On the Nature of Tintinnid Loricae (Ciliophora: Spirotricha: Tintinnina): a Histochemical, Enzymatic, EDX, and High-resolution TEM Study.

Tintinnids (Ciliophora: Spirotricha: Tintinnina) are occasionally the dominant ciliates in the marine plankton. The tintinnid loricae are minute artworks fascinating scientists for more than 230 years, but their chemical composition remained unclear, viz., chitinous or proteinaceous substances were discussed. Since sedimenting loricae contribute to the flux of elements and organic compounds in the oceans, knowledge about their nature is necessary in assessing their ecological role. Previous techniques and new methods, e.g. enzymatic digestion and high-resolution transmission electron microscopy, are applied in the present study. A chitinous nature of the loricae is rejected by the Van-Wisselingh test and failure of chitinase digestion. Only proteins might show a resistance against strong hot bases (KOH at 160°C for ~ 40 min. in tintinnid loricae) similar to that of chitin. Actually, the presence of nitrogen in the EDX analyses and the digestion of at least some loricae by proteinase K strongly indicate a proteinaceous nature. Furthermore, the crystal lattice revealed by high-resolution TEM in Eutintinnus loricae is similar to the proteinaceous surface layer (S-layer) of archaea, and the striation recognizable in transverse sections of Eutintinnus loricae has a periodicity resembling that of the crystalline proteins in the extruded trichocysts of Paramecium and Frontonia. The proteolytic resistance of some loricae does not reject the idea of a proteinaceous nature, as proteins in S-layers of some archaea and in most naturally occurring prions show comparable reactions. The data from the present study and the literature indicate proteins in the loricae of thirteen genera. Differences in the proteolytic resistance and staining properties between genera and congeners are probably due to deviations in the protein composition and the additional substances, e.g. lipids, carbohydrates. At the present state of knowledge, correlations between lorica structure, wall texture, ultrastructure of the lorica forming granules, and the histochemical and enzymatic findings are not evident. Therefore, further studies are required to estimate the taxonomic significance of these features and the ecological role of sedimenting loricae.