Lange, Skadi (2012) Mechanisms of Shell Repair in the Blue Mussel Mytilus edulis. (Diploma thesis), Christian-Albrechts-Universität, Kiel, Germany, 75 pp.

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Abstract

The bivalve mollusc shell comprises one to two major phases of calcium carbonate polymorphs, externally covered with organic, uncalcified periostracum. The two polymorphs predominant in most bivalve mollusc shell layers are (i) calcitic crystals forming an outer prismatic shell layer, and (ii) tabloid aragonitic crystals forming an inner nacreous shell layer. Shell-formation involves the introduction of an organic matrix framework on which nucleation and growth of calcium carbonate polymorphs is initialized. Formation of either of the polymorphs depends on specific interactions with the organic matrix constituents, with the functional properties of matrix proteins determining the characteristics of the formed mineral. The origin of proteins and carbohydrates involved in mineralization processes has been attributed to different parts of the mollusc mantle. In bivalves, this organ is divided into an inner- and outer mantle section. While the inner mantle (i.e. central zone) exclusively accounts for the formation of aragonite, the outer mantle (i.e. marginal and pallial zone) is involved in calcite and aragonite formation and furthermore propagates periostracum formation.
The aim of this study was to better understand the biomineralization process by analyzing alterations in gene expression patterns in the inner mantle that were elicited by shell damage. Prior to a four-week experiment, left valves of Mytilus edulis specimens from Kiel Fjord were exposed to a drilling treatment. Based on an existing M. edulis mantle tissue transcriptome, seven calcification-related genes were chosen, that were exclusively expressed at high levels in the outer or in the outer- and inner mantle. Gene expression patterns during re-calcification were analyzed after one and four weeks of shell repair using PCR and quantitative real-time PCR. Additionally, stereomicroscopy and SEM were utilized for structural observations of the inner shell surface to define repair stages and phenotypic correlation with gene expression patterns over time. Dry weight and length of the animals was determined to estimate the energetic impact of shell damage and repair on somatic growth. Strongly upregulated gene expression was demonstrated in inner mantle tissues for two out of the seven selected genes. Both of these genes were exclusively expressed in the outer mantle under control conditions. A tyrosinase gene, probably involved in periostracum formation, was most highly expressed in the inner mantle after one week of shell repair, while expression levels after four weeks were highest in the outer mantle. Expression of a gene for a nacrein-like protein, probably involved in nacre organisation, was highest in the inner mantle after four weeks, while after one week expression was predominant in the outer mantle. These results prove, for the first time in a mollusc, tissue-related shifts in gene expression during mineralization. As a response to shell damage, expression of genes for the synthesis of required proteins was specifically upregulated close to the site of repair. Shell re-calcification status was assessed by classifying shells according to six repair stages that were defined according to our observations within the experiment and allowed for a comprehensive scheme during shell analysis. At the first stage no signs of the repair process were detectable. The following stages were characterized by (i) initial formation of an organic sheet, (ii) enhancement of sheet rigidity and formation of calcite crystals, (iii) formation of calcite and aragonite crystals on the sheet (≤ 50% of the surface), (iv) progressed expansion of both polymorphs (> 50% of the surface), (v) complete mineralization of the inner shell surface. SEM analysis reflected the observed progress in mineralization over time, with calcite being the predominant polymorph present and aragonite accounting for ca. 20-30% of the mineralized surface after four weeks of shell-repair. Thus, the repair process was not completed at the end of the experiment. However, as the shell repair mechanism is characterized by formation of the same three structural components (i.e. periostracum – like layer, prismatic layer, nacreous layer) as in de novo shell synthesis, re-calcification assays can be powerful tools to better understand the genes involved in shell formation. No significant differences in dry weight and length-weight relationships between animals exposed to the treatment and control animals were detected. This suggests a minor energetic impact of the repair process on somatic growth.

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