Electron microscopic and preparative methods for the analysis of isopod cuticle

The crustacean cuticle consists of a complex organic matrix and a mineral phase. The physical and chemical properties of the cuticle are corellated to the specific functions of cuticular elements, leading to a large variety in its structure and composition. Investigation of the structure-function relationship in crustacean cuticle requires sophisticated methodological tools for the analysis of different aspects of the cuticular architecture. In the present paper we report improved preparation methods that, in combination with various electron microscopic techniques, have led to new insights of cuticle structure and composition in the tergite cuticle of Porcellio scaber. We used thin sections of non-decalcified tergites and decalcified resin embedded material for transmission electron microscopy and scanning transmission electron microscopy. Etched sagittal planes of bulk tergite samples were analysed with field emission scanning electron microscopy. We have found a distinct distal region within the exocuticle that differs from the subjacent proximal exocuticle in the arrangement of fibres. Within this distal exocuticle chitin-protein fibrils assemble to fibres with diameters between 15 and 50 nm that are embedded in a mineral matrix. In the proximal exocuticle and the endocuticle fibrils do not assemble to fibres and are surrounded by mineral individually. Furthermore, we show that the pore canals are filled with mineral, and demonstrate that mild etching of polished sagittal cuticle surfaces reveals regions containing mineral of diverse solubility.