Under review for J. R. Soc. Interface Foamy oysters: vesicular microstructure production in the Gryphaeidae via emulsification Journal: Journal of the Royal Society Interface Manuscript ID rsif-2020-0505.R2 Article Type:ForResearch Review Only Date Submitted by the n/a Author: Complete List of Authors: Checa, Antonio; Universidad de Granada, Estratigrafía y Paleontología; Linares, Fátima; Universidad de Granada, Centro de Instrumentación Científica Maldonado-Valderrama, Julia; Universidad de Granada, Departamento de Física Aplicada Harper, Elizabeth; University of Cambridge, Earth Sciences Categories: Life Sciences - Physics interface Biomaterials < CROSS-DISCIPLINARY SCIENCES, Biophysics < CROSS- Subject: DISCIPLINARY SCIENCES biomineralization, oysters, vesicular microstructure, solid foam, Keywords: emulsion, micro-CT http://mc.manuscriptcentral.com/jrsi Page 1 of 43 Under review for J. R. Soc. Interface 1 2 3 Author-supplied statements 4 5 6 Relevant information will appear here if provided. 7 8 Ethics 9 10 Does your article include research that required ethical approval or permits?: 11 This article does not present research with ethical considerations 12 13 Statement (if applicable): 14 CUST_IF_YES_ETHICS :No data available. 15 16 17 Data 18 19 It is a condition of publication that data, code and materials supporting your paper are made publicly 20 available. Does your paper present new data?: 21 My paper has no data For Review Only 22 23 Statement (if applicable): 24 25 CUST_IF_YES_DATA :No data available. 26 27 Conflict of interest 28 29 I/We declare we have no competing interests 30 31 Statement (if applicable): 32 CUST_STATE_CONFLICT :No data available. 33 34 35 Authors’ contributions 36 37 This paper has multiple authors and our individual contributions were as below 38 39 Statement (if applicable): 40 AGC conceived and designed the study, acquired and analysed data, wrote the paper. FL acquired 41 and analysed data, and revised the paper. JMV analysed data and revised the paper. EMH conceived 42 and designed the study, acquired and analysed data, revised the paper. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/jrsi Under review for J. R. Soc. Interface Page 2 of 43 1 2 3 Foamy oysters: vesicular microstructure production in the Gryphaeidae via 4 5 emulsification 6 7 8 Antonio G. Checa1,2,*, Fátima Linares3, Julia Maldonado-Valderrama4, Elizabeth M. Harper5 9 1 10 Departamento de Estratigrafía y Paleontología, Universidad de Granada, 18071 Granada, Spain 11 2Instituto Andaluz de Ciencias de la Tierra, CSIC-Universidad de Granada, 18100 Armilla, Spain 12 3Centro de Instrumentación Científica, Universidad de Granada, 18071 Granada, Spain 13 14 4Departamento de Física Aplicada, Universidad de Granada, 18071 Granada, Spain 15 5Department of Earth Sciences, Cambridge University, CB2 3EQ Cambridge, UK 16 17 18 iD AGC 0000-0001-7873-7545, FL 0000-0003-2205-5953, JMV 0000-0001-6372-723X 19 For Review Only 20 21 *Corresponding author: 22 23 e-mail: [email protected] 24 25 Tel. +35 958243201 (office) 26 +34 635332245 (mobile) 27 28 29 Keywords: biomineralization, oysters, vesicular microstructure, solid foam, emulsion, micro-CT 30 31 32 Running head: vesicular microstructure of oysters 33 34 35 The vesicular microstructure is a very distinctive arrangement of calcite, consisting of hollow 36 37 cavities (vesicles) of diverse sizes and shapes, usually elongated in the direction of shell thickening. 38 It is uniquely found among living bivalves in a single oyster family, Gryphaeidae. The vesicles are 39 distributed in lenses interleaved with compact foliated layers. We have studied the morphology 40 and distribution of vesicles within the lenses using optical and electron microscopy, and micro- 41 computed tomography. At a small scale, vesicles do not follow a classical von Neumann-Mullins 42 43 route typical of ideal foams. At a larger scale, the initiation and evolution of a vesicular layer 44 statistically proceed like a foam, with vesicles becoming more numerous, larger, and more even in 45 size. In summary, the vesicular material follows a foam-like coarsening to reduce the number of 46 energetically costly interfaces. However, a steady state is never reached because the animal 47 permanently introduces energy in the system by creating new vesicles. The fabrication of the 48 49 vesicular material is mediated by the production of an emulsion between the extrapallial fluid and 50 the precursor PILP of the calcitic walls within the thin extrapallial space. For this mechanism to 51 proceed, the mantle cells must perform highly sophisticated behaviours of contact recognition and 52 secretion. Accordingly, the vesicular material is under mixed physical-biological control. 53 54 55 1. Introduction 56 57 58 59 60 http://mc.manuscriptcentral.com/jrsi Page 3 of 43 Under review for J. R. Soc. Interface 1 2 3 Calcifying invertebrates have a large repertoire of shell forming microstructures. Molluscs, in 4 particular, show a broad range. These microstructures are a valuable source of inspiration for 5 6 materials scientists. Most studies have focused on calcified biomaterials with high biomechanical 7 performance (e.g. crossed-lamellar, prismatic calcite) and, sometimes also with economic interest 8 (nacre) [1-3]. Other microstructures have received very little attention, despite raising intriguing 9 fabricational issues. Oysters of the superfamily Ostreoidea comprise several such microstructures. 10 Their shells are entirely calcitic, with the exception of aragonitic myostracal layers and fibres 11 12 within the ligament [4]. The predominant microstructure is foliated (made of thin continuous 13 layers of sheet-like folia), combined with a thin external columnar prismatic layer (which is thicker 14 on the right than left valve) [5]. In addition though the shells often contain discontinuous lenses of 15 less compact, lower density material, the character of which is different in the two families which 16 make up the Ostreoidea. These intriguing microstructures appear to allow the manufacture of 17 18 thick shells while reducing either production costs or final weight. In the Ostreidae these patches 19 consist of ‘chalk’, made withFor the same Review type crystalline units Only (calcitic laths) of units as the foliated 20 material [5], while in the Gryphaeidae the material consists of a highly porous vesicular 21 microstructure (VM) that at least superficially resembles a froth or foam [6]. The focus of this 22 paper is characterizing the VM and building a model for its fabrication. 23 24 VM consists of lenses of hollow cavities (vesicles), tens of µm wide, usually elongate, separated 25 by relatively thick (several µm) calcitic walls with polygonal or quasi-polygonal outlines, forming a 26 sort of honeycomb structure (figure 1) [6]. The VM was described already in the early 19th century 27 28 by DeFrance [7], followed by Douvillé [8] and Ranson [9] in the first half of the past century. 29 Nevertheless, the number of later studies dealing with it is very reduced (see review in [6]). Today 30 there are three surviving genera of Gryphaeidae, all belonging to the subfamily Pycnodonteinae: 31 Hyotissa, Neopycnodonte, and Pycnodonte (which contain a total of nine recognized species [10]). 32 All extant taxa possess VM [11]. Gryphaeid oysters have a long evolutionary history and were 33 34 particularly diverse and abundant during the Mesozoic [12,13]. Recent evidence suggests that the 35 Pycnodonteinae originated in the Early Cretaceous, and with VM present since at least the Late 36 Cretaceous [14]. There is no suggestion that other extinct gryphaeid subfamilies also secreted VM 37 and hence the character appears to have evolved within the pycnodonteines. Fossil gryphaeids 38 39 have been important archives for the reconstruction of past climate and environment (e.g. [15- 40 17]). Thus, it is important to understand skeletal formation as it relates to the chronology of 41 skeletal archives. 42 Hitherto, the only explanation advanced about the mechanism for the fabrication of VM 43 44 suggests that it is preceded by froth produced by gas bubbles with mucus walls trapped within the 45 extrapallial space (EPS), i.e. between the mantle and the forming shell [6]. The mucus would be 46 later replaced by calcite, with crystallization progressing from the shell wall side towards the 47 mantle. However, this hypothesis has a number of problems. First, the EPS would need to be large 48 49 to accommodate entire bubbles, whereas we know that it is usually exceedingly thin (<1 µm; e.g. 50 [18]), even during the formation of comparable porous materials, such as the oyster chalk [5]. 51 Second, the vesicles display growth lines (e.g. [19]) which indicate progressive, not the 52 instantaneous growth implied by [6]. Additionally, the vesicles are most often elongated, whereas 53 bubbles are typically spherical to equidimensional polyhedral. 54 55 Despite its restricted distribution among the diverse range of microstructures produced by 56 bivalves, VM represents an interesting case from the fabricational viewpoint. How can foam-like 57 58 59 60 http://mc.manuscriptcentral.com/jrsi Under review for J. R. Soc. Interface Page 4 of 43 1 2 3 material be produced sequentially by the oyster across an extremely thin liquid film (the EPS)? In 4 order to answer this question, we have carried out a study of the vesicular lenses secreted by 5 6 species of two pycnodonteine genera using scanning electron microscopy (SEM) and micro 7 computed tomography (micro-CT). We have characterized the morphology of the vesicles within 8 the layers and studied their evolution during growth. The results allow us to compare the growth 9 pattern of vesicles in the VM with those of a liquid foam and to propose a model for their 10 fabrication by the oyster, taking into account the space restrictions imposed by the dimensions of 11 12 the EPS. 13 14 15 2. Material and methods 16 2.1.