Clam shell repair from the brown ring disease: a study of the organic matrix using Confocal Raman micro-spectrometry and WDS microprobe

Abstract

Since 1987, the Manila clam Ruditapes philippinarum has been regularly affected by the brown ring disease (BRD), an epizootic caused by the bacterium Vibrio tapetis. This disease is characterized by the development of a brown deposit on the inner face of valves. While most of the clams die from the BRD infection, some of them are able to recover by mineralizing a new repair shell layer, which covers the brown deposit by a process of encapsulation. The purpose of this work was to study the organic matrix of the shells of Manila clams in the inner shell layer before, during and after the brown deposit and during the shell repair process by confocal Raman micro-spectrometry and wavelength dispersive spectrometry (WDS) microprobe. In addition, the organic matrix of the repaired shell layer was extracted and quantified, by using standard biochemical shell matrix extractions protocols. The brown deposit exhibited high luminescence intensity in Raman spectra, and an increase of S, C, Sr (forming two peaks) and a decrease of Ca, Na concentrations (% w/w), using WDS microprobe mapping and cross-sectional transects. The signature of these trace elements was similar to that recorded on periostracal lamina (% w/w). The high S concentration likely corresponds to the presence of a high amount of sulfated organic compounds. Interestingly, on cross-sectional transects, before the brown deposit, a thin layer of the shell showed also a high luminescence, which may suggest that this layer is modified by bacteria. After the brown deposit, at the beginning of the shell repair process, the luminescence and the S concentration remain high, before declining the level found in non-BRD-affected shells. Quantification of the organic matrix shows that the shell repair layer zone is significantly different from non-BRD-affected shell layer, in particular with a much higher amount of insoluble matrix.