The mineral phase of shell repair in the Manila clam Venerupis philippinarum affected by brown ring disease (BRD) was characterised at various scales and at various stages of shell repair by confocal Raman microspectrometry and scanning electron microscopy. Spherulitic and quadrangular aragonite microstructures associated with polyene pigments were clearly observed. Von Kossa staining showed that at the beginning of shell repair, hemocytes are filled with insoluble calcium carbonate salts in all fluids and then are transported toward the extrapallial fluids and the repair sites. Our analyses suggest that after a Vibrio tapetis attack and BRD deposit some clams rapidly cover the deposit, resulting in a modification in the microstructure, which could be produced by the participation of both the mantle and hemocytes.KEY WORDS: Aragonite · Hemocytes · Shell microstructure · Calcium carbonate · Biomineralization · Mollusc · Raman microspectrometry · Scanning electron microscopy
Resale or republication not permitted without written consent of the publisherDis Aquat Org 93: 149-162, 2011149-162, al. 1989149-162, , Flassch et al. 1992. Landeda was the first Manila clam production site in France (Paillard et al. 1989, Flassch et al. 1992.Vibrio tapetis colonises the periostracum and inhibits the normal process of shell biomineralisation. The external response of the clam to the bacterial attack consists of a secretion of a brown organic matrix on the inner face of the shell, the well-known 'brown ring'. Immunological or PCR studies on the different physiological fluids of the clam have shown that, at later stages of the infection, V. tapetis invades the circulatory system (hemolymph) and the extrapallial fluids (Allam 1998, Allam et al. 2002, Paillard et al. 2006. At this advanced stage, clams often die from a hemolymphatic septicaemia. In some cases, clams recover by secreting a white shell layer on the brown organic matrix (Paillard & Maes 1995a,b). The bulk organic matrix associated with the shell repair has been recently characterised using Raman microspectrometry, wavelength-dispersive spectrometry (WDS) microprobe analysis and organic matrix quantification (Trinkler et al. 2009(Trinkler et al. , 2010a. However, the mineral phase and the microstructure of repaired Manila clam shells, including those that exhibit a fully repaired shell, have not yet been studied. In the present study, experimental investigations have been performed on the microstructure and the nature of the calcium carbonate repair layer, using Raman microspectrometry and scanning electron microscopy (SEM). In particular, Raman microspectrometry, a nondestructive and noninvasive technique, provides qualitative and quantitative information on organomineral composites (Urmos et al. 1991, Kontoyannis & Vagenas 2000, Withnall et al. 2003, Hedegaard et al. 2006, Carteret et al. 2009). A complementary characterisation using the Von Kossa stain, which is a stain specific for insoluble calcium salts, was performed on hemolymph and extrapallia...