An Integrative Investigation of Parabistichella variabilis (Protista, Ciliophora, Hypotrichia) Including Its General Morphology, Ultrastructure, Ontogenesis, and Molecular Phylogeny.

Hypotrichs are a highly differentiated and very diverse group of ciliated protists. Their systematics and taxonomy are challenging and call for detailed investigations on their general morphology, ultrastructure, ontogenesis, and molecular phylogeny. Here, a comprehensive study is conducted on a brackish water population of Parabistichella variabilis using light and electron microscopy and phylogenetic analyses based on small subunit ribosomal DNA sequence data. Its morphology, including the infraciliature, pellicle, nuclei, buccal seal, and extrusomes, is documented. The present findings indicate that in P. variabilis: (i) the cortical granules are extrusomes, which differ from those of other hypotrichs; (ii) the buccal seal is bounded by the plasma membrane and contains a single layer of longitudinal microtubules; (iii) two contractile vacuoles might be present rather than one; and (iv) the pharyngeal disks are bounded by a single membrane. Early-to-middle stages of ontogenesis are described for the first time, enabling the complete characterization of this process. Phylogenetic analyses indicate that Parabistichella variabilis is closely related to several species from different genera, such as Orthoamphisiella breviseries, Uroleptoides magnigranulosus, and Tachysoma pellionellum. However, ultrastructural and gene sequence data for more taxa are needed in order to resolve the systematics of Parabistichella.

Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite.

Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbot.

Chemical Defense by Erythrolactones in the Euryhaline Ciliated Protist, Pseudokeronopsis erythrina.

Pseudokeronopsis erythrina produces three new secondary metabolites, erythrolactones A2, B2 and C2, and their respective sulfate esters (A1, B1, C1), the structures of which have been recently elucidated on the basis of NMR spectroscopic data coupled to high resolution mass measurements (HR-MALDI-TOF). An analysis of the discharge of the protozoan pigment granules revealed that the non-sulfonated erythrolactones are exclusively stored in these cortical organelles, which are commonly used by a number of ciliates as chemical weapons in offense/defense interactions with prey and predators. We evaluated the toxic activity of pigment granule discharge on a panel of free-living ciliates and micro-invertebrates, and the activity of each single purified erythrolactone on three ciliate species. We also observed predator-prey interactions of P. erythrina with unicellular and multicellular predators. Experimental results confirm that only P. erythrina cells with discharged pigment granules were preferentially or exclusively hunted and eaten by at least some of its predators, whereas almost all intact (fully pigmented) cells remained alive. Our results indicate that erythrolactones are very effective as a chemical defense in P. erythrina.

Cold-shock based method to induce the discharge of extrusomes in ciliated protists and its efficiency.

Extrusomes are ejectable organelles in protists, which are able to discharge their contents to the outside of the cell in response to external stimuli. It is known that a large number of extrusomes functions as organelles for offense or defense in predator-prey interactions among protists and/or microinvertebrates. To date, the main approach to study these interactions was to compare artificially-induced extrusome-deficient cells with normal cells as prey for predators. Commonly applied methods to obtain extrusome-deficient cells use external chemicals, which could alter the viability of cells and/or interfere with the subsequent analysis of the substances (secondary metabolites) contained in the extrusomes. The cold-shock based method here presented has proven to be effective to remove different kinds of extrusomes from several protist species without harming the treated cells and without adding external reagents. This method could be also useful to simplify the related analysis of the chemical nature of the secreted secondary metabolites.

Chemical offense by means of toxicysts in the freshwater ciliate, Coleps hirtus.

Coleps hirtus is a small common freshwater ciliate belonging to the protostomatid group, its body covered by calcified plates assembled to form an armor. Coleps feeds on bacteria, algae, flagellates, living and dead ciliates, animal and plant tissues. To assist its carnivorous feeding the ciliate is equipped with offensive extrusomes (toxicysts), clustering mainly in and around its oral aperture. In this study, we isolated the discharge of the toxicysts from living cells, evaluating its cytotoxic effects against various ciliate species, and demonstrating that it is essential for the effectiveness of Coleps' predatory behavior. The analysis of the toxicyst discharge performed by liquid chromatography-electrospray-mass spectrometry and gas chromatography-mass spectrometry, revealed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids with the addition of a minor amount of a diterpenoid (phytanic acid).

Pseudocnidae of ribbon worms (Nemertea): ultrastructure, maturation, and functional morphology.

The fine structure of mature pseudocnidae of 32 species of nemerteans, representatives of 20 genera, six families, and two classes was investigated with scanning and transmission electron microscopy. Pseudocnidae are composed of four layers (cortex, medulla, precore layer, and core) in most species investigated, but the degree of development and position of each layer can vary between different species. The secretion products comprising immature pseudocnidae segregate into separate layers: a thin envelope, which subsequently separates into the cortex and medulla and an extensive internal layer. We distinguish two pseudocnida types: type I is characterized by a two-layered core and type II by a three-layered core. Type I pseudocnidae are present in archinemertean species, Carinoma mutabilis, and in all pilidiophoran species, except Heteronemertea sp. 5DS; type II pseudocnidae occur in all studied species of Tubulanidae and the basal Heteronemertea sp. 5DS. Based on the structure of the discharged pseudocnidae observed in eleven species of palaeonemerteans and in eight species of pilidiophorans, we distinguish three different mechanisms (1-3) of core extrusion/discharge with the following characteristics and distribution: (1) the outer core layer is everted simultaneously with the tube-like layer and occurs in type I pseudocnidae of most species; (2) the extruded core is formed by both eversion of the outer core layer and medullar layer, and occurs in type I pseudocnidae of Micrura cf. bella; (3) the eversion of the outer core layer begins together with the core rod and core rod lamina and occurs in type II pseudocnidae. Morpho-functional comparison with other extrusomes (cnidae, sagittocysts, rhabdtites, and paracnids) confirm that pseudocnidae are homologous structures that are unique to nemerteans.

Evolutionary Trends of Perkinsozoa (Alveolata) Characters Based on Observations of Two New Genera of Parasitoids of dinoflagellates, Dinovorax gen. nov. and Snorkelia gen. nov.

Parasites are one of the ecologically most relevant groups of marine food webs, but their taxonomic and biological complexity hampers the assessment of their diversity and evolutionary trends. Moreover, the within-host processes that govern parasitoid infection, development and reproduction are often unknown. In this study, we describe a new species of a perkinsozoan endoparasitoid that infects the toxic dinoflagellate Dinophysis sacculus, by including observations of its morphology, ultrastructure, life-cycle development and phylogeny. The SSU rDNA sequence and main morphological features were also obtained for a second parasitoid species infecting the bloom-forming dinoflagellate Levanderina fissa. Phylogenetic analyses including the sequences obtained show that all known Perkinsozoa species infecting dinoflagellates cluster together. However, sequences of Parvilucifera prorocentri and those obtained in this study cluster at the base of the clade, while the rest of Parvilucifera representatives form a separated highly-supported cluster. These results, together with differing morphological characters like the formation of a germ-tube, the presence of trichocysts, or the heterochromatin presence in zoospores nucleus justify the erection of Dinovorax pyriformis gen. nov. et sp. nov., and Snorkelia prorocentri gen. nov. et comb. nov. (=Parvilucifera prorocentri). The morphological features and phylogenetic position of these parasitoids represent ancestral characters for the Perkinsozoa phylum, and also for Dinozoa clade, allowing the inference of the evolutionary framework of these Alveolata members.

Multivariate morphometric analyses of the predatory ciliate genus Semispathidium (Ciliophora: Litostomatea), with description of S. longiarmatum nov. spec.

We studied morphometrical variation, species boundaries, and importance of morphometric features for a reliable separation of five African Semispathidium taxa. Altogether, 20 features traditionally used in alpha-taxonomy of the predatory genus Semispathidium were measured or scored on 85 protargol-impregnated interphase specimens, and were analyzed using hierarchical clustering as well as principal component and canonical discriminant analyses. This multivariate approach confirmed that a population found in Botswanan floodplain soil represents a distinct taxon. The new species is described here as S. longiarmatum, using live observation, protargol impregnation, and scanning electron microscopy. Semispathidium longiarmatum strongly resembles S. armatum and S. breviarmatum but it is clearly distinguished from these species by the extrusome pattern. The reliability of S. longiarmatum is also strengthened, according to the canonical discriminant analysis, by several quantitative features, viz., the number of ciliary rows, the length:width ratio of the macronucleus, and the number of dikinetids in brush row 1. Moreover, the present study documents the distinctness of all African Semispathidium species which can be separated by a combination of both qualitative and quantitative (morphometric) features. Consequently, Semispathidium species do not form a continuous complex but fairly discrete clusters in the phenotypic space.