Sabine Agatha

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.

We isolated an encysted ciliate from a geothermal field in Iceland. The morphological features of this isolate fit the descriptions of Dexiotricha colpidiopsis (Kahl, 1926) Jankowski, 1964 very well. These comprise body shape and size in vivo, the number of somatic kineties, and the positions of macronucleus and contractile vacuole. Using state-of-the-art taxonomic methods, the species is redescribed, including phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) gene as molecular marker. In the phylogenetic analyses, D. colpidiopsis clusters with the three available SSU rRNA gene sequences of congeners, suggesting a monophyly of the genus Dexiotricha. Its closest relative in phylogenetic analyses is D. elliptica, which also shows a high morphological similarity. This is the first record of a Dexiotricha species from a hot spring, indicating a wide temperature tolerance of this species at least in the encysted state. The new findings on D. colpidiopsis are included in a briefly revision of the scuticociliate genus Dexiotricha and an identification key to the species. 

The oligotrichid ciliate Strombidium coronatum (Leegaard, 1915) Kahl, 1932 is redescribed from plankton samples taken in the Irish Sea, using live observation, protargol impregnation, and scanning electron microscopy. The species is characterized by a conspicuous, uniquelly shaped peristome, which is flat and roughly triangular and extends in the sagittal plane. The Irish Sea specimens measure ~ 45 × 25 µm in vivo and ~ 40 × 24 µm after protargol impregnation. The girdle kinety is equatorial, ostensibly continuous, and composed of ~ 100 dikinetids. The ventral kinety extends longitudinally on the posterior fifth of the cell and is composed of about five dikinetids. The adoral zone of membranelles is widely open and composed of ~ 18 collar and ~ 12 buccal membranelles; the collar portion is disconnected from the buccal portion. The shape and orientation of the opisthe’s adoral zone of membranelles are apparently extraordinary, i.e., the membranelles form an inverted L-shaped stripe extending longitudinally in the elongated posterior cell portion of dividers.

Tintinnopsis parvula Jörgensen, 1912 has apparently a cosmopolitan distribution in the pelagial of marine and brackish coastal waters. The species is redescribed based on material from the Irish Sea off the Isle of Man, using live observation, protargol impregnation, and scanning electron microscopy. The agglomerated and stiff lorica measures 38–60 × 24–31 μm and is composed of a usually broadly obovate bowl and a slightly narrowed cylindroidal collar with an inner diameter of ~ 20 μm. The somatic ciliary pattern is of the most complex type, viz., it comprises a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field. The left ciliary field comprises four kineties, the lateral field about ten kineties, and the right field five kineties. The oral primordium develops apparently apokinetally posterior to the lateral ciliary field and generates ~ 15 collar membranelles and one buccal membranelle. Two further populations were studied: one from the North Sea off the Island of Sylt, the other from brackish polder basins at the German North Sea coast; they match the Irish Sea specimens in all main features. The loricae formed in almost particle-free cultures have a thin wall composed of an irregular network of fibres and very few attached or interwoven particles. This matrix type differs from the other three types found in congeners. Hence, the matrix ultrastructure might represent a promising feature for a reliable subdivision of the species-rich genus Tintinnopsis Stein, 1867 in the future.

The first transmission and scanning electron microscopical studies in combination with freeze-fracture technology have disclosed some important morphological and ultrastructural features in the freshwater oligotrichid Limnostrombidium viride. (I) The dikinetids (paired basal bodies) of the girdle kinety have a club-shaped cilium associated only with each left basal body. The electron-dense (paraflagellar) body on one side of its “92+2”-axoneme and the regular array of intramembranous particles indicate a sensory, perhaps photoreceptor function of these club-shaped cilia. (II) The stichomonad endoral membrane is proximally covered by a cytoplasmic fold and distally by multiple membranous layers. Thus entirely covered, the endoral is probably no longer involved in food capture; none the less, its associated microtubules might stabilise the cytopharynx. (III) Instead of a contractile vacuole, a horizontal ring-canal with supposed osmoregulatory function occurs. (IV) The extrusive nature of the trichites is not only observed in  electron micrographs, but the attachment sites of these organelles also display a rosette of “8+1”-particles in the P-face of freeze-fracture replicas typical for ciliate extrusomes. (V) The neoformation organelle, the subsurface tube in which stomatogenesis takes place, shows short basal bodies and normal axonemes about 1 μm long. It is accompanied by numerous membrane vesicles, which might provide membrane material for the outgrowing cilia.

Molecular phylogenies of Oligotrichea currently do not contain all genera and families and display topologies which are often incongruent with morphological findings. In ciliates, the somatic kinetids are rather conserved, i.e., their ultrastructures, particularly the fibrillar associates, often characterise the main groups, except for the choreotrichids. Four  different kinetid types are found in protargolstained choreotrichids and used for reconstructing the taxon’s evolution (the “Kinetid Transformation Hypothesis”). Proof for this hypothesis requires transmission electron microscopic studies, which are very rare in the choreotrichids and oligotrichids. Such an approach provides insights into the ultrastructural variability of somatic kinetids in spirotrichs and may also detect apomorphies characterising certain choreotrichid families. In the model tintinnid Schmidingerella meunieri, the ultrastructure of the three kinetid types in the somatic ciliature is studied in cryofixed cells. The data support the “Kinetid Transformation Hypothesis” regarding  tintinnids with a ventral kinety. This first detailed study on kinetids in tintinnids and choreotrichids in general reveals totally new kinetid types in ciliates: beyond the three common associates, they are characterised by two or three conspicuous microtubular ribbons extending on the kinetids’ left sides. These extraordinary ribbons form together with the overlapping postciliary ribbons a unique network in the cortex of the anterior cell portion. The evolutionary constrains which might have fostered the development of such structures are discussed for the Oligotrichea, the choreotrichids, and tintinnids as their first occurrence is currently uncertain. Additionally, the kinetids in tintinnids, aloricate  choreotrichids, oligotrichids, hypotrichs, and euplotids are compared.

The evolution of the dominant marine plankton ciliates, the oligotrichids and choreotrichids, is analysed for morphologic and genetic convergences and apomorphies based on literature and our own data. These findings have taxonomic implications. Within the oligotrichid genus Parallelostrombidium two subgenera, Parallelostrombidium Agatha, 2004 nov. stat. and Asymptokinetum nov. subgen., are established, using the courses of the ventral and girdle kineties as a distinguishing feature. Likewise, a different arrangement of extrusome attachment sites is used for a split of the oligotrichid genus Novistrombidium into the subgenera Novistrombidium Song and Bradbury, 1998 nov. stat. and Propecingulum nov. subgen.; Novistrombidium (Propecingulum) ioanum (Lynn and Gilron, 1993) nov. comb. and Novistrombidium (Propecingulum) platum (Song and Packroff, 1997) nov. comb. are affiliated. Based on discrepancies in the somatic ciliary pattern and the presence of conspicuous argyrophilic inclusions, the aloricate choreotrichid species Pelagostrobilidium kimae nov. spec. is distinguished from P. conicum. The diagnosis for the tintinnid family Eutintinnidae Bachy et al., 2012 is improved by including cell features. The co-operation of taxonomists and molecular biologists is strongly recommended to prevent misinterpretations of gene trees due to incorrectly identified species and for better species circumscriptions.