Pinctada Margaritifera Research Articles

Influence of the Depth on the Shape and Thickness of Nacre Tablets of Pinctada margaritifera Pearl Oyster, and on Oxygen Isotopic Composition

Nacre, or mother of pearl, is composed of aragonite tablets and is produced by some mollusks. Because of the highly organized internal structure, chemical complexity, mechanical properties and optical effects of nacre, its formation is among the best-studied examples of calcium carbonate biomineralization. The pearl oyster Pinctada margaritifera is harvested in French Polynesia for pearl farming. The quality of the pearl depends on the quality of the nacre on its surface and its iridescent colors are affected by the thickness of the layers. Here we report on an experimental study conducted to influence the shape and the thickness of nacre tablets by keeping pearl oysters at four different depths (7, 20, 30 and 39 m) for one week. Scanning electron microscopy was used to measure the thickness of the nacre tablets and to analyze their final shape. The shape of the tablets changed from hexagonal to rhomboid at a depth of 39 m. The change in shape led to a change in size. The thickness of the tablets was reduced by between 16 and 30% on average. We also measured the oxygen isotopic composition using Secondary Ion Mass Spectrometry. In this study, we demonstrated that depth can modify the size, shape and thickness of nacre tablets, but not the d18O. This environmental modification is important for the biomineralization of the shell of the pearl oyster Pinctada margaritifera.

Transcriptome analysis of biomineralisation-related genes within the pearl sac: Host and donor oyster contribution

Cultured pearl production is a complex biological process involving the implantation of a mantle graft from a donor pearl oyster along with a bead nucleus into the gonad of a second recipient host oyster. Therefore, pearl production potentially involves the genetic co-operation of two oyster genomes. Whilst many genes in the mantle tissue have been identified and linked to shell biomineralisation in pearl oysters, few studies have determined which of these biomineralisation genes are expressed in the pearl sac and potentially linked to pearl biomineralisation processes. It is also uncertain whether the host or donor oyster is primarily responsible for the expression of biomineralisation genes governing pearl formation, with only two shell matrix proteins previously identified as being expressed by the donor oyster in the pearl sac. To further our understanding of pearl formation, the pearl sac transcriptome of Pinctada maxima and Pinctada margaritifera was each sequenced to an equivalent 5× genome coverage with putative molluscan biomineralisation-related genes identified. Furthermore, the host and donor contribution of these expressed genes within the pearl sac were quantified using a novel approach whereby two pearl oyster species harbouring unique genomes, P. maxima or P. margaritifera, were used to produce xenografted pearl sacs.A total of 19 putative mollusc biomineralisation genes were identified and found to be expressed in the pearl sacs of P. maxima and P. margaritifera. From this list of expressed genes, species-diagnostic single nucleotide polymorphisms (SNP) were identified within seven of these genes; Linkine, N66, Perline, N44, MSI60, Calreticulin and PfCHS1. Based on the presence/absence of species diagnostic gene transcripts within xenografted pearl sacs, all seven genes were found to be expressed by the species used as the donor oyster. In one individual we also found that the host was expressing Linkine. These results convincingly show for the first time that the donor mantle tissue is primarily responsible for the expression of biomineralisation genes in the pearl sac.

Multi-scale distribution and dynamics of bivalve larvae in a deep atoll lagoon (Ahe, French Polynesia)

Bivalve larvae and hydrographic parameters were sampled over a range of spatio-temporal scales in a deep atoll lagoon. Bivalve larvae abundances were very high throughout the year: 18,550m−3 in average. Larvae were (i) concentrated at mid-depth with nocturnal ascent and diurnal descent, (ii) heterogeneously dispersed at the lagoon scale, (iii) subject to day-to-day variation in abundance and (iv) transferred between different parts of the lagoon providing evidence of intra-lagoonal connectivity. The primacy of physical factors was seen on large spatial scale with the diluting effect of water renewal and transfers by hydrodynamics. On smaller spatial scale, the primacy of biological processes was recognised, with larval swimming activity leading to dial vertical migration correlated with food concentration. Variations in larval abundance were driven by bivalve reproductive activity correlated with meteorological conditions (i.e. windy periods). Finally, relationship between bivalve larvae patterns and pearl oyster (Pinctada margaritifera) settlement structuring is discussed.

Aragonite crystal orientation in mollusk shell nacre may depend on temperature. The angle spread of crystalline aragonite tablets records the water temperature at which nacre was deposited by Pinctada margaritifera

Nacre, or mother-of-pearl, is a lamellar composite of aragonite (CaCO3) tablets with a broad distribution of angular orientations. The angle spread is the full-width of this distribution. Here we analysed the angle spread as a function of position in the nacre layer of one Pinctada margaritifera shell, compared the results with temperature data, and found these two parameters to be highly correlated. This result suggests that one could calculate the temperature at which nacre formed by measuring only its angle spread. Validation of the correlation in modern and ancient nacre from other species is necessary, but if confirmed, nacre could provide a physical proxy for temperature in modern and ancient climates.

Influence of plankton concentration on gametogenesis and spawning of the black lip pearl oyster Pinctada margaritifera in Ahe atoll lagoon (Tuamotu archipelago, French polynesia)

Pearl culture industry represents one of the dominant business sector of French Polynesia. However, it still entirely relies on unpredictable spat collection success. Our aim was to assess the influence of natural plankton concentration fluctuations on maturation and spawning of the black lip pearl oyster Pinctada margaritifera, during a 4months survey conducted in Ahe atoll lagoon. Plankton concentration was assessed by chlorophyll a extraction and by microscope counts while gonadic index, gonado-visceral dry weights and histology were used to measure pearl oysters reproduction activity. We found that (i) plankton concentration fluctuations were mainly related to wind regime, (ii) gametogenesis rate was mainly related to plankton concentration, (iii) spawning occurred when maximal gonad storage was reached, (iv) plankton concentration was the main spawning synchronizing factor. These results contribute explaining P. margaritifera spat collection variability in French Polynesian atoll lagoon.

Pearl oysters Pinctada margaritifera grazing on natural plankton in Ahe atoll lagoon (Tuamotu archipelago, French Polynesia)

In atoll lagoons of French Polynesia, growth and reproduction of pearl oysters are mainly driven by plankton concentration. However, the actual diet of black-lip pearl oysters Pinctada margaritifera in these lagoons is poorly known. To fill this gap, we used the flow through chamber method to measure clearance rates of P. margaritifera in Ahe atoll lagoon (Tuamotu Archipelago, French Polynesia). We found: (i) that pearl oysters cleared plankton at a rate that was positively related to plankton biovolume, (ii) that nanoflagellates were the main source of carbon for the pearl oysters, and (iii) that the quantity and origin of carbon filtrated by pearl oysters was highly dependent on the concentration and composition of plankton. These results provide essential elements for the comprehension of growth and reproduction variability of pearl oysters in atoll lagoons of French Polynesia.

Colour or no colour in the juvenile shell of the black lip pearl oyster, Pinctada margaritifera?

Pinctada margaritifera mollusc is cultivated in French Polynesia for the production of black pearls. For this study, the colour of juvenile samples (48 days old) was investigated in the visible range spectra (430–700 nm) using spectrophotometry. A first measurement was done with the soft parts still inside the shell (entire animal). Then, the soft parts were removed in order to do a second measurement on the growing edge of the shell. Comparison of the two measurements shows that the estimation of the living animal colour with unaided eye is strongly influenced by the colour of the soft parts. The use of the International Commission on Illumination (ISI) chromaticity diagram shows that at this growth stage, the shells are “white”; i.e. present no absorptions to the visible part. Multivariate statistical analyses based on the intensities of 6 wavelengths show that the shell colour is less variable than the colours of the entire animals. Wavelength intensities of the shells are similar, so no colour trend is visible. On the other hand, the colours of the entire animals are darker, and more variable. At this growth stage, the shell colour is not predictable for a potential selection of receiver or donor.

Motility activation and metabolism characteristics of spermatozoa of the black-lip-pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media.

Is the pearl layer a reversed shell? A re-examination of the theory of pearl formation through physical characterizations of pearl and shell developmental stages inPinctada margaritifera

A series of physical characterization methods (UV fluorescence microscopy, X-ray microdiffraction, backscattered electron imaging and X-ray absorption spectroscopy) were applied to Polynesian pearls collected after different cultivation periods, varying from three weeks to eighteen months. Through this rigorous time-based sampling, 120 pearls produced by 20 different donor oysters were compared. Results show that the structure of the pearl layer can be understood as a sequence of distinct secretion processes whose progressive occurrence through time may lead to variously arranged and sometimes aberrant mineralized structures. By making comparisons with the structure and growth mode of the Pinctada margaritifera shell, this study shows that the currently accepted theory that views the pearl-bed as a “reversed shell” cannot account for the diversity of the microstructural patterns and mineralogical properties observed in the pearl layers. From a practical and economic view point, it appears that development of these pre-nacreous materials superposed onto a perfectly round-shaped nucleus is the main cause of shape irregularities in pearls and the consequent decrease in their value.

Determination of Gender in the Pearl OysterPinctada margaritifera

The pearl industry in French Polynesia is based on exploitation of natural stocks of the blacklip pearl oyster (Pinctada margaritifera). It generates an annual turnover of €90 million. Improvements in pearl quality need genetic studies to improve the populations. This pearl oyster is a protandric species, in which the sex ratio normally is biased toward males. There is an increasing interest in gender control to find the mechanisms to augment female proportions for management purposes. This review summarizes information on exogenous and endogenous factors regulating gender in this and other bivalves, and concludes that P. margaritifera is a protandric hermaphrodite, developing as a male during the first 2 y, and without evidence of an effect from abiotic and biotic factors on gender during this phase. Later, pearl oysters progressively change to females, reaching a sex ratio close to 1:1 in specimens older than 8 y. At this stage, gender is apparently influenced by environmental parameters, but particularly by stress. Future research should seek to determine accurately the effect of temperature and food on sex ratios. Studies should be performed to characterize genes responsible for expression of gender. The use of hormones is a path that might be explored to influence the gender of pearl oysters.

Pmarg‐Pearlin is a Matrix Protein Involved in Nacre Framework Formation in the Pearl OysterPinctada margaritifera

The shell of pearl oysters is organized in multiple layers of CaCO(3) crystallites packed together in an organic matrix. Relationships between the components of the organic matrix and mechanisms of nacre formation currently constitute the main focus of research into biomineralization. In this study, we characterized the pearlin protein from the oyster Pinctada margaritifera (Pmarg); this shares structural features with other members of a matrix protein family, N14/N16/pearlin. Pmarg pearlin exhibits calcium- and chitin-binding properties. Pmarg pearlin transcripts are distinctively localized in the mineralizing tissue responsible for nacre formation. More specifically, we demonstrate that Pmarg pearlin is localized within the interlamellar matrix of nacre aragonite tablets. Our results support recent models for multidomain matrix protein involvement in nacreous layer formation. We provide evidence here for the existence of a conserved family of nacre-associated proteins in Pteriidae, and reassess the evolutionarily conserved set of biomineralization genes related to nacre formation in this taxa.

Growth and gonad development of the tropical black-lip pearl oyster, Pinctada margaritifera (L.), in the Gambier archipelago (French Polynesia)

The growth and reproductive cycle of cultured black-lipped pearl oysters, Pinctada margaritifera (L.), were studied in the Gambier Islands (134°52′ W, 23°07′ S) from September 2002 to August 2003. Temperatures were recorded throughout the year, revealing seasonal temperature variations between 22.3 and 27.8°C. The mean annual chlorophyll a value, as computed from satellite data, was 0.188 ± 0.075 μg L−1. To study growth and reproduction, 720 two-year-old individuals were ear hung on long-lines suspended at a depth of 7 m. Samples were taken twice a month to obtain the following measurements: shell height; wet weight of flesh and total oyster; dry weight of adductor muscle, mantle and visceral mass; and glycogen content. Gonad development was also studied by histology on parallel samples. Growth was relatively fast during the first 6 months of the study: average shell height increased from 89.1 ± 9.1 to 119.7 ± 10.8 mm and total weight from 93.4 ± 24.5 to 155.1 ± 33.6 g, between September and the end of March. Subsequently, from April to August, no significant growth was observed for shell and flesh, while the muscle weight decreased significantly. Condition index (CI), defined as the ratio of wet weight of the visceral mass to shell weight, and histological changes in the gonad revealed 3 significant reproductive events of different intensities. The analysis of correlations revealed a specific effect of the chlorophyll a concentration on the growth of shell and soma, and one of the temperature on tissue glycogen content. This study also showed also that CI could be an efficient indicator of reproductive events in pearl oyster. It thus appears that the development of gonads goes on throughout the year in the Gambier Islands, without any detectable phase of sexual rest.

Application of a bioenergetic growth model to larvae of the pearl oyster Pinctada margaritifera L.

We applied, for the first time, a dynamic energy budget (DEB) growth model to the larval phase in the pearl oyster Pinctada margaritifera var. cumingii (Linnaeus, 1758) to evaluate the impact of spatio-temporal variation in the atoll lagoon environment on its capacity for development. The specific parameters of the model, which represent ingestion, temperature effect and the relationship between length and biovolume of the larvae, were determined from experiments or taken from the literature. The interpretation of the values of these parameters allowed us to identify the underlying adaptive character trait: P. margaritifera larvae have a good capacity to exploit low food concentrations and a narrow range of thermal tolerance restricted to hydrobiological conditions found in the tropical oligotrophic waters of its distribution zone. Growth simulations show a good fit with the observations made on reared larvae under different conditions: fed on either cultured algae or natural plankton, and with growth data from a natural cohort. Finally, a first application of the model to a pearl-culture lagoon reveals the predominant effect of the vertical structure of trophic resources in determining spatial variation in larval growth.

Hatchery-scale trials using cryopreserved spermatozoa of black-lip pearl oyster,Pinctada margaritifera

Cryopreservation is a valuable tool for genetic improvement programs. Several bivalve mollusc species have already been the subject of such programs and the Tahitian black pearl oyster industry is now planning the development of selective breeding for desirable traits in Pinctada margaritifera. The ability to cryopreserve spermatozoa would, therefore, offer significant benefits to the cultured black pearl industry. Spermatozoa were cryopreserved with cryoprotectant agent (CPA) 0.7 M trehalose in 0.8 M Me2SO and a two-step freezing process was used: straws were first maintained in nitrogen vapour for 10 minutes, then directly plunged into liquid nitrogen and stored for one week before use. The viability of thawed sperm was 23% lower than that of fresh sperm. When using thawed sperm, therefore, a higher sperm/egg ratio of 100 000:1 was required to reach 80% oocyte fertilization, compared with 100:1 for fresh sperm. Nevertheless, this first demonstration of cryopreserved sperm fertility in black pearl oyster confirms the hatchery applicability of the cryopreservation technique defined here. Monitoring for larval viability during the first 23 days of life revealed no significant differences between the progeny produced with cryopreserved sperm and that produced using fresh sperm.

Coupling Proteomics and Transcriptomics for the Identification of Novel and Variant Forms of Mollusk Shell Proteins: A Study with P. margaritifera

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Shell layers of the black-lip pearl oyster Pinctada margaritifera: Matching microstructure and composition

The nacre-prism transition of the mollusc shell Pinctada margaritifera was studied using scanning electron microscopy, electron probe micro-analysis (EPMA) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Mineralogical change is correlated with a change in organic matrix. Previous analyses had shown that sugars were involved in the transition layer (fibrous aragonite). The new Energy Dispersive Spectrometry (EDS) and TOF-SIMS maps show that the modifications at the layer boundary are complex, and that proteins and lipids are also involved. Detailed TOF-SIMS maps show that the thick organic envelopes surrounding the prisms, and between the prisms and the fibrous aragonitic layer, are not composed by regular layers, but are a patchwork of various molecules. The amino acid compositions of the nacreous and prismatic layer are compared thanks to the TOF-SIMS localized analyses.

Calcein staining of calcified structures in pearl oyster Pinctada margaritifera and the effect of food resource level on shell growth

Marine mollusc shell growth has been widely measured using fluorochrome marking. In order to test the efficiency and reliability of calcein staining on Pinctada margaritifera shells and pearls, the present study examined two administration methods, different concentrations and several immersion times. Immersion in a 150 mg L −1 calcein solution for 12 h to 24 h appeared to be the best method for marking P. margaritifera shells. For pearl marking, injection of a 200 mg L −1 calcein solution into the pearl pouch was the optimal method. Calcein marking was then used to measure the influence of food resource levels on the shell growth. Groups of 23-month-old P. margaritifera were fed at three trophic levels for two months. The two highest food levels tested (6000 cell mL −1 and 15 000 cell mL −1) induced uniform growth between the dorsal and ventral sides of shell, whereas the lowest food level (800 cell mL −1) induced greater growth on the dorsal side. Shell deposits from the ventral side were observed using a scanning electron microscope, revealing that the difference of the trophic level over two months had modified the thickness of the aragonite tablets formed. These results showed that the trophic level is a major factor conditioning P. margaritifera development.

Development of in situ hybridisation using 16S rRNA gene to monitor black-lip pearl oyster,Pinctada margaritifera, larvae in plankton samples

In French Polynesia, the black-lip pearl oyster Pinctada margaritifera has been farmed to produce pearls since the 1980s, forming the basis of a major industry. The sustainability of this activity relies on spat collection in the lagoons. However, pearl oyster spat can be difficult to identify for the evaluation of stock variations. It is especially hard to distinguish Pinctada spp. larvae at a very early stage of development. In the present study, a whole-mount in situ hybridisation (ISH) technique was developed to allow the discrimination of larvae of closely-related pearl oyster species found in the French Polynesian atolls. Using specific ribosomal 16S-DNA sequence data, we were able to successfully differentiate between Pinctada margaritifera and Pinctada maculata larvae from 5 to 13 days old. This is the first description of a non-destructive method allowing bivalve larvae discrimination between species within this genus. The method allowed us to successfully identify P. margaritifera larvae in natural plankton samples. This result is a key step needed to develop monitoring of P. margaritifera larval distribution in French Polynesian lagoons, a procedure which will increase spat collection efficiency and ensure sustainable development of pearl oyster farming.

Correction Method for Null Alleles in Species with Variable Microsatellite Flanking Regions, A Case Study of the Black-Lipped Pearl Oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.

Wild Black-lip Pearl Oyster ( Pinctada margaritifera ) Spat Collection in Tanzania

Pearl farming is a growing aquaculture activity in Tanzania but requires sufficient young pearl oysters to make it feasible. Collection of spat in the wild is the most viable way of doing this and was tested to establish whether it would yield sufficient juvenile pearl oysters to support an industry. A total of 4263 Pinctada margaritifera spat were collected over a year at sites considered suitable for spat collection: Bweleo and Nyamanzi on Zanzibar, and Tawalani in Northern Tanzania. Spat "yield" exhibited seasonal variations at these sites in a pattern similar in annual trend at Tawalani and Bweleo but different at Nyamanzi. Generally, the dry season (June-November) yielded a higher number of spat than the wet season (December-May). During the dry season, Tawalani, Bweleo and Nyamanzi produced 877, 942 and 1176 P. margaritifera spat respectively, while, during the wet season, these numbers were 503, 730 and 35. A few other pearl oysters such as Pteria penguin were inadvertently collected at Nyamanzi during the study. Three different types of spat collectors were used during the study, comprising coconut shells, spat bags and rubber tiles. There was significant variation in the number of spat collected on these materials (P < 0.01). The yield was always greater on spat bags and rubber tiles than on coconut shells. The study showed that it is possible to collect sufficient numbers of wild spat for the culture of black-lip pearl oysters at some sites along the coast of Tanzania.