Aquatic Living Resources. Jean-Pierre Cuif 1,a, Yannicke Dauphin 1, Lauren Howard 2, Julius Nouet 1, Stéphan Rouzière 3 and Murielle Salomé 4 - PDF

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Aquat. Living Resour. 24, (2011) EDP Sciences, IFREMER, IRD 2011 DOI: /alr/ Aquatic Living Resources Is the pearl layer a reversed shell? A re-examination of the

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Aquat. Living Resour. 24, (2011) EDP Sciences, IFREMER, IRD 2011 DOI: /alr/ Aquatic Living Resources 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 in Pinctada margaritifera Jean-Pierre Cuif 1,a, Yannicke Dauphin 1, Lauren Howard 2, Julius Nouet 1, Stéphan Rouzière 3 and Murielle Salomé 4 1 UMR 8148 IDES, bât. 504, Sciences de la Terre, Université Paris Sud, Orsay, France 2 Natural History Museum, Cromwell Road, London, SW7 5BD, UK 3 UMR 8502, Physique des Solides, Bât. 510, Université Paris Sud, Orsay, France 4 ID21, European Synchrotron Radiation Facility, BP 220, Grenoble, France Received 4 June 2011 ; Accepted 4 October 2011 Abstract 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. Key words: Pearl-oyster / Pinctada margaritifera / Biocrystallization / Microstructural sequence / Organic matrix distribution / Layered growth mode 1 Introduction: the concept of pearls as reversed shell It is commonly accepted that formation of the pearl layer in cultivated pearls follows a mineralization pathway comparable, in many respects, to the shell formation of pearl oysters themselves. Taylor and Strack (2008) summarized this concept of pearl layer viewed as a reversed shell : sections of pearls reveal that their structure is similar to the structure of the shell, the difference lies only in the reversed sequence of layers, such that the inside of the shell corresponds in its layers to the outside of the pearl. Such a view basically relies on the observations carried out by Kawakami in the early 1950s. Mostly working with Pinctada martensi, Kawakami noted that, in contrast to the simple scheme of pearl as a layer of nacre surrounding the a Corresponding author: spherical nucleus, the presence of non-nacreous materials is very frequent. In the method of pearl production initiated by Mise and Nishikawa in the first decade of the 20th century, the sequence of grafting operations begins by preparation of the grafts, small pieces of living tissue (about 3 3 square millimetres) cut from the nacre producing area of the mantle of a donor oyster. In a second step, each graft is deposited into the body cavity of a receiver oyster, with a nucleus (a sphere of biogenic calcium carbonate, usually prepared from the nacreous layer of a fresh water mollusc shell). Care must be taken to place the mineralizing surface of the graft (i.e., the external epithelium of the mantle) against the nucleus surface. Then the receiver oyster is returned to sea water and reared for two years under carefully-surveyed living conditions in order to promote optimal growth of the pearl layer. Within the visceral cavity of the receiver oyster, the grafted fragment of mantle tissue undergoes cellular proliferation that Article published by EDP Sciences 412 J.-P. Cuif et al.: Aquat. Living Resour. 23, (2010) leads to complete wrapping of the nucleus. This results in formation of the pearl-sac, a complex structure whose essential component is the internal mineralizing epithelium, directly in contact with the nucleus surface. With respect to the origin of the newly formed mineralized structures, recent studies have shown that cells involved in mineralization by the pearl-sac epithelium are actually derived from the outer cell layer of the graft (Arnaud-Haond et al. 2007). Thus, formation of a nacreous layer surrounding the nucleus could be expected to be a simple continuation of the mineralizing activity of the graft before it was cut from the mantle. In reality, however, this pattern of development rarely occurs. Kawakami (1952a, 1952b) therefore made the hypothesis that during wrapping of the nucleus by the expanding graft, cells of the mineralizing epithelium undergo important metabolic changes referred to as regeneration, leading to formation of non-nacreous materials. Kawakami compared the organic layers visible at the base of the pearl layers with the periostracum of the shells and calcite prismatic structures frequently produced during initial mineralization of pearls, with the outer prismatic layer of the Pinctada shells, and finally with the nacreous layers of both pearl and shells. The results led Kawakami to conclude that, during its transformation into a pearl sac, metabolic changes in the mineralizing epithelium resulted in formation of a new mineralization sequence, comparable to the structure of the shell itself. As this hypothetical new sequence started from the nucleus surface, it led to the concept of the pearl layer as a reversed shell. As pearls are of high economic importance, formation of nacre has been intensively investigated for decades, from both structural and biochemical points of view (e.g., Tsukamoto et al. 2004; Nudelman et al. 2008; Suzuki et al. 2009; Inoue et al. 2011). Recent studies on genomic expression associated with nacre production have underlined the high complexity of this biomineralization mechanism (see Joubert et al for review). With respect to the validity of Kawakami s hypothesis though, formation of the calcite prisms, which make up the outer layer of the Pinctada shell, must also be taken into account. Although calcite prisms have been relatively less studied than nacreous tablets, recent studies have emphasized the leading role of organic components (proteins, polysaccharides and lipids) in their formation (Dauphin 2003; Baronnet et al. 2008; Okumura et al. 2010; Farre et al. 2011). Based on Polynesian pearls, provided by a number of different producers, a first examination of the basal pearl layers was made in order to assess their actual level of microstructural diversity (Cuif et al. 2008). An on-going long term experimental program in French Polynesia offers the opportunity to make a developmental investigation through a series of samples taken over time. This comparative study of sizes, shapes, spatial arrangements and crystallographic status of the surprisingly diverse pearl components, each of which varies through time, provides a large database for re-examination of the current concept of pearl formation. 2 Materials and methods 2.1 Materials Pearls were produced at the Polynesian Centre des Métiers de la Nacre et de la Perliculture at Rangiroa (Tuamotu). Receiver oysters (n=1952) were grafted on November 20 21, All the grafting operations were made by two professional grafters only, in order to reduce influence of diversity in grafting technique. Grafts were prepared from 40 different donor oysters belonging to the Pinctada margaritifera (the black lip pearl oyster) sub-species cumingi, which is used in all production sites in Polynesia. All receiver oysters were grown in similar conditions in the Rangiroa lagoon. Pearls were collected after growth periods ranging from 21 days to 18 months, resulting in parallel growth series of pearls, each of known growth duration. Of these growth series, 20 were dedicated to microstructural investigations. Owing to nucleus rejections, which occurred at the usual rate, microstructure of the pearl bed was studied on 120 pearls. At the Orsay laboratory, pearls were cut using a 0.3 mm thick 150 mm diameter diamond saw at very low grinding speed (about 2 rotations/min) with a water-cooled grinding surface. Polishing of the resulting surfaces was carried out using the decreasing-grade series of the Buehler products, up to colloid silica. Only water was used as a suspension medium during grinding and polishing processes. The polished surfaces were studied using the methods described below, performed as far as possible in such an order that data could be obtained from the same area of the pearl bed. 2.2 Methods Optical microscopy included observation by transmitted light (natural and crossed-nicol polarization), reflected natural light, UV epifluorescence by mercury lamp light filtered by UV (365 nm) and blue (435 nm) filters using both Zeiss Standard Universal epi-illumination and a Leica TCS-NT SP inverted microscope. Microbeam X-ray diffraction measurements were carried out at the Laboratoire de Physique des Solides (Paris-Sud University) using a laboratory-built micro-diffraction instrument (Rouzière et al. 2010). In this equipment, X-rays are produced by a rotating-anode generator working at 40 kv and monochromatized at Cu-Kα energy (8.04 kev). The microbeam is defined by a pinhole of 20 µm diameter placed 2 mm in front of the sample, resulting in a 25 µm circular surface of the sample being submitted to X-ray for the whole measurement. Micro-diffraction mapping over the sample is performed with two motorized translation stages along the horizontal and vertical axes, with a 50 µm incremental step. The diffracted signals are collected by a CCD camera positioned behind sample and beamstop. Micro-XANES mapping (X-ray Absorption Near Edge Structure spectroscopy), Biochemical mapping was carried out J.-P. Cuif et al.: Aquat. Living Resour. 23, (2010) 413 at the ID21 beamline of the European Synchrotron Radiation Facility. The ID21 Scanning X-ray Microscope uses Fresnel zone plates as focusing optics to generate a submicron X-ray probe. An energy-dispersive high-purity Ge detector (Princeton, Gamma-Tech) mounted in the horizontal plane, perpendicular to the beam, collects the fluorescence emission photons. This geometry minimizes the contribution of elastic scattering. An energy range between 2 and 7 kev is available, which gives access to the sulphur K-edge at ev (sulphur in amino acids) and ev (sulphated sulphur in polysaccharides). For methodological examples of identification in biominerals, see Cuif et al. 2003, Dauphin et al. 2003a, 2003b. The XANES energy scan around the sulphur K-edge is achieved using a fixed-exit double-crystal Si (111) monochromator located upstream from the microscope, which provides the required energy resolution. The present experiment required the X-ray microscope to be operated under vacuum to avoid the strong absorption of the sulphur emission lines by air. Scanning electron microscope (SEM) observations were carried out using a Philips XL30 equipped with a secondary electron and backscattered electron (BSE) detectors. In this technique, imaging contrast is due to the difference in BSE ratio, which depends on the atomic number of the atoms composing the substrate material. Thus, using a 30 kv acceleration voltage and a maximum detector contrast, BSE method provides very readable pictures emphasizing the distribution of organic envelopes and calcium carbonate phases. X-ray computed tomography (CT) of pearl samples was performed at the Natural History Museum, London. In this method, data on pearl layers are obtained through a three-step process: (1) The collection of a series of 2D X-ray projection images, by rotating the sample in the X-ray beam (cone-beam) by 0.1 degree increments over 360 degrees. The Metris X-Tek (Nikon Metrology) HMX ST 225 micro-ct system was used, with a 4M pixel Perkin-Elmer detector (voxel size 7.8 µm). X-rays were produced by focusing a 190 kv, 225 µa electron beam onto a molybdenum target. (2) The 2D X-ray projection images are reconstructed into a stack of serial 2D slices using the cone-beam back projection algorithms in Nikon Metrology CT-Pro software. (3) Rendering of the three dimensional structure of the sample is done by assembling the separate 2D slices into a single 3D volume using VGstudiomax 2.1 software. This software allows adjustment of the original rotation axis of the sample and acquisition of X-ray sections at any chosen orientation. 3 Results 3.1 The early mineralization patterns observed on the D+21 pearl layers Most of the nuclei collected 21 days after grafting were completely covered by the very first secretions of the mineralizing epithelium, indicating that wrapping of the nuclei was already completed after three weeks (Figs. 1a f). Many of these 20 to 30 µm thick organic coatings (Fig. 1g) exhibited colour heterogeneity: brown areas of varying colour intensity had developed over different proportions of the surface. Fragments of these early deposits could be detached from the nucleus surface (Fig. 1h). Interestingly, polarized light revealed the presence of mineral materials within these mostly organic layers. Some samples (Figs. 1i k) clearly show two distinctly different areas with respect to polarization. Rotation of the sample (Fig. 1i) shows that some mineral components are rather consistently oriented from crystallographic view point (Figs. 1j, k). Simultaneously, neighbouring areas of the same coating fragment do not show consistent extinction. There was considerable diversity in these early mineral depositions. Sometimes, rather large areas are covered by well crystallized spots (Figs. 1l q), with closely comparable morphological pattern and micro-granular structure. Contrastingly, other sectors showed a patchwork of very distinctly polarizing and irregularly distributed areas (Fig. 1r). As optical identification of Ca-carbonate polymorphs using polarizing microscope is not possible, microbeam X-ray diffraction was used to precisely identify the location of the mineral phases produced at these early developmental stages. 3.2 Localized characterization of the mineral phases by microbeam X-ray diffraction Linear series of X-ray diffraction diagrams were produced to give precise information about the diversity of the crystallization status in the mineralized areas in the 21-day-old pearl layers. Detached fragments of the nucleus cortex were fixed at the tip of a needle sample-holder and moved in front of the beam source (Figs. 2a, b). The resulting series of X-ray diffraction diagrams are obtained from interaction between the µm diameter X-ray beam and the mineralized areas of the nucleus cortex (Figs. 2c to 2i). In this example, which is fully representative of several tens of measurements, the six recorded diagrams form a series, starting with aragonite, in which diffraction of randomly-oriented microcrystals results in circular rings of small spots (Fig. 2d), then, the three successive steps in crystals of calcite (Figs. 2e g). In these calcite areas, diffracted beam spots are well grouped showing that calcite crystalline units are more consistently oriented (see also Fig. 3). The simultaneous occurrence of calcite and aragonite, produced by the mineralizing epithelium in its early secretion stages, is very surprising. The validity of this observation can be assessed by examining the microbeam X-ray diffraction diagrams in more detail. Figure 3a shows one of these diagrams, in which calcite and aragonite materials are simultaneously present in the 25 µm diameter analysis spot of the microbeam diffraction instrument. The centre of the diagram shows the area where the incident beam hits the sample. For a given X-ray wavelength, crystallized minerals present in the X-ray incident area produce a diffracted beam each time their atomic lattice plans are in the Bragg position with respect to the direction of 414 J.-P. Cuif et al.: Aquat. Living Resour. 23, (2010) Fig. 1. Very first mineral depositions 21 days after nucleus grafting. 1a f: surface of six nuclei illustrating colour heterogeneity of the coating. 1g, h: coating can be separated from nucleus surface (specifically the brown parts). Thickness between tips of the arrows is about 30 µm (1g). Detached fragments from a single nucleus, illustrating colour diversity (1h). 1i k: mineralized areas of the early coating. This fragment (1i) comprises areas with distinct polarization behaviour. The mineralized sector of the upper-left part of the field view is sensitive to rotation of the microscope stage (Figs. 1j, k: arrows): mineral units of this sector change from clear to black, whereas lower part of the sector remains apparently insensitive to rotation (see below: X-ray microbeam characterization). 1l, m: example of mineralizing areas producing patchy microcrystalline units. 1l n: rotation of a mineralized fragment observed under polarized light. Extinction of the unit (1m: arrow) during rotation from 1lto1n reveals its cristallographic unity. 1o: surface morphology. The flat round-shaped spots are clearly visible. 1p, q: part of the same area observed in natural transmitted light (1p) between cross nicols (1q). Despite extinction under polarized light, each round-shaped unit seems to be microcrystalline. 1r: example of mineral deposition with a patchwork pattern: each of these distinct mineralized areas can be characterized by microbeam X-ray diffraction. the incident X-ray beam and the used X-ray wave-length. The resulting diffracted beams diverge from the initial beam direction and hit the camera screen. Figures produced by positions of the resulting black dots provide information about two basic properties of the minerals. Distances of the spots to the diagram centre (e.g., Fig. 3a: R1, R2, R3) allow calculation of the angle between incident and diffracted beams, taking account of the distance between the sample and the screen. Compared to reference diagrams, each angle can be related to a specific lattice plan for a given mineral (see Fig. 3b). Calcite and aragonite exhibit very different status with respect to orientation of the crystalline units (Fig. 3a). Diffracted beams located at the R3 distance (calcite) are grouped into discrete spots, whereas diffracted beams at the R1 and R2 distances from the centre are oriented in different ways, forming two quite distinct circles. These two distinct diffraction patterns indicate that in the sample area submitted to incident X-ray beam (about 25 µm in diameter) calcite crystals were rather well oriented, leading to formation of small angle crescents, whereas aragonite was made of randomly oriented microcrystalline units. This differential result for calcite and aragonite confirms previous observations carried out using polarized light. For instance, contrast was noted between areas that were visually sensitive or insensitive to rotation of the sample between crossed nicols (Figs. 1j, k). X-ray diffraction showed that rotation sensitive zones are made of larger crystals, now known to be calcite, whereas apparently insensitive areas are built by randomly oriented microcrystals of aragonite. Here, owing to random orientation of micro-crystals, a rather constant proportion of these microcrystals were extinct or clear during rotation, causing apparent insensitivity to rotation. However, even in calcite areas, diffraction spots may not be so well grouped (e.g., Fig. 2g), forming more or less extended crescents. In this short series, the 2h diagram shows both calcite and aragonite diffraction spots. Keeping in mind a diameter of µm for the incident X-ray beam, we can see that it was certainly located on the limit between two distinct cry
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