Introduction

During grafting, a live tissue fragment or graft prepared from the mantle of a donor pearl mollusc is implanted into the gonad of a recipient pearl mollusc together with a small inorganic bead, often referred to as the nucleus, in contact with the graft. Cultured black pearls from Pinctada margaratifera show a wide range of color and luster, which are the most important characteristics determining their commercial value. This variation is likely to be influenced by both environmental and genetic factors. The latter may depend on the recipient oyster, but professional grafters commonly consider that the color and luster of pearl are often related to the phenotypic properties of the donor (rather than of recipient) oyster (Wada and Komaru, 1996). The grafting process induces a great upheaval in the recipient organism, including an immunological reaction and cell differentiations that make it difficult to track the different cells until the development around the bead of a follicle called a “pearl-sac” and the beginning of nacreous secretion. For that reason, none of the histological studies performed during this early stage were able to determine clearly the initiation of the graft cells (Herbaut et al., 2000).

In the absence of evidence, opinions diverge as to the role and fate of the mantle allograft in the recipient. On one hand it may influence the very first steps of pearl sac formation and the secretion of the very first nacreous layers of the pearl before being rejected by the immunological response of the host. On the other hand, if it persists in the host during the whole course of pearl formation, it would imply an exceptional rate of graft success, as well as a unique biological cooperation between cells from distinct individuals with distinct genotypes in the nacreous secretion process. In an attempt to clarify its role and presence throughout the course of biomineralization, we tried to detect its presence using molecular methods, to screen for the occurrence of a foreign genotype in the pearl sac of the recipient oyster. We compared the genomes in the muscles and pearl sacs of 80 black-lipped pearl oysters, Pinctada margaritifera, at the day of harvesting (about 18 months after the graft), using three codominant polymorphic molecular markers (Arnaud-Haond et al., 2002, 2003a,b).

Materials and Methods

Sampling and Extraction

Two series of 50 and 30 pearl oysters were collected 18 month after grafting, at the time of pearl collection. The pearl sac was extracted with the pearl inside, and the cells layer around the pearl were carefully split until a layer of one cell width surrounding the pearl was obtained. This layer and a piece of the abductor muscle of the recipient oyster were both labeled identically and preserved in 80% ethanol. The second series of 30 pearl oysters was sampled and processed after the first series of 50 had been entirely analyzed. This was done to make sure that the discrepancies observed between the genotypes of pairs of muscles and pearl sacs was a repeatable observation, and could not be attributed to some incident mixing of the sample during the sampling or genotyping processes.

The procedures of DNA extraction, precipitation, and storage were similar to those described in Sambrook et al. (1989), using approximately 0.5 g of chopped and subsequently air-dried tissue. The nucleic acid pellet obtained after precipitation in 100% ethanol was washed with 70% ethanol, air-dried, resuspended in 100 to 200 μl of deionized water, and preserved at −20°C. Concentration of DNA extraction was estimated via fluorometry, and all extractions were rediluted to obtain a standardized concentration of DNA of 50 ng/μl for each extract.

Polymerase Chain Reaction (PCR) and Genotyping.

Three markers developed (Arnaud-Haond et al., 2002) via the direct amplification of length polymorphism (DALP; Desmarais et al., 1998) and the exon primers intron crossing (EPIC; Palumbi, 1995) methods were used: pinucl2, pinucl3, and pinaldo (Table 1).

Table 1 Detail of the Loci Used, with the Sequence of Each Primer, Range of Size of the Fragments on the Basis of Sequences, and Names of Alleles Observed Polynesian Archipelagoes
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PCR was performed in a 20-μl reaction volumes with final concentrations of 300 μM each dNTPs, 1.8 mM MgCl2, 0.4 μM of each primer, about 100 ng of template DNA, 1× Taq buffer, and 0.75 U of Taq polymerase. For all PCR reactions, a negative control was used, replacing DNA with nanopure water, to ascertain the absence of contamination of the PCR reaction. This negative control would always lead to the absence of PCR product. PCR products were separated through 6% denaturing polyacrylamide gels (acrylamide-bisacrylamide, 29:1, 7 M urea) using 1× Tris-borate-EDTA buffer, running each pair of samples (the DNA extracted from pearl sac and from the corresponding adductor muscle) in neighboring lanes to facilitate comparison of the genotypes revealed by PCR in each tissue. The gels were then silver stained according to Bassam et al. (1991).

Results and Discussion

For the three loci pinucl2, pinucl3, and pinaldo, among the respectively five, five, and six alleles observed over all the samples analyzed in Polynesia (Arnaud-Haond et al., 2002, 2003b, 2004), respectively three, three, and six were observed in the two series of grafted oysters analyzed. In the first series of 50 oysters analyzed at the time of pearl collection, differences in the genotype of the muscle and pearl sac cells were revealed in respectively 43%, 44%, and 7% of the sample pairs successfully genotyped. Owing to the high sensitivity of PCR, it is very probable that an admixture of host and donor genotypes were amplified from the isolated pearl sac, but in any case the discrepancies between the genomes from muscle and pearl sac demonstrate the presence of donor cells as well as their corresponding genome in the pearl sac at the moment of pearl collection, 18 months after grafting. Indeed in some cases, three bands appeared in the lane corresponding to the pearl sac, resulting from the simultaneous amplification of distinct alleles from donor and recipient oysters that were both present in the pearl sac. Similar results were obtained on the second series of 30 oyster analyzed after. Joint analyses of both series distinguished about 65% pairs of distinct genotypes in pearl sacs versus muscle tissue (Table 2). In most cases where differences were highlighted, two bands were observed in the lane corresponding to the amplification product from pearl sac extract (Figure 1), one band being absent in the corresponding muscle PCR product. This implies most recipient oysters were homozygotes, and donors may have been homozygote for the distinct allele appearing in the pearl sac, or heterozygote with only one allele in common with the host oyster.

Table 2 Expected Numbers of Undistinguishable Genotypes Estimated on the Basis of Allele Frequencies and Consequent Pairs of Possible Multilocus Genotypes*
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Fig. 1
figure 1

Electrophoresis gel showing differences between PCR products for the locus pinucl2 amplified from genomic DNA extracted from muscle (M) and corresponding pearl sac (PS) tissues from five oysters. From left to right, each pair of samples shows a different genotype with pinucl2 (M homozygote for the allele 250 and PS heterozygote with the alleles 210 and 250), pinucl3(M homozygote for the allele 110 and PS heterozygote with the alleles 100 and 110), and pinaldo (M homozygote for the allele 100 and p heterozygote with the alleles 090 and 100).

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Once we established the presence of donor cells in the pearl sac 18 months after grafting by the recognition of a foreign genotype present together with the host genotype in the pearl sac, we wanted to screen for the extent of this process. The next question we aimed to answer was therefore whether the grafted cells from the donor systematically persisted in the pearl sac during the entire process of pearl formation, or only occasionally. The probability of occurrence of each possible monolocus genotype was estimated according to allelic frequencies at each locus, assuming Hardy-Weinberg equilibrium and random mating. The expected probabilities that the donor and recipient oyster genotypes were distinguishable were computed on the basis of the multilocus genotype probabilities. We did expect to observe distinct genotypes while comparing PCR products issued from pearl sac and muscle extract in all the cases in which the donor oyster bore at least one allele that was not present in the recipient oyster genome. This probability made it possible to estimate the number of cases in which we expected to observe distinguishable genotypes for at least one locus, under the assumption the donor cells would systematically contribute to pearl sac formation, and the genome of the donor would therefore always be present in the pearl sac. This value was compared to the observed number of cases in which distinguishable genotypes were indeed amplified from extracts of pearl sac and its corresponding adductor muscle (Table 2). A χ2 test was then performed and showed that V obs and V exp were not significantly different (χ2 = 0.58, with a ddl = 2; P>0.25), supporting the hypothesis that the pearl sac was systematically bearing cells and genome from the donor oyster.

The pearl sac, the formation of which is induced by the graft process, is composed of a single layer of epithelial cells, the origin of which remained unclear so far because of the process of cell dedifferentiation and proliferation following the grafting (Machii, 1968; Herbaut et al., 2000) that did not allow tracing the fate of the graft cells via classical histological methods. We found a high number (about 65%) of differences between the muscle and corresponding pearl sac genotypes (Figure 1). The proportion of indistinct muscle-pearl sac genotypes is not significantly different from the expected proportion of nondistinguishable genotypes between distinct individuals of the population that contained the host oyster. These results support the systematic survival of grafted mantel cells in the recipient oyster during the whole process of pearl formation, and their participation in the constitution of the pearl sac that secretes nacreous layers. In agreement with empirical observations of professional grafters, this study supports the idea that the part of the pearl properties determined by genetic factors can be influenced by the donor genome, thus making pearl formation a unique example of cooperation among distinct genomes in a biomineralization process. In future genetic selection programs, efforts may therefore be focused on selection not only in the recipient, but also in the donor oyster.