design the most appropriate strategy for the distribution of hatchery-produced phenotypes. A large and standardized grafting experiment using the same donor phenotype was designed and carried out over six grow-out locations, covering the three archipelagos. Results revealed significant differences in commercial pearl quality traits among archipelagos, giving these groups of growing sites distinctive ‘‘signatures’’: (1) more color, less circles, and higher overall pearl grade in Gambier; (2) larger size with paler pearls in Tuamotu; and (3) darker pearls with intermediate size in Society. Characteristic differences in the environment and seasonal temperature ranges among the three archipelagos, corresponding to their distinct environmental conditions, can explain the specific variations between pearl quality traits among the sites. The strong disparities at archipelago scale should be taken into account in selective breeding programs for P. margaritifera so as to choose the most appropriate pearl oyster donor phenotype for use in each environment and thus enhance site-specific qualities for pearl production.

International audience; 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.

Using advanced intermated populations has been proposed as a way to increase the accuracy of mapping experiments. An F3 population of 300 lines and an advanced intermated F3 population of 322 lines, both derived from the same parental maize inbred lines, were jointly evaluated for dry grain yield (DGY), grain moisture (GM), and silking date (SD). Genetic variance for dry grain yield was significantly lower in the intermated population compared to the F3 population. The confidence interval around a QTL was on average 2.31 times smaller in the intermated population compared to the F3 population. One controversy surrounding QTL mapping is whether QTL identified in fact represent single loci. This study identifies two distinct loci for dry grain yield in the intermated population in coupling phase, while the F3 identifies only a single locus. Surprisingly, fewer QTL were detected in the intermated population than the F3 (21 vs. 30) and

Ageing is defined as the progressive decline in tissue and organ functions over time. This study aims to evaluate the ageing effect on cultured pearl quality phenotypes (including size and quality traits) in the graft-recipient animal model: Pinctada margaritifera. For this, eight uniform grafting experiments were designed using two hatchery-produced pearl oyster families as donors, which were followed through time, between 7 and 30 months in age. For each age category, 20 donors were studied for each culture site giving a total of 2400 grafted oysters. Several phenotypic measurements were made: 1) donor family growth performance from shell size records, 2) pearl size and corresponding quality traits, and 3) expression of some genes related to biomineralization processes on both the mantle graft and on pearl sac tissues. Results showed that: 1) donor age has an impact on pearl size, with grafts coming from the youngest donors yielding the biggest pearls; and 2) grafts from donors between 12 and 18 months in age produced pearls of the highest quality (grade and surface quality), a result supported by an analysis where the level of expression for a panel of genes associated with biomineralization was greatest in donors within the 12 to 18 months age group. These results indicate that donors aged between 12 and 18 months have high potential for biomineralisation and nacre deposition, and likely produce larger and higher quality cultured pearls than older donors.

International audience; Breeding strategies and parental effects play a major role in genetic selection programmes for quality trait improvement. The black-lip pearl oyster, Pinctada margaritifera, is a protandrous hermaphrodite species in which the number of mature females is low, and these only appear after 4 years of culture. This difference underlines the importance of studying the contributions of dams and sires in the production and selection of donor pearl oyster families to choose the most appropriate breeding strategies for cultured pearl quality trait improvement. Laboratory crosses were made using wild dams and sires to produce half-sib families from polyandry and polygyny mating designs. The resulting half-sib families were used as donor oysters in a large grafting experiment of 900 grafts with random recipient oysters, using nuclei of the same size and brand, the same operating technician and the same grow-out site. After 18 months of culture, 497 cultured pearls were harvested. Comparison between half-sib pairs revealed that for nacre thickness, weight and shape, a polygyny effect exists, whereas no significant effect was observed for the polyandry mating. By contrast, no evidence of dam- or sire-related benefits was found for the presence/absence of circles, darkness level and colour categories or grade and its components: lustre and surface defects. These results should have important implications for the selective breeding of donor pearl oysters for hatchery management.

International audience; The present study investigated the effect of electrolysis on the biomineralization capacities of juveniles of the mollusk Pinctada argaritifera for the first time. Size-selected individuals from two groups, “Medium” and “Large”, from a multi-parental family produced in a hatchery system were subjected to electrolysis under a low voltage current over a nine-week experimental period. The growth of the juveniles was individually monitored and assessed weekly by wet weight and shell height measurements. At the end of the experiment, mantle tissue was sampled for biomineralization-related gene expression analysis. Electrolysis significantly increased pearl oyster growth in terms of shell height and wet weight for Large juveniles from the 5th and the 2nd week, respectively, until the end of the experiment. However, differences were only significant for Medium individuals from the 7th week for shell height and from the 9th week for wet weight. Furthermore, transcriptional analysis of six known biomineralization genes coding for shell matrix proteins of calcitic prisms and/or nacreous shell structures revealed that five were significantly overexpressed in the mantle mineralizing tissue under electrolysis: three in common between the two size class groups and two that were expressed exclusively in one or the other group. Finally, we found no statistical difference of the shell thickness ratio between individuals undergoing electrolysis and control conditions. Taken together, our results indicate, for the first time in a calcifying marine organism, that electrolysis influences molecular mechanisms involved in biomineralization and may stimulate some parameters of pearl oyster growth rate.

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II beta-antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.

Production of larger, rounder, high-quality cultured pearls with fewer circles is one of the main challenges of Pinctada margaritifera aquaculture faced by every pearl farm in French Polynesia. Although bigger pearl sizes can be achieved through surgreffe operations (implantation of a second nucleus following pearl harvest), control of the development of pearl circles and shapes still remains unclear, as illustrated by grafter’s empirical rules, where often the surgreffe process is only performed after production of uncircled and round pearl shapes. The present study was designed with a real pearl by pearl traceability to reveal for the first time the development of circles and shapes from graft and surgreffe, in relation to the size of the pearl sac. This was indirectly assessed by measuring the differences in diameter (DD) and weight (DW) between standardized surgreffe nuclei and the pearl that had been harvested following the initial graft. An experimental graft and surgreffe experiment was designed using the same criteria: grafter, location, nuclei brand and size for graft and surgreffe, and donor oysters from 10 bi-parental families produced in a hatchery system. We studied the differences between pearls harvested following graft and surgreffe on the same recipient oysters (N = 295 for both graft and surgreffe) in relation to three classes of DD and DW in which the surgreffe nuclei, were: 1) bigger / heavier, 2) equivalent to or 3) smaller/lighter than the harvested pearl. Results revealed that to increase the rate of uncircled pearls following surgreffe, insertion of a nucleus larger than the harvested pearl may be advisable. Indeed, the formation of uncircled pearls following surgreffe was enhanced by inserting bigger / heavier second nucleus, both in animals that had produced a uncircled pearl after the initial graft and in those that had produced a circled pearl . For pearl shape, significantly more round shape pearls were produced after surgreffe, following initial oval and baroque samples from graft, by inserting smaller/lighter and bigger/heavier second nucleus, respectively. Inserting a larger second nucleus will significantly increase the rate of both uncircled and round-shaped pearls. This finding has important implications for surgreffe practices, where recipient oysters with undesirable circle or baroque pearls could now be used in this second stage of production.