Coupling between Ribotypic and Phenotypic Traits of Protists across Life Cycle Stages and Temperatures.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation.

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.

Two new species of Euplotes with cirrotype-9, Euplotes foissneri sp. nov. and Euplotes warreni sp. nov. (Ciliophora, Spirotrichea, Euplotida), from the coasts of Patagonia: implications from their distant, early and late branching in the Euplotes phylogenetic tree.

Two new Euplotes species have been isolated from cold shallow sandy sediments of the extreme Southern Chilean coasts: Euplotes foissneri sp. nov., from a low-salinity site at Puerto Natales on the Pacific coast, and Euplotes warreni sp. nov., from a marine site at Punta Arenas on the Atlantic coast. Euplotes foissneri has a medium body size (53×36 µm in vivo), a dorsal surface marked by six prominent ridges, a double dargyrome, six dorsal and two ventrolateral kineties, a buccal field extending to about 3/4 of the body length, an adoral zone composed of 28-32 membranelles, and nine fronto-ventral, five transverse and two or three caudal cirri. The bulky, hook-, horseshoe- or 3-shaped macronucleus is associated with one sub-spherical micronucleus. The central body region hosts taxonomically unidentified endosymbiotic eubacteria. Euplotes warreni has a small body size (39×27 µm in vivo), a smooth dorsal surface marked by three deep grooves, a double dargyrome, four dorsal and two ventrolateral kineties, a buccal field extending to about 2/3 of the body length, an adoral zone composed of 23-25 adoral membranelles, and nine fronto-ventral, five transverse and three caudal cirri. The macronucleus is hook- or C-shaped and associated with one spherical micronucleus. Endosymbiotic bacteria belonging to the genus Francisella reside preferentially in the anterior cell region. Both species lack the fronto-ventral cirrus numbered 'V/2', whereby their cirrotype-9 conforms to the so-called 'pattern I', which is the basic distinctive trait of the genus Euplotopsis Borror and Hill, 1995. Phylogenetic analyses of small subunit rRNA gene sequences, however, classify E. warreni into its own early branching clade and E. foissneri into a late branching clade. This indicates a polyphyletic nature and taxonomic inconsistency of the genus Euplotopsis, which was erected to include Euplotes species with cirrotype-9 pattern I.

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco-Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a "Well-known" Euplotes Ciliate, Euplotes harpa Stein, 1859 (Ciliophora, Euplotida).

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco-Kernéis, and the previously well described Aspidisca lynceus (Müller, ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44-68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11-13 dorsal kineties with 17-25 dikinetids in the mid-dorsal row, and dorsal silverline system of the double-eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the "well-known" Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this "well-known" form in molecular databases requires further confirmation.

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al-Adaid Lagoon in Qatar.

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al-Adaid Lagoon in Qatar were investigated based on live observation as well as protargol- and silver nitrate-impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50-65 × 33-40 µm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral-transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans-Euplotes nobilii-Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.

Morphology and SSU rRNA gene-based phylogeny of two marine Euplotes species, E. orientalis spec. nov. and E. raikovi (Ciliophora, Euplotida).

The living morphology, infraciliature and silverline system of two small marine Euplotes species, E. orientalis spec. nov. and E. raikoviAgamaliev, 1966, isolated from a sandy beach near Qingdao, China, were investigated. Euplotes orientalis is characterized by a combination of features including their small size (35-45 microm long), five or six conspicuous dorsal ridges, two cilia-free basal plaques, eight normal-sized frontoventral cirri (FVC), and a double-patella-I type of silverline system. Euplotes raikovi is redescribed based on a Chinese population and includes the first detailed description of its morphology in vivo. It can be recognized by having one highly reduced and seven normal-sized frontoventral cirri, a single marginal cirrus, and a double-patella-I type of silverline system. For both species the small subunit rRNA (SSU rRNA) gene sequence was determined. Phylogenetic analyses based on these data indicate that E. orientalis is most closely related to E. plicatum and E. bisulcatus, whereas E. raikovi clusters with its conspecific strains, close to E. nobilii and E. elegans.

Heavy metal resistant freshwater ciliate, Euplotes mutabilis, isolated from industrial effluents has potential to decontaminate wastewater of toxic metals.

The ciliate, Euplotes mutabilis, isolated from industrial wastewater of tanneries of Kasur, Pakistan, showed tolerance against Cd2+ (22 microg ml(-1)), Cr6+ (60 microg ml(-1)), Pb2+ (75 microg ml(-1)) and Cu2+ (22 microg ml(-1)). The heavy metals, Cr and Pb, were randomly selected for determining the capability of the ciliate to reduce the concentration of these metal ions in the medium and to evaluate its potential use as bioremediator of wastewater. The live protozoans could remove 97% of Pb2+ and 98% of Cr6+ from the medium, 96 h after inoculation of the medium containing 10 micro gml(-1) of metal ions. The acid digestion of ciliate showed 89% of Pb2+ and 93% of Cr6+ ions accumulated in the organism. When the ciliate was exposed to heavy metals at a larger scale viz., 10 l of water containing 10 micro gml(-1) of heavy metals, it removed 86% of Pb2+ and 90% of Cr6+ from the medium. The metal uptake ability of E. mutabilis, as evidenced by its survival and growth in 100ml and 10 l of water containing 10 microg ml(-1) of metal ions, reduction in the concentration of heavy metals in the medium and its increased uptake by the live cells, and no metal uptake by the heat killed ciliate can be exploited for metal detoxification of industrial wastes and environmental clean-up operations.

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia).

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.

Identification of the tubulin gene family and sequence determination of one beta-tubulin gene in a cold-poikilotherm protozoan, the antarctic ciliate Euplotes focardii.

Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly cold-adapted, Antarctic ciliate: one of 1,800 bp for alpha-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for beta-tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp beta-tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of alpha-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.