Variability of shell repair in the Manila clam Ruditapes philippinarum affected by the Brown Ring Disease: A microstructural and biochemical study Nolwenn Trinkler a,⇑ , Nathalie Guichard b,c , Maylis Labonne a , Laurent Plasseraud c,d , Christine Paillard a , Frédéric Marin b,c,⇑ a IUEM, UMR CNRS 6539, Technopôle Brest Iroise, 29280 Plouzané, France b UMR 5561 CNRS, Biogéosciences, 21000 Dijon, France c Université de Bourgogne, 21000 Dijon, France d ICMUB, UMR CNRS 5260, Faculté des Sciences Mirande, 21000 Dijon, France article info Article history: Received 1 July 2010 Accepted 10 December 2010 Available online 17 December 2010 Keywords: Biomineralization Mollusc Shell repair Calcifying matrix Microstructure Antibody Secretory regime abstract For more than two decades, the Manila clam Ruditapes philippinarum has been regularly affected by Brown Ring Disease (BRD), an epizootic event caused by the bacterium Vibrio tapetis and characterized by the development of a brown deposit on the inner face of valves. Although BRD infection is often lethal, some clams recover by mineralizing a new repair shell layer, which covers the brown deposit and fully isolates it from living tissues. In order to understand this specific shell repair process, the microstructures of repaired zones were compared to those of shells unaffected by BRD. In addition, the organic matrix associated with unaffected shells and to repair patches were extracted and compared by biochemical and immunological techniques. Our results show that the repaired zones exhibit microstructures that resemble the so-called homogeneous microstructure of the internal layer, with some marked differences, like the development of crossed-acicular crystals, which form chevron-like patterns. In the three tested batches of repaired layers, the matrices exhibit certain heterogeneity, i.e., they are partially to widely dif- ferent from the ones of shells unaffected by BRD, as illustrated by SDS–PAGE and by serological compar- isons. Our results strongly suggest a modification of the secretory regime of calcifying mantle cells during the shell repair process. Polyclonal antibodies, which were developed against specific protein fractions of the shell, represent relevant tools for localizing by immunohistology the cells responsible for the repair. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The molluscan shell is a remarkable example of calcium carbon- ate biomineralization, synthesized outside living tissues. According to classical views, the shell results from a subtle chemistry between the precursor mineral ions that are extruded in the extra- pallial space and the numerous macromolecular extracellular com- ponents produced by the mantle epithelial cells, i.e., proteins and polysaccharides. All these components react in a well-coordinated manner. From this self-assembling process, solid and compact microstructures such as nacre, prisms, crossed-lamellar or homo- geneous textures, emerge (Bøggild, 1930; Carter, 1980; Popov, 1992; Checa and Rodriguez-Navarro, 2005). One peculiarity that makes the molluscan shell an appealing model to study lies in its ability to self-repair after shell damage. Artificially induced repair processes have been studied in a number of cases, after shell cutting (Caseiro and Gauthier, 1997) or shell drilling (Kapur and Sen Gupta, 1970; Caseiro and Gauthier, 1998; Fleury et al., 2008; Kadar et al., 2008). In natural environments, the shell repair process frequently takes place after accidental shell breakage, shell boring by epibionts (Marshall and Day, 2001; Nollens et al., 2002; Huchette et al., 2006), or predation by fishes. One particular case of natural repair occurs when the mollusc has to overcome a bacterial infection of the shell, including the periostracal layer. The Manila clam Ruditapes philippinarum pro- vides such an example. In western Europe, this species is infected by a bacterium, Vibrio tapetis (Paillard and Maes, 1990), that colo- nizes the clam by, first, adhering to the periostracal lamina, then, by penetrating into the extrapallial fluids (Paillard, 2004). At this stage, the most visible effect of the infection is the secretion by the mantle tissues of a brown organic deposit – ‘‘the Brown Ring Disease’’, also called BRD – on the inner and peripheral surface of the shell (Paillard and Maes, 1990, 1995a,b). When the hemolymph and the soft tissues are colonized by the bacterium, the clam rap- idly dies. This leads to mass mortalities in cultured clam popula- tions, in particular during winter. Clams that overcome the infections are the ones that can isolate the brown ring deposit from 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.12.011 ⇑ Corresponding authors. Fax: +33 (0)2 98 49 86 45 (N. Trinkler); fax: +33 (0)3 80 39 63 87 (F. Marin). E-mail addresses: nolwenn.trinkler@univ-brest.fr (N. Trinkler), Frederic.mar- in@u-bourgogne.fr (F. Marin). Journal of Invertebrate Pathology 106 (2011) 407–417 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip