Revisiting the Organic Template Model Through the Microstructural Study of Shell Development in Pinctada Margaritifera, the Polynesian Pearl Oyster

minerals

Article Revisiting the Organic Template Model through the Microstructural Study of Shell Development in Pinctada margaritifera, the Polynesian Pearl Oyster

Jean-Pierre Cuif 1,*, Yannicke Dauphin 2 ID , Gilles Luquet 3, Kadda Medjoubi 4, Andrea Somogyi 4 and Alberto Perez-Huerta 5 ID

1 CR2P Centre de Recherche sur la Paléodiversité et les Paléoenvironnements, UMR 7207, Muséum National d’Histoire Naturelle CNRS MNHN Sorbonne-Université, 75005 Paris, France 2 ISYEB Institut de Systématique, Evolution, Biodiversité, UMR 7205 CNRS MNHN Sorbonne-Université EPHE Muséum National d’Histoire Naturelle, 75005 Paris, France; [email protected] 3 BOREA Biologie des Organismes et Ecosystèmes Aquatiques, UMR 7208 CNRS MNHN Sorbonne-Université UA UCN IRD 207, Muséum National d’Histoire Naturelle, 75005 Paris, France; [email protected] 4 Nanoscopium beamline, Synchrotron Soleil, 91190 Saint Aubin, France; [email protected] (K.M.); [email protected] (A.S.) 5 Department of Geological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA; [email protected] * Correspondence: [email protected]

Received: 21 June 2018; Accepted: 21 August 2018; Published: 25 August 2018

Abstract: A top-down approach to the mineralized structures and developmental steps that can be separated in the shells of Pinctada margaritifera was carried out. Detailed characterizations show that each of the two major layers usually taken into account (the outer prismatic layer and the inner nacreous layer) is actually the result of a complex process during which the microstructural patterns were progressively established. From its early growing stages in the deeper part of the periostracal grove up to the formation of the most inner nacreous layers, this species provides a demonstrative case study illustrating the leading role of speciﬁcally secreted organic structures as determinants of the crystallographic properties of the shell-building units. Gathering data established at various observational scales ranging from morphology to the nanometer level, this study allows for a reexamination of the recent and current biomineralization models.

Keywords: biomineralization; Mollusca; shell development; biocrystallization model

1. Introduction Three decades only after the pioneering observations of shell microstructures by Bowerbank [1] and Carpenter [2,3], the paradoxical patterns of the calcareous hard parts built by many invertebrate groups were clearly apparent. Formation and structure of calcareous shells are not due to the calcification of the cells themselves; they should be interpreted as a secretion process [4]. This interpretation was not easily accepted, as illustrated by a famous one-decade long controversy with the opposing views of von Heider [5] and von Koch [6] about the origin of stony coral skeletons. Ten years later, Ogilvie [7] was still supporting a cellular calcification process (the von Heider’s view), until the publication of Bourne’s high-resolution observations [8] that definitely validated von Koch’s conclusions. The progress in characterization methods (such as polarization microscopy and X-ray diffraction) has continuously increased the evidence of the paradoxical character of the skeletal units that build calcareous shells. Although crystallization always occurs outside the cells of the mineralizing organ (mantle of the mollusks, basal epithelium of the coral polyps, etc.), the calcareous units exhibit

Minerals 2018, 8, 370; doi:10.3390/min8090370 www.mdpi.com/journal/minerals Minerals 2018, 8, 370 2 of 20 species-specific well-defined morphologies, various three-dimensional arrangements, and individual crystalline behaviors with the additional particularity that the morphology of the crystalline units never corresponds to the idiomorphic pattern of the selected carbonate polymorph. The first conceptual response to this intriguing blend of physical and biological properties relies on a parallel made between the arrangement of the crystalline units of the shells and the oriented development of crystals that may grow on the surface of some organic films, as exemplified by the Langmuir-Blodgett experiments. This process enables the production of single- or multilayer films with precise control of molecular orientations. As a result, thin organic polymers with specific structures and topologically controlled functional groups can be used as experimental frameworks, allowing the formation of layers of oriented crystalline particles when placed in appropriate chemical conditions. Mann [9] emphasized the possible transposition of this organic-inorganic process of crystallization to the living world, in which epithelial cell layers produce obviously controlled calcareous crystal-like units. However, long before the occurrence of the template model, the presence of organic molecules associated with the mineral phase was recognized not only at the surface of the calcareous structures but within their whole thickness [10]. The following decades were marked by a series of biochemical analyses, mostly focusing on proteins, due to the rapid technical improvement of analytical devices. This analytical approach resulted in models emphasizing the possible relationships between molecular chains of acidic amino-acids acting as attractive substrates for Ca-carbonate ions [11,12]. In a reciprocal view, explaining the species-specific morphologies of Ca-carbonate crystals in living systems, the molecular attraction between Ca-carbonate lattices and organic compounds was hypothesized as a potential “crystal modeler” mechanism. In this concept, specifically secreted sets of organic compounds may act as growth modifiers through their fixation onto the normally growing crystal faces, inducing the modification of crystal shape and size. Thus, the concept of a continuous relationship between organic and mineral components was substituted to the template model to explain the formation and growth of calcareous biocrystals. This hypothesis was summarized as “shaping crystals with biomolecules” [13]. A new paradigm emerged when it was shown that, at the infra-micrometer level, the microstructural units made of aragonite or calcite do not exhibit the continuous mineral lattices that were suggested by both optical microscopy and usual scanning electron microscopy (Figure1)[ 14,15]. Rare observations (e.g., [16,17]) had already shown that a granular structure of the mineral phase can be observed within some fossil samples. A series of secondary electron microscopy (SEM) views of growing surfaces and internal structures found in Pinna prisms (the reference model for biocrystals since Bowerbank [1] and Carpenter [2,3]) provided evidence of an unexpected fine structure and mode of growth, quite different from the structural patterns of a chemically produced crystal [18–21] (Figure1c,d). Figure1e presents the first atomic forces microscopy (AFM) picture obtained from a calcareous prism of the outer shell layer of Pinna nobilis (Pelecypod Mollusca), fully confirming the SEM views. The dimensions of the granular units clearly establish the discontinuous organization of the mineral phase revealed in the 1980s by surface etching of the same type of material. Additionally, AFM phase contrast imaging (Figure1f) shows the regular presence of a highly interactive layer around the grains, an interpretation supported by a previous series of pictures resulting from etching experiments in the 80s. Slight etching of the mineral phase produced a reticulate network of residual organic filaments ([14], Figure 1.6). First published for the description of the nacreous tablets of Cephalopods [20], this basically distinct and specific type of calcareous structure was extended to all biologically controlled Ca-carbonate [21]. These evidences contrast with the recently published model by De Yoreo and 13 associated physicists or chemists, in which particles forming the basic units of the crystallization process (including biominerals) exhibit a faceted morphology (mesocrystal style) at the end of their formation, whatever the diversity of their chemical trajectories ([22], Figure1). Minerals 2018, 8, 370 3 of 20 Minerals 2018, 8, x FOR PEER REVIEW 3 of 20

Figure 1. The earliest data on the reticulate structure of the calcareous biocrystals illustrated from Figure 1. The earliest data on the reticulate structure of the calcareous biocrystals illustrated from Pinna Pinna shells (Pteriomorphid Bivalve). (a) Polarization microscopy (transmitted light) of a thin shells (Pteriomorphid Bivalve). (a) Polarization microscopy (transmitted light) of a thin section in the section in the prismatic structure (outer layer of the shell). Prisms are obliquely cut with respect to prismatic structure (outer layer of the shell). Prisms are obliquely cut with respect to their growth their growth axis. (b) Surface of a fractured prismatic layer. Note the perfectly compact aspect of the axis. (b) Surface of a fractured prismatic layer. Note the perfectly compact aspect of the prismatic prismatic units consistent with polarized thin section. (c) Granular aspect of the growing surface of units consistent with polarized thin section. (c) Granular aspect of the growing surface of a prism a prism whereas lateral faces show the layered mode of growth. (d) Evidence of the granular whereas lateral faces show the layered mode of growth. (d) Evidence of the granular structure of the structure of the growth layers. (e) AFM view of the calcite prism ultrastructure. (f) Phase contrast growth layers. (e) AFM view of the calcite prism ultrastructure. (f) Phase contrast imaging of the prism imaging of the prism mineralized material. Credit pictures: (b,c) [15]; (d) [14]. mineralized material. Credit pictures: (b,c) [15]; (d) [14]. Far from secondary, this difference is essential with respect to further steps in the process of theFar formation from secondary, of crystalline this difference units at the is essential microstructural with respect level. to In further chemically steps driven in the processes, process of a the formationdirect association of crystalline of the units basic at particles the microstructural through forces level. acting In between chemically their driven lateral processes, faces allows a directfor associationthe development of the basic of mesocrystals, particles through as schematized forces acting in the between Cölfen-Antonietti their lateral model faces [23,24] allows (Figure for the developmentA1). of mesocrystals, as schematized in the Cölfen-Antonietti model [23,24] (Figure A1). Contrastingly,Contrastingly, in in thethe casecase of the the granular granular units units forming forming the themicrostructural microstructural components components of the of theshells shells (and (and other other calcareous calcareous biominerals), biominerals), the the absence absence of of faceted faceted surfaces surfaces implies implies a aspecific specific developmentaldevelopmental process process driving driving the the production production and assemblage assemblage of of these these spheroidal spheroidal particles particles to to produce the species-specific morphologies of the microstructural units. Major evidence for this produce the species-specific morphologies of the microstructural units. Major evidence for this biological control is given by the repeated observations (also exemplified below by the biological control is given by the repeated observations (also exemplified below by the development development of Pinctada margaritifera shell) that within a given species simultaneously producing of Pinctada margaritifera shell) that within a given species simultaneously producing two distinct two distinct shell layers built by morphologically different units, the basic grains are similar within shell layers built by morphologically different units, the basic grains are similar within each of these each of these microstructural units. In other words, no morphological feature of the elementary microstructuralgrains can explain units. the In morphological other words, nospecificity morphological of the skeletal feature units of the forming elementary the shell grains layers. can explain the morphologicalHere, through specificity a top-down of the approach skeletal units of shell forming microstructure the shell layers. in Pinctada margaritifera, the PolynesianHere, through pearl oyster, a top-down an attempt approach is made of shell to describe microstructure this controlled in Pinctada growth margaritifera mode throughout, the Polynesian the pearldevelopmental oyster, an attempt sequence. is made Particular to describe attention this controlledis paid to growththe presence mode and throughout potential the role developmental of organic sequence.structures Particular as it can attention be inferred is paid from to the the presence time-based and sequence potential of role events of organic that theystructures control as theit can bethree-dimensional inferred from the growth time-based of the sequence shell. of events that they control the three-dimensional growth of the shell. 2. Materials and Methods 2. Materials and Methods Micro- and nanostructures were studied using thin sections, fractures, and polished etched surfacesMicro- under and nanostructuresscanning electron were microscopy studied (SEM) using and thin atomic sections, force fractures, microscopy and (AFM). polished Electron etched surfacesmicroprobes under (energy scanning and electron wavelength microscopy dispersive (SEM) spectrometry) and atomic were force used microscopy for quantitative (AFM). elemental Electron microprobeschemical composition (energy and and wavelength distribution dispersive maps. Chemical spectrometry) distribution were used maps for were quantitative also performed elemental chemicalusing secondary composition ion andmass distribution spectrometer maps. (NanoSIMS), Chemical time distribution of flight (TOF-SIMS), maps were alsoand fluorescence performed using X,

Minerals 2018, 8, 370 4 of 20 secondary ion mass spectrometer (NanoSIMS), time of flight (TOF-SIMS), and fluorescence X, while the chemical speciation was investigated by X-ray absorption near edge structure (microXANES). Details about the origin of the samples, preparative process, and setup of the diverse used techniques are given in the relevant publications listed in References [14,15,18–21,25–27]. Some sections were fixed, decalcified, and stained using Alcian blue solution; pieces of shells were first incubated for 15 min in a fixative solution containing 50% methanol and 7% acetic acid, then carefully washed in bi-distilled water before staining for 1 h in a solution containing 0.2% Alcian blue, 3% acetic acid, and 0.05 M MgCl2 (pH 2.5). They were then incubated for 15 min in a destaining solution (3% acetic acid, 0.05 M MgCl2), quickly washed, and air-dried before transmission light observations and X ray fluorescence (XRF) analysis. The high analytical sensitivity XRF imaging was performed at the Nanoscopium beamline of SOLEIL Synchrotron (St. Aubin, France). The incident X-ray beam of 10 keV energy was focused by a Kirckpatrick-Baez mirror to a size of 0.3 × 0.3 µm2 at the sample position. The elemental distribution maps were collected in continuous scanning (FLYSCAN) mode by two Si-drift detectors (Ketek) [28]. Information on the sample morphology was obtained by scanning transmission X-ray microscopy (STXM) by using a XPAD fast pixel-detector having 120 × 560 pixels with a pixel size of 130 µm.

3. Results In contrast to the usual static description of the three distinct superimposed layers that make up Pinctada shells (periostracum, prismatic, and nacreous layers), a developmental investigation drew attention to the importance of the initial and transitional phases during which the characteristics of the microstructural units are established. This results in a different view of the shell, emphasizing the biological continuity between the activity of the mineralizing epithelium and the resulting microstructural growth steps. From the very beginning of shell formation in the deeper parts of the outer mantle grove up to the secretion of the nacreous tablets, these organic structures are key players in the determination of the structural patterns of shell components.

3.1. Origin and Growth Mode of the Flexible Shell That Predates the Prismatic Layer The scaly aspect of the Pinctada shell is due to repeated retractions of the mantle within the shell, followed by a new onward growth phase. During these back-and-forth movements, the periostracum, this organic film that ensures continuity between the animal mantle and the shell, is fractured. One part remains attached to the animal and is withdrawn within the shell, whereas the other part remains attached to the outer surface of the shell. As a result, the outer side of the shell is marked by successive traces of the repeated shell closures (Figure2b: arrows). A closer observation of these remains of the periostracum (Figure2c,d) shows that they contain distant mineralized elements (Figure2d), better visible at the growing edge of the shells, allowing the remains of the very last shell closure to be observed (Figure2e–g). Without exception, these irregularly circular elements exhibit a distinct nodule in their center (Figure2h–k). The mode of growth of these structures is very specific. The mineral phase is deposited around the central nodule as concentric growth circles (Figure3a–d), resulting in the formation of flat disks whose thickness does not exceed 3–4 µm (Figure3e). The granular structure of the mineral phase is well visible in SEM view, on both natural and fractured surfaces (Figure3d–f). The observation in transmitted polarized light clearly shows the crystallographic homogeneity of disks (Figure2k). Attention must be drawn to the thickness of approximately 3 µm of these disks with respect to the polarization color. In spite of the high refractive index of calcite, the observed grey polarizing color (primary color) is in agreement with a thickness of 3 µm (see Figure3e). More importantly, each disk behaves as a crystal-like unit in spite of its concentric growth mode, a pattern that has been previously established by a series of X-ray diffractions [25] (Figure A2). As the disks grow on the internal side of the periostracum, they are usually viewed perpendicularly to their surfaces, resulting in weak differences in grey colors, due to their slight differences in crystallographic orientations (Figure3g). Minerals 2018, 8, 370 5 of 20 Minerals 2018, 8, x FOR PEER REVIEW 5 of 20 Minerals 2018, 8, x FOR PEER REVIEW 5 of 20

Figure 2. Pinctada margaritifera: discoid mineralized structures on the internal side of the FigureFigure 2. Pinctada 2. Pinctada margaritifera: margaritifera:discoid discoid mineralized mineralized structures structures on the internal on the side internal of the side periostracum. of the periostracum. (a) Scaly aspect of the shell due to the series of back-and-forth movements of the (a)periostracum. Scaly aspect of (a the) Scaly shell aspect due to of the the series shell of due back-and-forth to the series movements of back-and-forth of the mantle. movements (b,c) Remainsof the mantle. (b,c) Remains of the periostracum on the outer surface of the shell. (d) Enlarged view of a ofmantle. the periostracum (b,c) Remains onthe of the outer periostracum surface of theon shell.the outer (d) Enlargedsurface of viewthe shell. of a periostracal(d) Enlarged remain: view of notea periostracal remain: note the presence of the polarizing disks in the enrolled membrane. (e,f)Flat theperiostracal presence of remain: the polarizing note the disks presence in the of the enrolled polarizing membrane. disks in ( e the,f) Flatenrolled growing membrane. edge of (e the,f)Flat shell growing edge of the shell showing the round shaped disks at the forefront. (g) SEM view of an showinggrowing the edge round of shapedthe shell disks showing at the the forefront. round shaped (g) SEM disks view at of the an forefront. equivalent (g area.) SEM (h view–k) Different of an equivalent area. (h–k) Different equivalent views of the periostracal disks at the growing edge of the equivalent views of the periostracal disks at the growing edge of the shell growth lamella. (h) Disks shell growth lamella. (h) Disks and central nodule viewed by SEM outer surface of the and central nodule viewed by SEM outer surface of the periostracum. (i) Optical microscopy (polarized periostracum. (i) Optical microscopy (polarized episcopy). (j) SEM view of the folded periostracum episcopy). (j) SEM view of the folded periostracum showing the different growth statuses of the disks. showing the different growth statuses of the disks. (k) Equivalent area in transmitted polarized (k) Equivalent area in transmitted polarized light. Note that the young disks (arrows) are apparently light.light. Note Note that that the the young young disks disks (arrows) (arrows) are are apparently apparently superposed superposed to to the the older older ones, ones, due due to to superposed to the older ones, due to periostracum folding. Credit pictures: (g,j) [25]. periostracum folding. Credit pictures: (g,j) [25].

Figure 3. Pinctadamargaritifera:: disksdisks developmentdevelopment andand structure.structure. ((a) Early mineralization stages of Figure 3. Pinctada margaritifera: disks development and structure. (a) Early mineralization stages the disks. Note the parallel growth striation of the periostracum. (b) Almost final growth stages. of the disks. Note the parallel growth striation of the periostracum. (b) Almost ﬁnal growth stages. (c,,d) Evidence of concentric mineralization. Note the central nodule. (e) Growth of the disks is only (c,d) Evidence of concentric mineralization. Note the central nodule. (e) Growth of the disks is only bi-directional. Up to their last stages they remain thin structures about 3µmthick. (f) Granular bi-directional. Up to their last stages they remain thin structures about 3 µm thick. (f) Granular structure of the disks. (g) Differences in polarized transmitted light between neighbor disks (similar structure of the disks. (g) Differences in polarized transmitted light between neighbor disks (similar thickness) suggest distinct crystallographic orientations. (h) A partially decayed disk shows the thickness)empty place suggest of the distinct initial crystallographicnodule. Credit pictures: orientations. (b,d) [25]. (h) A partially decayed disk shows the empty place of the initial nodule. Credit pictures: (b,d) [25].

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It shouldshould bebe noted noted that that the the central central nodules nodules are are always always visible visible in transmitted in transmitted light but,light when but, when using usingSEM, theSEM, central the nodulescentral nodules and concentric and concentric growth lines growth are visible lines are only visible when lookingonly when at the looking external at sidethe externalof the periostracum. side of the periostracum. On the internal On sides the ofinternal the disks sides (opposite of the disks to the (opposite periostracum) to the central periostracum) nodules centraland growth nodules circles and are growth generally circles not are visible. generally not visible.

3.2. From Flexible to Rigid Shell: Integration of Periostracal Disks into a Polygonal Prismatic Network At the end end of of the the transit transit within within the the outer outer mantle mantle grove, grove, the the periostracal periostracal flexible ﬂexible shell shell appears appears as aas cluster a cluster of of juxtaposed juxtaposed and and rather rather irregular irregular units units (Figures (Figures 3h3h and4 4a,b).a,b). Due Due to to the the continuous continuous stepping growth of the periostracum, the mineralized units that have been produced in the deeper part ofof thethe grovegrove and and have have grown grown during during their their transit transit arrive arrive at theat the junction junction between between the epithelialthe epithelial cells cellsof the of grove the grove and the and outer the mantle outer mantle surface surface ([25], (Figure ([25], 8 (Figure and Figure 8 and A3 Figure). There, A3). they There, are subjected they are subjectedto an upside to movementan upside movement of the periostracum, of the periostracum, leading the rawleading disk the to becomeraw disk the to new become growing the edge new growingof the shell. edge At of this the turning shell. At point this ofturning shell formation, point of shell the mineralizationformation, the ismineralization no longer ensured is no longer by the ensuredcells of the by outerthe cells mantle of the grove outer but mantle by the grove external but by cell the layer external of the cell mantle. layer of This the leadsmantle. to twoThis majorleads toconsequences two major with consequences respect to shell with structure respect to and shell formation: structure the and development formation: of thepolygonal development envelopes of polygonalof the prismatic envelopes units of and the the prismatic correlated units setting and the of a correlated new mode setting of growth. of a new mode of growth.

Figure 4. First step of the formation of the rigid shell of Pinctada. (a,b) SEM view of the periostracal Figure 4. First step of the formation of the rigid shell of Pinctada.(a,b) SEM view of the periostracal units close to the end of their transit within the outer mantle grove; each is surrounded by an units close to the end of their transit within the outer mantle grove; each is surrounded by an organic peripheral bulge formed by the mineralizing gel secreted by the mantle grove epithelium. organic peripheral bulge formed by the mineralizing gel secreted by the mantle grove epithelium. (c) Synchrotron X-ray fluorescence shows evidence of Zn concentration in the individual bulge of (c) Synchrotron X-ray fluorescence shows evidence of Zn concentration in the individual bulge of the the discs; see also Figure A4. (d) Beginning and progressive thickening (arrows 1 to 3) of the discs; see also Figure A4.(d) Beginning and progressive thickening (arrows 1 to 3) of the polygonal polygonal organic framework (Alcian Blue staining, decalcified preparation, transmitted light). Note organic framework (Alcian Blue staining, decalcified preparation, transmitted light). Note in the in the background the parallel striation due to the pulsed periostracal growth. (e) Example of background the parallel striation due to the pulsed periostracal growth. (e) Example of inclusion of inclusion of two periostracal units (note their concentric individual development).(f)Transmitted two periostracal units (note their concentric individual development). (f) Transmitted polarized light. polarized light. (g) Alcian Blue staining decalcified, transmitted light. Note the remains of the (g) Alcian Blue staining decalcified, transmitted light. Note the remains of the periostracal units inside periostracal units inside polygons. (h) SEM view of an equivalent area. The calcified surface still polygons. (h) SEM view of an equivalent area. The calcified surface still bears the remains of the bears the remains of the previous growth stage. previous growth stage.

The diversity of sizes and irregularity of disk shapes contrast with the rather regular dimensionsThe diversity of the ofprisms sizes andin the irregularity adult shells. of diskA regularization shapes contrast of withthe mean the rather sizes regularand shapes dimensions occurs whenof the theprisms calcified in the units adult of shells. the flexible A regularization periostracal of shellthe mean are included sizes and in shapes an organic occurs polygonal when the networkcalcified unitssecreted of the by flexiblethe external periostracal cell layer shell of the are mantle. included Figure in an 4 organic shows polygonalvarious images network obtained secreted by SEM,by the optical external microscopy cell layer of (transmitted the mantle. Figure natural4 shows and polarized various images lights), obtained and synchrotron by SEM, optical X-ray fluorescence,microscopy (transmitted providing natural evidence and that polarized two or lights), three and of thesynchrotron neighbor X-ray periostracal fluorescence, disks providing may be includedevidence that in a two polygonal or three of organic the neighbor framework periostracal to form disks a may newbe mineral included unit in a withpolygonal much organic more dimensionalframework to statistical form a new regularity. mineral unit with much more dimensional statistical regularity.

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3.3. From Extensive Growth to Prism Thickness Increase: The Potential Role of the Marsh and Sass Membrane The production of the polygonal organic framework not only regularizes the sizes and shapes of the shell building units but, most importantly, forms a key step in formation of the rigid shell. Firstly, the remaining spaces between the periostracal disks are sealed by the deposition of calcareous material at the periphery of the disks (Figure 5a, arrows), clearly marking the passage from flexible to rigid shell. From this point the shell is no longer flexible, allowing the shell Minerals 2018, 8, 370 7 of 20 thickening step to begin. The microstructure of the calcareous units that grow onto the regularized surface of the shell 3.3.edge From reveals Extensive that the Growth epithelial to Prism cell Thickness layer of Increase:the mantle The follows Potential a Rolemineralization of the Marsh mode and Sass quite Membrane distinct from the previous calcification process by which the periostracal disks have been produced (Figure The production of the polygonal organic framework not only regularizes the sizes and shapes of 5). Instead of an individual mode of growth typical for the periostracal disks, the mineral phase of the shell building units but, most importantly, forms a key step in formation of the rigid shell. these units is synchronically deposited within the newly produced polygonal framework (Figure Firstly, the remaining spaces between the periostracal disks are sealed by the deposition of 5b). Superposition of micrometer-thick growth layers ensures the inward growth of these units, calcareousincreasing materialthe thickness at the of periphery the shell.of As the a result, disks (Figurethese calcified5a, arrows), units clearly must markingbe called the prisms passage from from this ﬂexiblepoint owing to rigid to shell. their From growth this mode, point the although shell is noat longerthis stage ﬂexible, their allowing diameter the largely shell thickening exceeds their step tothickness. begin.

Figure 5. The newly formed prisms and the Marsh and Sass membrane on the internal side of the Figure 5. The newly formed prisms and the Marsh and Sass membrane on the internal side of the shell of Pinctada. (a) Sealing of the remaining spaces between disks. (b) Two neighbor prisms shell of Pinctada.(a) Sealing of the remaining spaces between disks. (b) Two neighbor prisms starting starting their inward growth by synchronic mineral deposition. On their outer surfaces, early decay their inward growth by synchronic mineral deposition. On their outer surfaces, early decay of the of the periostracum reveals the positions of the initial nodules. Note the Marsh membrane on the periostracum reveals the positions of the initial nodules. Note the Marsh membrane on the internal internal side of the prisms. (c,d) Enlarged views of some prisms showing their internal side covered side of the prisms. (c,d) Enlarged views of some prisms showing their internal side covered by the by the Marsh membrane. (e) Close adherence of the Marsh membrane to the mineralized material of Marsh membrane. (e) Close adherence of the Marsh membrane to the mineralized material of the the prism. Note the parallel striation of the outer side of the Marsh membrane (arrows). (f) Granular prism. Note the parallel striation of the outer side of the Marsh membrane (arrows). (f) Granular morphology of the Marsh membrane. morphology of the Marsh membrane.

A close observation of the basis of these newly formed prisms reveals the presence of a common membraneThe microstructure covering the ofwhole the calcareous internal surface units that of the grow growing onto the structure regularized (Figure surface 5b–e, of thearrows). shell edgeThis revealsmembrane that is the in epithelialclose contact cell layerto the of mineral the mantle phase follows of the a prism. mineralization The limits mode of the quite growing distinct prisms from theare previousvisible on calcification the external process surface by of whichthe membrane the periostracal (Figure disks5c,d, haveblue arrows). been produced A striking (Figure parallel5). Instead can be ofmade an individual between these mode two of growth figures typical and the for observation the periostracal of the disks, “lamella” the mineral made phase by Bevelander of these units and is synchronicallyNakahara [29] (Figure deposited A6). within the newly produced polygonal framework (Figure5b). Superposition of micrometer-thickTwo additional growthpatterns layers are also ensures visible: the a inwardparallel growth striation of of these the membrane units, increasing (Figure the 5e: thickness arrows) ofand the the shell. granular As a result, organization these calcified of the membrane units must clearly be called visible prisms from from the this SEM point observations. owing to their This growthgranular mode, pattern although was already at this emphasized stage their diameterby Marsh largely and Sass exceeds in their their pioneering thickness. paper [30]. A close observation of the basis of these newly formed prisms reveals the presence of a common membrane covering the whole internal surface of the growing structure (Figure5b–e, arrows). This membrane is in close contact to the mineral phase of the prism. The limits of the growing prisms are visible on the external surface of the membrane (Figure5c,d, blue arrows). A striking parallel can be made between these two figures and the observation of the “lamella” made by Bevelander and Nakahara [29] (Figure A6). Two additional patterns are also visible: a parallel striation of the membrane (Figure5e: arrows) and the granular organization of the membrane clearly visible from the SEM observations. This granular pattern was already emphasized by Marsh and Sass in their pioneering paper [30]. Minerals 2018, 8, 370 8 of 20 Minerals 2018, 8, x FOR PEER REVIEW 8 of 20

It isIt important is important to note to note that, that, owing owing to its to position its position at the basisat the of basis the growing of the growing prisms, this prisms, membrane this formsmembrane a permanent forms intermediatea permanent intermediate structure between structure the between outer surface the outer of the surface mineralizing of the mineralizing epithelium of theepithelium mantle and of thethe mineralmantle and phase the ofmineral the prisms. phase of the prisms.

3.4.3.4. An An Overall Overall View View of of the the Layered Layered Growth Growth ofof thethe PrismsPrisms from Micrometer Micrometer to to Nanometer Nanometer Range Range FigureFigure6a, 6a, obtained obtained by by polarization polarization microscopy microscopy (transmitted(transmitted light), light), provides provides us us with with colorful colorful evidenceevidence of of what what occurs occurs at atthe the shellshell growinggrowing edge during the the early early stages stages of of the the prismatic prismatic layer layer formation.formation. At At the the upper upper part part of the of view, the view, the front the front line of line the of prismatic the prismatic units exhibits units exhibits slightly slightly different different grey colors, corresponding to a thickness of 3–4 µm of the calcite units, with distinct grey colors, corresponding to a thickness of 3–4 µm of the calcite units, with distinct crystallographic crystallographic orientations. The SEM view of the same area (Figure 6b,c) reveals their early orientations. The SEM view of the same area (Figure6b,c) reveals their early developmental steps. developmental steps. The first occurrence of the Marsh membrane is visible on the inner side of the The ﬁrst occurrence of the Marsh membrane is visible on the inner side of the polygonal units, whereas polygonal units, whereas on its outer side the regularly spaced lines illustrate the pulsed growth on its outer side the regularly spaced lines illustrate the pulsed growth mode of the periostracum. mode of the periostracum.

Figure 6. Layered growth of the prisms and morphology of the elementary mineralized units of Figure 6. Layered growth of the prisms and morphology of the elementary mineralized units of Pinctada. (a) Transmitted polarized light on a large area of the shell growing edge. Thickness Pinctada.(a) Transmitted polarized light on a large area of the shell growing edge. Thickness increase increase is assessed by the changing colors from the upper to lower part of the picture. Note the is assessed by the changing colors from the upper to lower part of the picture. Note the statistical statistical dimensional regularity of the prism sections. (b,c) Inner (b) and outer (c) sides of the dimensionalprisms at the regularity shell growing of the prism edge. sections. Note on (b the,c) outer Inner side (b) and the outer parallel (c) stripessides of of the the prisms periostracum, at the shell growingindicating edge. the Note pulsed on secretion the outer of side the thespecialized parallel stripescells in ofthe the deeper periostracum, part of the indicating outer grove the of pulsed the secretionmantle. of (d the) Internal specialized surface cells of inthe the shell deeper prismatic part of layer; the outer displaced grove Marsh of the membrane mantle. (d )(blue Internal arrows). surface of( thee) Slightly shell prismatic oblique surface layer; displacedin the prismatic Marsh layer membrane showing (blue the synchronic arrows). ( egrowth) Slightly of the oblique envelopes surface in(laser the prismatic confocal layermicroscopy). showing (f the) Layered synchronic growth growth mode ofof thethe envelopesmineralized (laser phase. confocal (g) Simultaneous microscopy). (f)growth Layered of growth envelopes mode and of mineral the mineralized layers. (h phase.) Discontinuous (g) Simultaneous structure growth of the ofmineral envelopes material and (SEM mineral layers.view). ( h ()i– Discontinuousk) AFM views structure of the mineralized of the mineral material. material (j) Amplitude (SEM view). image. (i– k ()k AFM) Phase views contrast of the mineralizedimaging. Credit material. pictures: (j) Amplitude (b,c,j,k) [21]. image. (k) Phase contrast imaging. Credit pictures: (b,c,j,k) [21].

AtAt the the shell shell growing growing edge,edge, the growth growth of of the the prisms prisms is isillustrated illustrated by the by therapid rapid color color change change of polarized light, passing from grey to the upper primary colors. Note the persistency of color of polarized light, passing from grey to the upper primary colors. Note the persistency of color diversity among prisms of the same ages (same distance from shell edge), suggesting that distinct diversity among prisms of the same ages (same distance from shell edge), suggesting that distinct crystallographic orientations visible among newly formed prisms are preserved during prism axial growth. Minerals 2018, 8, x FOR PEER REVIEW 9 of 20 crystallographicMinerals 2018, 8, 370 orientations visible among newly formed prisms are preserved during prism 9axial of 20 growth. For prisms about 100 µm in length, the organic envelopes and the Marsh membrane (blue arrows)For prisms are clearly about visible 100 µm onin length, the internal the organic surface envelopes of the and shell the Marsh (Figure membrane 6d). Note (blue the arrows) slow morphologicalare clearly visible changes on the internal in prism surface morphology, of the shell also (Figure visible6d). in Note Figure the slow 6e. Enlarged morphological views changes of the mineralizedin prism morphology, material also confirm visible the in Figure layered6e. Enlarged mode of views mineralization of the mineralized assessed material by conﬁrmthe perfect the synchronismlayered mode of of mineral mineralization singularities assessed between by the neighbor perfect synchronism prisms (Figure of mineral6f). As a singularities rule, envelopes between and mineralizedneighbor prisms material (Figure grow6f). Aswith a rule, the envelopessame layered and mineralizedmode as shown material by the grow similar with the growth same layeredrhythm visiblemode asin shownthe slightly by the decalcified similar growth fractured rhythm surface visible (Figure in the6g). slightlyThe discontinuity decalciﬁed of fractured the mineralized surface (Figurematerial6 g).is visible The discontinuity in the SEM ofpictures, the mineralized but the true material morphology is visible of in mineral the SEM particles pictures, can but be the clearly true illustratedmorphology by of AFM mineral pictures particles only can(Figure be clearly 6i–k). illustrated by AFM pictures only (Figure6i–k). Attention must must be be drawn drawn to to the the complete complete absence absence of of crystal crystal faces faces on on these these basic basic components components of theof the prisms. prisms. Phase Phase contrast contrast imaging imaging (Figure (Figure 6k)6k) emphasizes emphasizes the the distinct distinct properties of thethe materialmaterial located between the spheroidal units.

3.5. Time-Based Change of Prism Internal Structure Prisms of of PinctadaPinctada margaritifera margaritifera exhibitexhibit two two distinct distinct and and successive successive structural structural status status (Figure (Figure 7).7 In). µ theirIn their early early stages, stages, up up to to a alength length of of about about 120 120 to to 150 150 µm,m, they they grow grow as single-crystal-like units µ (calcite) by the superposition of about 2–3 µmm thick growth layers that also comprise the formation of the polygonal organic network (prism envelopes) that grows accordingly. Figure 77bb showsshows thethe successive growthgrowth layerslayers after after acidic acidic etching. etching. Longitudinal Longitudinal (Figure (Figure7c) and7c) and transversal transversal (Figure (Figure7d) thin 7d) thinsections sections of these of these young young prisms prisms indicate indicate a monocrystalline a monocrystalline behavior. behavior.

Figure 7. Shift from single-crystal-like units to polycrystalline organization of the calcite prisms in Figure 7. Shift from single-crystal-like units to polycrystalline organization of the calcite prisms Pinctada margaritifera. (a) The shell stepping growth at the macro-scale; three scales of various in Pinctada margaritifera. (a) The shell stepping growth at the macro-scale; three scales of various thicknesses due to three mantle withdraws. (b) The stepping growth at the microscale seen by the thicknesses due to three mantle withdraws. (b) The stepping growth at the microscale seen by the synchronic growth of the envelopes and the mineral phase of the prisms. (c,d) Overall similarity in synchronic growth of the envelopes and the mineral phase of the prisms. (c,d) Overall similarity in crystallographic orientation during early elongation of the prisms (about 200 µm). (e,f) The surface crystallographic orientation during early elongation of the prisms (about 200 µm). (e,f) The surface fracture of the shell reveals the morphological changes in prisms. (g) Diversity of crystal fracture of the shell reveals the morphological changes in prisms. (g) Diversity of crystal orientations orientations in the second growth phase of the prisms. in the second growth phase of the prisms.

Figure 7e shows the second stage, characterized by the presence of several crystalline units withinFigure a single7e shows envelope. the second This stage,microstructural characterized change by the is presenceclearly visible of several by crystallinesimple examination units within of prisma single morphologies envelope. This on a microstructuralfractured shell. Instead change of is flat clearly prism visible surfaces by simplein contact examination with each ofother prism by regularmorphologies angles on(Figure a fractured 7f, upper shell. part), Instead in this of stage ﬂat prism the surfaces surfaces of in the contact prisms with are each marked other by by irregular regular nodulesangles (Figure (Figure7f, 7f, upper lower part), part). in thisThin stage sections the surfaces made in of such the prisms areas areand marked observed by in irregular polarized nodules light (Figurereveal 7 thatf, lower multiple part). crystalline Thin sections orientations made in such can areas be found and observed within in a polarized given prism light (Figure reveal that 7g), multiple crystalline orientations can be found within a given prism (Figure7g), contrasting with the single-crystal appearance of the thin sections made in the upper part of the same prism (Figure7d).

Minerals 2018, 8, x FOR PEER REVIEW 10 of 20 contrasting with the single-crystal appearance of the thin sections made in the upper part of the Minerals 2018, 8, 370 10 of 20 same prism (Figure 7d). Also remarkable is the synchronism of the passage from single to polycrystalline status within a singleAlso shell remarkable scaly unit. is Figure the synchronism 7f shows the of theperfectly passage regular from singlegrowth to striation polycrystalline parallel status to the within shell surfacea single visible shell scaly on the unit. upper Figure part7 off shows four neighbor the perfectly prisms regular and the growth simultaneity striation of parallel the microstructural to the shell changesurface leading visible on to thethe upperproduction part of of four polycrystalline neighbor prisms units. and the simultaneity of the microstructural change leading to the production of polycrystalline units. 3.6. Sequence of Microstructural Events in the Formation of the Nacreous Layer 3.6. Sequence of Microstructural Events in the Formation of the Nacreous Layer In contrast to a widely shared view, the spreading of the nacreous layer onto the prismatic internalIn contrast surface tois not a widely the cause shared of view,the end the of spreading prism growth. of the Various nacreous converging layer onto thedata prismatic suggest internalthat the endsurface of prism is not growth the cause is ofalso the a endtime-based of prism process. growth. An Various SEM view converging of the upper data suggest surface that of the the prisms end of prismshows growtha regression is also of a time-basedthe mineralizing process. area An before SEM viewthe deposition of the upper of surfacearagonite of the(Figure prisms 8a,b). shows This a correlatesregression of with the mineralizing biochemical area changes before the in deposition the prism of mineralizing aragonite (Figure matrices8a,b). This (Figure correlates 8c) with [26]. Simultaneously,biochemical changes biochemical in the prism changes mineralizing occur in the matrices composition (Figure 8ofc) the [ 26 prism]. Simultaneously, envelope, leading biochemical to the appearancechanges occur of inholes the composition(Figure 8d). of the prism envelope, leading to the appearance of holes (Figure8d).

Figure 8. Pinctada: structural and biochemical changes during the major transitional phase in shell Figure 8. Pinctada: structural and biochemical changes during the major transitional phase in shell formation: occurrence of aragonite in a pre-nacreous phase. (a,b) Regression of the mineralizing formation: occurrence of aragonite in a pre-nacreous phase. (a,b) Regression of the mineralizing membranes at the top of the prisms in the terminal growth phase. (c) Biochemical changes in the membranes at the top of the prisms in the terminal growth phase. (c) Biochemical changes in the composition of the mineralizing layers (XANES mapping). (d) Holes in the prism envelopes (UV composition of the mineralizing layers (XANES mapping). (d) Holes in the prism envelopes (UV fluorescence). (e) Patchy aspect and specific composition of the organic (glycine) deposition onto the fluorescence). (e) Patchy aspect and specific composition of the organic (glycine) deposition onto final surface of the prisms (TOF-SIMS mapping); note the difference from the prism envelopes. (f) the final surface of the prisms (TOF-SIMS mapping); note the difference from the prism envelopes. Granular structure of early aragonite deposition on the organic membrane covering the prisms. (f) Granular structure of early aragonite deposition on the organic membrane covering the prisms. (g,h) Spherulitic crystallization of aragonite on the initial aragonite deposition. (i,j) Progressive (g,h) Spherulitic crystallization of aragonite on the initial aragonite deposition. (i,j) Progressive occurrence of nacreous membranes viewed in SEM backscattered mode and (i) NanoSIMS (j). Credit occurrence of nacreous membranes viewed in SEM backscattered mode and (i) NanoSIMS (j). pictures: (c,j)[26], (e) [27]. Credit pictures: (c,j) [26], (e) [27].

The biochemical specificity of the organic deposition that covers the upper surface of the prismaticThe biochemical layer when specificity the growth of the of organic this layer deposition is finished that covers has the long upper been surface ignored. of the TOF-SIMS prismatic investigationslayer when the have growth revealed of this layer its patchy is finished deposition has long and been locally ignored. variable TOF-SIMS composition investigations (Figure have 8e: arrows)revealed [27]. its patchy deposition and locally variable composition (Figure8e: arrows) [27]. In this transitionaltransitional area,area, thethe occurrenceoccurrence of of such such a a specific specific organic organic deposition deposition is is far far from from neutral neutral as asit provides it provides a substrate a substrate for the for primary the primary deposition deposition of aragonite. of aragonite. Noticeably, Noticeably, aragonite aragonite is not deposited is not depositedimmediately immediately as nacre (i.e., as nacre mineral (i.e., tablets mineral surrounded tablets surrounded by organic by envelopes). organic envelopes). Rather, the Rather, very early the veryaragonite early deposition aragonite consists deposition of circular consists patches of circular of very patchessmall granules of very (Figure small8 f) granules followed (Figure by fibrous 8f) followeddiverging by fascicles fibrous or diverging spherulites fascicles (Figure or8 g,h).spherulites (Figure 8g,h).

Minerals 2018, 8, 370 11 of 20 Minerals 2018, 8, x FOR PEER REVIEW 11 of 20

The following step towards nacre formation is the occurrence of parallel organic membranes. µ Initially discontinuous,discontinuous, these these parallel parallel membranes membranes (with (with a distancea distance between between them them of about of about 1 m 1 orµm less) or becomeless) become rapidly rapidly continuous continuous (Figure (Figure8i). These 8i). membranes These membranes are essentially are essentially protein-made, protein-made, as shown by as NanoSIMSshown by mapping NanoSIMS (Figure mapping8j). Although (Figure it 8j). resembles Although a nacre it resembles organization, a nacre this microstructure organization, still this differsmicrostructure from true still nacre differs as careful from true observation nacre as ofcareful the interval observation between of the the interval organic between membranes the organic reveals thatmembranes the short reveals mineral that units the between short mineral the membranes units between maintain the membranes the orientation maintain of the the underlying orientation ﬁbers of (Figurethe underlying9a,b). fibers (Figure 9a,b).

Figure 9. The two-phase process of nacre formation: from pre-nacre to nacreous tablets and overall Figure 9. The two-phase process of nacre formation: from pre-nacre to nacreous tablets and overall crystallographic orientation of the nacreous layer of Pinctada. (a,b) Persistency of fibrous radial crystallographic orientation of the nacreous layer of Pinctada.(a,b) Persistency of ﬁbrous radial orientation between the parallel organic membranes. (c,d) Nacreous tablet growth and (e) Alcian orientation between the parallel organic membranes. (c,d) Nacreous tablet growth and (e) Alcian blue stainingblue staining of their of initial their initialnodules. nodules. (f) Regular (f) Regular spiral made spiral by made nacreous by nacreous tablets and tablets (g) most and common (g) most irregularcommon overall irregular arrangement. overall arrangement. (h,i) View of (h an,i) inner View surface of an of innerPinctada surfaceshell of showing Pinctada crystallographic shell showing orientationscrystallographic of the orientations nacreous tablets of the linked nacreous to the tablets growth linked direction to the (yellow growth arrows: directiona, blue:(yellowb, red arrows: dots: ca,perpendicular blue: b, red dots: to the c shellperpendicular surface).( jto)Reticulate the shell crystallizationsurface). (j) Reticulate of the grains crystallization necessarily of controlled the grains at thenecessarily overall shellcontrolled level. Creditat the overall pictures: shell (c,d,f) level. [21 Credit]. pictures: (c,d,f) [21].

True nacre is completely different in origin. It occurs when small organic nodules are deposited True nacre is completely different in origin. It occurs when small organic nodules are deposited onto the organic membranes and act as centers of calcium carbonate deposition. The protein and onto the organic membranes and act as centers of calcium carbonate deposition. The protein and sulfated proteoglycan composition of these nodules was shown by Nudelman et al. [31]. This sulfated proteoglycan composition of these nodules was shown by Nudelman et al. [31]. This implies implies the formation of a new specific generative area in the mantle epithelium producing flat the formation of a new specific generative area in the mantle epithelium producing flat crystals crystals expending up to enter in mutual contact (Figure 9e,g). It must be noted that the growth of expending up to enter in mutual contact (Figure9e,g). It must be noted that the growth of nacreous nacreous tablets is an oblique stepping process with respect to the direction of the nacreous layers. tablets is an oblique stepping process with respect to the direction of the nacreous layers. The initial The initial nodules are linearly deposited on the distal area of the nacreous layer, creating a series of nodules are linearly deposited on the distal area of the nacreous layer, creating a series of superposed superposed layers that simultaneously spread onward and cover the fibrous aragonite areas. Finally, layers that simultaneously spread onward and cover the fibrous aragonite areas. Finally, the complex the complex aragonitic structure covers the internal surface of the calcite prismatic units. aragonitic structure covers the internal surface of the calcite prismatic units. Evidence of the overall control of this crystallization process has long been remarked [32]. Evidence of the overall control of this crystallization process has long been remarked [32]. Pinctada margaritifera produces rather circular shells. Using X-ray diffraction, Wada was able to show Pinctada margaritifera produces rather circular shells. Using X-ray diffraction, Wada was able to that the crystallographic orientation of nacreous tablets within a given shell is by no means a show that the crystallographic orientation of nacreous tablets within a given shell is by no means randomly distributed property. At the growing edge of the nacreous area, the crystallographic a randomly distributed property. At the growing edge of the nacreous area, the crystallographic orientation of the nacreous tablets crystals is arranged in such way that the c axis is statistically orientation of the nacreous tablets crystals is arranged in such way that the c axis is statistically perpendicular to the shell surface, the a and b axes being respectively parallel and perpendicular to perpendicular to the shell surface, the a and b axes being respectively parallel and perpendicular to the the shell edge. In more internal areas, tablet groups in which the crystallographic orientations are shell edge. In more internal areas, tablet groups in which the crystallographic orientations are rather rather similar are produced, whatever the global figure of the geometrical distribution may be. similar are produced, whatever the global figure of the geometrical distribution may be. 4. Discussion: Ancient and Current Models in Light of Recent Microstructural Data Starting with the initial deposition of organic nodules in the deeper part of the outer mantle grove and ending with the remarkably controlled arrangement of the nacreous tablets in the most

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4. Discussion: Ancient and Current Models in Light of Recent Microstructural Data

MineralsStarting 2018, 8, x with FOR thePEER initial REVIEW deposition of organic nodules in the deeper part of the outer mantle 12 of 20 grove and ending with the remarkably controlled arrangement of the nacreous tablets in the most internal areas, areas, this this overview overview of Pinctada of Pinctadashell shell development development can be can summarized be summarized in Figure in 10 . Figure Attention 10. isAttention drawn to is thedrawn series to ofthe metabolic series of eventsmetabolic that events lead to that the lead formation to the offormation this shell, of which this shell, is the which typical is representativethe typical representative of the “prismatic/nacreous” of the “prismatic/nacreous” model, frequently model, used frequently as a general used as model a general for calciﬁcation model for incalcification Bivalves. in Bivalves.

Figure 10.Sequence of the organic structures acting during Pinctada shell development. Figure 10. Sequence of the organic structures acting during Pinctada shell development. Supported by the series of structural data from Section 3, this scheme allows for a reexaminationSupported of by the the literature series of structural current models data from (usually Section taking3, this schemeinto account allows a for limited a reexamination part of the ofdevelopmental the literature sequence current modelsonly), then (usually a comparison taking into of accountthe origin a limitedand growth part mode of the of developmental the prismatic sequenceand nacreous only), layers, then a apparently comparison very of the distinct origin under and growthmany respects, mode of but the showing prismatic also and surprising nacreous layers,similarities apparently when considering very distinct the under whole many developmental respects, but history. showing also surprising similarities when considering the whole developmental history. 4.1. Recent Views about Shell Growing Edge 4.1. Recent Views about Shell Growing Edge Although less thoroughly investigated than the nacreous layer, the growing edge of the shell Although less thoroughly investigated than the nacreous layer, the growing edge of the shell has has gained attention since the work of Wada ([32], Figure 8, p. 780), who presented a realistic gained attention since the work of Wada ([32], Figure8, p. 780), who presented a realistic approach approach of shell formation, observing a specific area predating the typical prismatic layer in of shell formation, observing a specific area predating the typical prismatic layer in Pinctada fucata. Pinctada fucata. Wilbur ([33], p. 161), using Wada’s data, then Taylor and Kennedy [34] considered Wilbur ([33], p. 161), using Wada’s data, then Taylor and Kennedy [34] considered these structures to these structures to be the result of a spherulitic crystallization process simultaneously producing be the result of a spherulitic crystallization process simultaneously producing the prismatic mineral the prismatic mineral structure and the organic envelopes through a single-phase mechanism. structure and the organic envelopes through a single-phase mechanism. Admitting that prisms become Admitting that prisms become polygonal by mutual contact, these authors considered that the polygonal by mutual contact, these authors considered that the growing mineral units produce the growing mineral units produce the prism envelopes by “squeezing” the remaining organic prism envelopes by “squeezing” the remaining organic components “as impurities to the edges of the components “as impurities to the edges of the growing crystals”. growing crystals”. In a more recent investigation dealing with Pinctada fucata (the Japanese pearl oyster), Suzuki et In a more recent investigation dealing with Pinctada fucata (the Japanese pearl oyster), al. [35] were able to characterize calcite in a thin section of disk still in place on the internal side of Suzuki et al. [35] were able to characterize calcite in a thin section of disk still in place on the the periostracum using transmission electron microscopy and selected area electron diffraction internal side of the periostracum using transmission electron microscopy and selected area electron (SAED. diffraction (SAED). The concentric crystallization described in the present paper modifies Wada’s scheme. The concentric crystallization described in the present paper modifies Wada’s scheme. Undoubtedly, every growing unit appears as a crystal-like unit but it does not develop by spherulitic Undoubtedly, every growing unit appears as a crystal-like unit but it does not develop by spherulitic crystallization. Starting from an organic nodule and growing by the addition of concentric rings, the crystallization. Starting from an organic nodule and growing by the addition of concentric rings, development of the periostracal disks appears as a remarkable example of organic/inorganic the development of the periostracal disks appears as a remarkable example of organic/inorganic relationships, i.e., a first illustration of the organic template model in shell development. To date no relationships, i.e., a first illustration of the organic template model in shell development. To date no data exists regarding molecular composition and the structure of the initial nodules that ensure the data exists regarding molecular composition and the structure of the initial nodules that ensure the initial crystallization of the first calcite ring. initial crystallization of the first calcite ring. Crystallization of the disks by accretion of concentric rings contrasts with their single-crystal behavior. Note that if the disk crystallization followed the classical ionic-type process, these individually distant and freely growing disks would exhibit visible traces of the typical calcite regular faces. It must be emphasized that this never occurs, providing a demonstrative example of the biological effect exerted on the crystallization process, preventing the formation of faceted crystals at the supra-micrometric dimensional scale.

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Crystallization of the disks by accretion of concentric rings contrasts with their single-crystal behavior. Note that if the disk crystallization followed the classical ionic-type process, these individually distant and freely growing disks would exhibit visible traces of the typical calcite regular faces. It must be emphasized that this never occurs, providing a demonstrative example of the biological effect exerted on the crystallization process, preventing the formation of faceted crystals at the supra-micrometric dimensional scale. In the mesocrystal model of crystallization, the mutual attachment of faceted particles leads to faceted massive crystals (Figure A1). Distant from each other during their growth and consequently growing without mutual interaction, every disk should grow as a faceted crystal if the mesocrystal growth mode was followed during disk growth. Contrastingly, no trace of crystalline surface is visible in the disks, whatever the observational level, clearly establishing that attachment by lateral sides, as suggested in the De Yoreo et al. model [22], is not the mechanism at work in the formation of the disks. Figure4c shows that the periphery of every growing disk is marked by an important bulge whose particular chemical composition is illustrated in this paper by a single example: the concentration of zinc detected by synchrotron X-ray ﬂuorescence mapping (Figures4c and A4). Conclusively, the growth of periostracal disks provides an accessible case study of a species-speciﬁc biochemical matrix in which biologically controlled crystallization occurs. The importance of data regarding the composition of the organic medium in which the disks are growing cannot be overestimated. Once started, the accretion of calcite rings increases the disk diameter and note must be made of the probable concomitance between the pulse growth of the disks and the stepping growth mode of periostracum acting as a conveyor-belt for these growing units. To date, no evidence has been found of a correlation between the pulse growth mode of the periostracum (assessed by parallel striation visible on the whole shell (see Figures2h,3c,d,4d and6c) and the stepping increases of disk diameters (Figure3c,d). However, further observations on the growth mode of prisms and nacreous tablets in Pinctada shell contribute to such an interpretation.

4.2. Formation and Structural Evolution of the Prisms Among the data gathered in Figure4, Figure4d offers a clear view of the second step in shell development. Viewed from the internal side of the periostracum (whose stepping growth striation is still visible in the background), this decalcified preparation clearly shows the occurrence of the polygonal organic framework that appears at the turning point of the periostracum (Figure A3). These polygons frequently gather two or three small irregular disks produced during the previous growth step. Figure4h exemplifies the solid counterpart of Figure4d; the calcareous surface still bears the peripheral remains of the periostracal calcification phase. Both pictures point to the fact that the formation of prismatic envelopes is not simply due to the mutual contact of early calcifying units, as hypothesized by researchers long ago. Polygonal organic frameworks are specifically produced at the growing edge of the shell to increase its lateral dimensions. The transfer of crystallization from the disks to the prisms is assessed by the repeated observation of the mean-size regulation of microstructural units at the very beginning of the prismatic layer. The formation of prisms by the inclusion of complex predating structures disproves the widely shared concept of a “self-assembly” model based on freely growing molecules moving in a fluid-filled extra-pallial space (exemplified in Figure A5). From Saleuddin and Petit [36] to Calvo-Iglesias [37], this concept avoids any microstructural investigation, the users being confident in the capability of the molecules to reach the right place at the appropriate time.

4.3. The Possible Function of the Marsh Membrane Following the Bevelander/Nakahara Model The elongation of the prisms through repeated and synchronic adjunctions of envelopes and mineralized material is an additional evidence of the pulsed biomineralization mechanism at the global scale in Pinctada shell growth. Additionally, the rigorously synchronized deposition of these growth layers points to the key-role of the Marsh membrane in the crystallization process of the prisms. Minerals 2018, 8, 370 14 of 20

It has long been noted that prisms maintain their individual crystallographic orientation during their axial growth. This is particularly obvious in Pinna prisms (the reference for the prismatic microstructures) as well as true for Pinctada prisms during their early developmental phase (Figure7). This remarkable property contrasts with the repeated back-and-forth movement of the mantle epithelium that provides the chemical and biochemical components ensuring prism growth. As it is quite improbable that such crystallographic continuity of the prisms could rely on an exact repositioning of the mantle epithelium after each withdrawing phase, this could be the role of the Marsh membrane. The point is that if the mantle epithelium withdraws at every shell closure, the Marsh membrane does not participate in this mantle retraction as it is closely adhered to the growing internal faces of the prisms. When the shell reopens and mineralizing activity restarts, it can be reasonably hypothesized that the Marsh membrane plays a key role as an intermediate active agent by ensuring the continuity of each prism crystallization. This provides an additional evidence that the microstructure of the prisms cannot rely on the properties of their nanometer-sized structural units (the spheroidal grains) and some chemical attractive forces between their lateral faces that simply do not exist (Figure6j,k). In contrast, the activity of the Marsh membrane in controlling the crystalline orientation of every prism suggests that the concept of an organic template should be adapted to the complexity of the molecular environments in which the layered crystallization of prisms occurs.

4.4. From Simple to Polycrystalline Prisms: A Counter Argument to the Concept of “Crystal-Growth Competition” The key role of the Marsh membrane can be inferred from the structural changes that occur in the growth of Pinctada prisms: the passage from single-crystal to polycrystalline status. This seems to be a rare phenomenon, possibly specific to Pinctada (see also [38]). The patterns illustrated in Figure7 clearly show the intriguing crystallographic changes that synchronically occur in the prisms after a given period of growth as a single crystal. Crystal-growth competition is a widely shared concept based on the argument that the growth of a given crystal is easier if its major growth axis (e.g., the c axis for calcite) is oriented in conformity with the geometrical organization of the shell (i.e., perpendicular to the shell surface). This concept obviously leads to a reduction of the number of prisms. What occurs in the prismatic layer of a Pinctada margaritifera does not support such an interpretation. After a “single-crystal” period of growth, the prisms become polycrystalline. It is difficult to understand this phenomenon in the concept of a moving mineralizing epithelium that simultaneously produces prisms with a single-crystal structure and others with different organization. A time-based modification of the biochemical activity of the Marsh membrane resulting in the production of polycrystal prisms (instead of single) could be the origin of this distinct crystallization pattern. Actually, what occurs at the end of the prismatic area clearly supports the interpretation that aging of the Marsh membrane modifies its activity and has a detectable influence on prism growth. It is a commonly shared view that the growth of the calcite prisms is interrupted by the onward spreading of the nacreous layer. Actually, converging data have shown that the end of prism biomineralization predates coverage of the prism layer surface [25]. Reduction of the Marsh membrane activity is assessed by the reduction of the prism mineralizing surfaces (Figure8a,b) with detectable modification of the composition of the mineralizing matrices (Figure8c) of envelope composition (Figure8d). Formation of nacre does not prevent prism growth because end of the biochemical and crystallization activity of the prismatic area opens the way to the major microstructural change that subsequently occurs in shell formation.

4.5. From Prisms to Nacre: The Increasing Control Exerted on Aragonite Mineralization It has long been noted that calcite and aragonite compartments of Pinctada shells are carefully separated by an organic membrane. Until the biochemical characterization of this membrane by Minerals 2018, 8, 370 15 of 20

TOF-SIMS mapping (Figure8e), it was considered as a simple expansion of the prism envelopes, carefully closing the calcite compartment before nacre deposition. Figure8e leads to a completely different interpretation. Instead of a simple separative role, the biochemical composition of this membrane suggests that it could be an active factor in occurrence of the first aragonite deposition. Figure8f–h provides evidence of aragonite localized precipitation onto this membrane, whose irregular composition corresponds to the patchy deposition of aragonite. Figure8f shows the granular structure of the very initial aragonite. There is no indication of a more precise biological control at this stage. Further steps consist in the repeated production of layered aragonite that takes a fibrous habitus, close to what can be seen in sedimentary precipitations. With respect to biomineralization models, there is little doubt that the prism covering membrane acts as a mineralization substrate, though no organizational factor is present at that time to drive the aragonite grains up to the formation of morphologically specific units. Even the occurrence of parallel membranes (mostly protein, Figure8i,j) does not influence aragonite deposition; Figure9a,b reveals that between the parallel membranes the direction of the diverging fibers is preserved. The phase of fibrous aragonite mineralization ends with the occurrence of small organic nodules, whose composition is now established [27]. With respect to mineralization models, the deposition of these nodules strikingly resembles the occurrence of the central nodules in the outer mantle grove (S3: oc), around which the calcite disks grow. From this viewpoint, the deposition of the central nodules of nacreous tablets can be interpreted as a second occurrence of crystallization centers, the role of which is potentially comparable in both prisms and nacre. However, the impressive results obtained by Wada [32], summarized here in Figures9f–i and A7, demonstrate that the control of the crystallographic orientation of the nacre tablets is much more extensive than that of prism crystallization. Whatever the complexity of the overall figures formed by the spatial arrangement of the nacreous tablets, Wada was able to show that their c axis is perpendicular to the surface of the nacreous layer, whereas the a and b axes are oriented with respect to the overall growth directions of the shell(Figure9i). The value of this result is shown by Wada’s investigations carried out on different nacre-producing species. No exception was found to this pattern, demonstrating a highly significant example of biological control exerted over crystallization. In spite of this difference, attention must be drawn to the gathered evidences that prisms and nacre, frequently pictured as directly produced shell building units (in a static description of shell microstructures), do not crystallize from freely moving molecules finding their way within a huge fluid-filled volume. Realistic reconstructions of shells must take into account the long and specifically organized preparative phases that predate the occurrence of the typical calcite and aragonite crystal-like units.

5. Conclusions 1. Microstructural investigation applied to the shell of Pinctada margaritifera reveals a sequence of growth steps in which organic structures play a key role by driving the formation and spatial arrangements of the mineralized units. With specific conditions for calcite prisms and nacreous tablets, structural observations suggest that the template interaction between organic structures and deposited minerals represents the organizational factor responsible for shell microstructures. 2. Up to the highest resolution levels, no trace of crystalline surface is visible in the shell building units, rendering irrelevant any model in which the formation of the microstructural units occurs by the side accretion of faceted pre-existing microcrystals. 3. In contrast to the “self-assembly” models, which consider that prisms and nacre are built through the movement of free molecules within a fluid-filled chamber, specific preparative structures can be identified, creating appropriate substrates for the development of prisms and nacre biomineralization.

Author Contributions: J.P.C. conceived, wrote the original draft and finalized the manuscript. All authors contributed to the writing and editing of the paper. A.S. and K.M. designed the synchrotron XFR experiments and Minerals 2018, 8, x FOR PEER REVIEW 16 of 20 Minerals 2018, 8, x FOR PEER REVIEW 16 of 20 MineralsXFR experiments2018, 8, 370 and interpreted the results. J.P.C., Y.D. and G.L. selected and prepared16 of the 20 XFR experiments and interpreted the results. J.P.C., Y.D. and G.L. selected and prepared the samples. J.P.C. performed SEM observations. Y.D. performed AFM observations. samples. J.P.C. performed SEM observations. Y.D. performed AFM observations. interpretedFunding: This the results. research J.P.C., was Y.D. supported and G.L. by selected a SYNTHESYS and prepared grant, the samples. the financial J.P.C. support performed of SEM the French observations. ANR Funding: This research was supported by a SYNTHESYS grant, the financial support of the French ANR Y.D.through performed contract AFM ANR-06-BLAN-0233 observations. (biocrystal), ESRF grants (EC24, EC208), EU-IHP programme through contract ANR-06-BLAN-0233 (biocrystal), ESRF grants (EC24, EC208), EU-IHP programme Funding:SYNTHESYS,This researchGB-TAF was NHM, supported and by by the a SYNTHESYS ESF-EuroCores-EuroMinScI-BioCalc grant, the financial support project, of the andFrench a SOLEIL ANR through grant SYNTHESYS, GB-TAF NHM, and by the ESF-EuroCores-EuroMinScI-BioCalc project, and a SOLEIL grant contract(20170784). ANR-06-BLAN-0233 (biocrystal), ESRF grants (EC24, EC208), EU-IHP programme SYNTHESYS, GB-TAF NHM,(20170784). and by the ESF-EuroCores-EuroMinScI-BioCalc project, and a SOLEIL grant (20170784). Acknowledgments: The authors are grateful to several Polynesian people who contribute to this investigation Acknowledgments: The authorsauthors are gratefulgrateful toto severalseveral PolynesianPolynesian peoplepeople who contribute to this investigationinvestigation by providing us with Pinctada specimens collected alive, then immediately preserved and sent to us in relevant by providing us with Pinctada specimens collected alive, then immediately preserved and sent to us in relevant conditions.conditions. Dominique Dominique DEVAUX, DEVAUX, pearl pearl producer producer at Rikitea at Rikitea (Gambier (Gambier archipelago), archipelago), Gabriel Gabriel HAUMANI, HAUMANI, DRMM conditions. Dominique DEVAUX, pearl producer at Rikitea (Gambier archipelago), Gabriel HAUMANI, biologicalDRMM biological station at station Takapoto at (Tuamotu Takapoto archipelago), (Tuamotu archipelago), and Cedrik LO, and Research Cedrik LO, and Research innovation and in perliculture innovation atin DRMM biological station at Takapoto (Tuamotu archipelago), and Cedrik LO, Research and innovation in theperliculture Direction at des the Ressources Direction Marinesdes Ressources et Mini èMarinesres de Polyn et Minièresésie and de Mereani Polynésie BELLAIS and Mereani (DRMM BELLAIS station at (DRMM Vairao) perliculture at the Direction des Ressources Marines et Minières de Polynésie and Mereani BELLAIS (DRMM havestation made at Vairao) this research have made possible. this research possible. station at Vairao) have made this research possible. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study;Conflicts in theof interpretationInterest: The authors of data; declare in the writing no conflict of the of manuscript, interest. The and funders in the decisionhad no role to publish in the design the results. of the study; in the interpretation of data; in the writing of the manuscript, and in the decision to publish the results. study; in the interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Appendix A Appendix A Appendix A

Figure A1. Scheme of mesocrystal formation by accretion of small crystalline units. Attraction due Figure A1. SchemeScheme of of mesocrystal mesocrystal formation formation by by accretion accretion of ofsmall small crystalline crystalline units. units. Attraction Attraction due to chemical forces between their crystalline faces results in formation of the massive crystal. Credit dueto chemical to chemical forces forces between between their crystalline their crystalline faces results faces results in formation in formation of the massive of the massive crystal. crystal.Credit picture [23]. Creditpicture picture [23]. [23].

Figure A2. Disk at the growing edge of a Pinctada margaritifera shell showing the 3 × 9 X-ray Figure A2. Disk at the growing edge of a Pinctada margaritifera shell showing the 3 × 9 X-ray Figurediffraction A2. spotsDisk (a at) the resulting growing in a edge single-crystal of a Pinctada like margaritifera diagram (b).shell Origin showing ESRF Grenoble,the 3 × 9 ID X-ray 13, diffraction spots (a) resulting in a single-crystal like diagram (b). Origin ESRF Grenoble, ID 13, diffractionoperator: M. spots Burghammer.(a) resulting Credit in apicture single-crystal [25]. like diagram (b). Origin ESRF Grenoble, ID 13, operator: M. Burghammer. Credit picture [25]. operator: M. Burghammer. Credit picture [25].

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Figure A3. Scheme of the outer mantle grove (a) at growing edge of aPinctada margaritifera shell cut Figure A3. Scheme of the outer mantle grove (a) at growing edge of aPinctada margaritifera shell perpendicular to shell surface. All along the animal life, periostracum is produced by a group of cutFigure perpendicular A3. Scheme to of shell the surface.outer mantle All alonggrove the(a) at animal growing life, edge periostracum of aPinctada is margaritifera produced byshell a groupcut perpendicularspecialized cells to ( bshell). Through surface. a Allpulsed along secretion the animal mode life, assessed periostracum by parallel is produced striation (Figureby a group 6c), the of of specialized cells (b). Through a pulsed secretion mode assessed by parallel striation (Figure6c), specializedresulting membrane cells (b). Throughacts as a conveyor-belta pulsed secretion for disks mode that assessed grow by parallelaccretion striation of calcite (Figure rings 6c),around the the resulting membrane acts as a conveyor-belt for disks that grow by accretion of calcite rings around resultinginitial organic membrane nodules acts deposited as a conveyor-belt onto the for internal disks sidethat ofgrow the by periostracal accretion of membrane. calcite rings The around black initial organic nodules deposited onto the internal side of the periostracal membrane. The black curved initialcurved organic line shows nodules the point deposited where ontothe disks the internalare incorporated side of the into periostracal the shell, becoming membrane. the Thefore-front black line shows the point where the disks are incorporated into the shell, becoming the fore-front of the solid curvedof the solidline shows shell. the Credit point pictures where the (a) disks Cuif andare incorporated Dauphin, redrawn into the fromshell, Proceedingsbecoming the of fore-front the 14th shell. Credit pictures (a) Cuif and Dauphin, redrawn from Proceedings of the 14th Biomineralization ofBiomineralization the solid shell. meeting, Credit pictures Tsukuba, (a) Japan, Cuif in and press; Dauphin, b: [39]. redrawn from Proceedings of the 14th meeting, Tsukuba, Japan, in press; b: [39]. Biomineralization meeting, Tsukuba, Japan, in press; b: [39].

Figure A4. (a) Total XRF spectrum from Figure 4c. (b) Elemental peaks deconvolution showing the Figurepresence A4. of (Cua) Total and ZnXRF in spectrumthe growing from edge Figure of Pinctada 4c. (b). Elemental peaks deconvolution showing the Figure A4. (a) Total XRF spectrum from Figure4c. ( b) Elemental peaks deconvolution showing the presence of Cu and Zn in the growing edge of Pinctada. presence of Cu and Zn in the growing edge of Pinctada.

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MineralsMinerals 2018 2018, ,8 8, ,x x FOR FOR PEER PEER REVIEW REVIEW 18 18 of of 20 20

Figure A5. Models of fluid-filled extra-pallial chambers in which moving molecules go to the right Figure A5. Models of ﬂuid-ﬁlled extra-pallial chambers in which moving molecules go to the right Figureplaces toA5. build Models the shellof fluid-filled layers. (a extra-pallial) Model from chambers Saleuddin in andwhich Petit moving [36]. ( bmolecules) Model from go to Saleuddin the right placesFigure to build A5. Models the shell of layers.fluid-filled (a) Model extra-pallial from Saleuddin chambers andin which Petit [moving36]. (b) Modelmolecules from go Saleuddin to the right and placesand Wilbur to build [37]. the Credit shell pictures layers. ((a):a) Model [36]; (b): from [37]. Saleuddin and Petit [36]. (b) Model from Saleuddin Wilburplaces [37 to]. build Credit the pictures shell layers. (a): [36 (a];) (b):Model [37 ].from Saleuddin and Petit [36]. (b) Model from Saleuddin andand Wilbur Wilbur [37]. [37]. Credit Credit pictures pictures (a): (a): [36]; [36]; (b): (b): [37]. [37].

Figure A6. Comparison of the Bevelander and Nakahara figure (a) Transmission electron microscopy) with a SEM view of a fractured shell of Pinctada margaritifera, showing prisms and FigureFigure A6. A6. ComparisonComparison of of the the Bevelander Bevelander and and Nakahara Nakahara figure figure ( (aa)) Transmission Transmission electron electron Figureinner A6. surfaceComparison of the prismatic of the Bevelanderlayer. Secretions and Nakaharaof the mantle ﬁgure epithelial (a) Transmission cell layer (m.e.c electron) are separated microscopy microscopy)microscopy) with with a a SEM SEM view view of of a a fractured fractured shell shell of of PinctadaPinctada margaritifera, margaritifera, showingshowing prisms prisms and and withfrom a SEM the mineralized view of a fractured prisms by shell the of“lamella”Pinctada (L margaritifera in [29]), i.e.,, showingexactly the prisms place andof the inner Mash surface and Sass of the innerinner surface surface of of the the prismatic prismatic layer. layer. Secretions Secretions of of the the mantle mantle epithelial epithelial cell cell layer layer ( (m.e.cm.e.c)) are are separated separated prismaticmembrane. layer. (b Secretions) Equivalent of the membrane mantle epithelial is clearly cell visible layer at ( m.e.c the ) basis are separated of the prismatic from the layer mineralized in P. fromfrom thethe mineralizedmineralized prismsprisms byby thethe “lamella”“lamella” (L(L inin [29]),[29]), i.e.,i.e., exactlyexactly thethe placeplace ofof thethe MashMash andand SassSass prismsmargaritifera. by the “lamella” Credit (L pictures in [29]), (a):i.e., exactly [29]; (b): the place Cuif of and the Mash Dauphin, and Sass Proceedings membrane. of ( b) the Equivalent 14th membrane.membrane. ( (bb)) Equivalent Equivalent membrane membrane is is clearly clearly visible visible at at the the basis basis of of the the prismatic prismatic layer layer in in P. P. membraneBiomineralization is clearly meeting, visible at Tsukuba the basis Japan, of the in prismatic press. layer in P. margaritifera. Credit pictures (a): [29]; margaritifera.margaritifera. CreditCredit pictures pictures (a): (a): [29]; [29]; (b): (b): Cuif Cuif and and Dauphin, Dauphin, Proceedings Proceedings of of the the 14th 14th (b): Cuif and Dauphin, Proceedings of the 14th Biomineralization meeting, Tsukuba Japan, in press. BiomineralizationBiomineralization meeting, meeting, Tsukuba Tsukuba Japan, Japan, in in press. press.

Figure A7. The Wada’s figure showing large scale figures made by arrangement of nacreous tablets (a) in which parallel crystal orientations is globally maintained (b). Note that looking at the FigureFigure A7.A7. TheThe Wada’sWada’s figurefigure showingshowing largelarge scalescale figuresfigures mademade byby arrangementarrangement ofof nacreousnacreous tabletstablets Figurewhole-shell A7. The level Wada’s orientation ﬁgure showingof nacreous large tablets scale follows ﬁgures the made morphology by arrangement of the shell of nacreous growing tablets edge. (a) ((aa)) in in which which parallel parallel crystal crystal orientations orientations is is globally globally maintained maintained ( (bb).). Note Note that that looking looking at at the the inCredit which pictures: parallel crystal[32]. orientations is globally maintained (b). Note that looking at the whole-shell whole-shellwhole-shell levellevel orientationorientation ofof nacreousnacreous tabletstablets followsfollows thethe morphologymorphology ofof thethe shellshell growinggrowing edge.edge. level orientation of nacreous tablets follows the morphology of the shell growing edge. Credit CreditCredit pictures: pictures: [32]. [32]. Referencespictures: [ 32]. References References

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