The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria . The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria , and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

a b s t r a c tThe Nassophorea and Prostomatea are two of the key classes in understanding the morphological diversification and higher classification of the phylum Ciliophora. However, their phylogenetic relationships with other ciliate groups within the subphylum Intramacronucleata remain elusive. In this study, we investigated the small and large subunit (SSU and LSU) rRNA gene-based phylogeny of these groups with sequences of additional taxa including several key species. The results show that: (1) the class Nassophorea remains polyphyletic, with the microthoracids clustering with the Phyllopharyngea, whereas the nassulids represent a basal group of the CONthreeP superclade in the SSU tree; (2) the Prostomatea is not depicted as a monophyletic group in phylogenetic trees, and the monophyly of this class is marginally rejected by statistical tree topology tests; (3) the nassulid genus Parafurgasonia is more closely related to the family Colpodidiidae than to Furgasonia; (4) Paranassula, which was previously thought to be a nassulid, is phylogenetically related to the oligohymenophorean peniculids in both the SSU and LSU trees; (5) the microthoracid genus Discotricha does not group with the other microthoracids in either SSU or LSU trees; (6) the family Plagiocampidae is closely related to the prostome parasite Cryptocaryon irritans and to the family Urotrichidae in the order Prorodontida; and (7) the family Placidae, represented by Placus salinus, is sister to the family Holophryidae in the order Prorodontida. Based on the present data, we consider the genus Discotricha to be an unclassified taxon within the CONthreeP. We also propose resurrecting the order Paranassulida and classifying it within the subclass Peniculia, class Oligohymenophorea. Primary and secondary structure signatures for higher taxa within Phyllopharyngea and Nassophorea are supplied.

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The encystment of many ciliates is an advanced survival strategy against adversity and the most important reason for ciliates existence worldwide. However, the molecular mechanism for the encystment of free-living ciliates is poorly understood. Here, we performed comparative transcriptomic analysis of dormant cysts and trophonts from Pseudourostyla cristata using transcriptomics, qRT-PCR and bioinformatic techniques. We identified 2565 differentially expressed unigenes between the dormant cysts and the trophonts. The total number of differentially expressed genes in GO database was 1752. The differential unigenes noted to the GO terms were 1993. These differential categories were mainly related to polyamine transport, pectin decomposition, cytoplasmic translation, ribosome, respiratory chain, ribosome structure, ion channel activity, and RNA ligation. A total of 224 different pathways were mapped. Among them, 184 pathways were upregulated, while 162 were downregulated. Further investigation showed that the calcium and AMPK signaling pathway had important induction effects on the encystment. In addition, FOXO and ubiquitin-mediated proteolysis signaling pathway jointly regulated the encystment. Based on these findings, we propose a hypothetical signaling network that regulates Pseudourostyla cristata encystment. Overall, these results provide deeper insights into the molecular mechanisms of ciliates encystment and adaptation to adverse environments.of Strepkiella histriomuscorum has been studied in the encystment and trophont stages through biochip technology, which suggested that cyst formation is associated with Ribosomal L7, Ribosomal acidic P2, Cathepsin B, Cathepsin H, Ubiquitin, Ca 2+ -ATPase and Actin1 7 . Other studies investigated the differential proteins of the cysts and the trophonts from Pseudourostyla cristata (P. cristata) using shotgun LC-MS/MS, finding the association of fibrillarin-like rRNA methylase, methylmalonyl-coenzyme mutase, ADP ribosylation factor, Rab12, MAPK-related kinase and KR multi-domain proteins with cyst formation 8 . However, the reports of systematically studying the genes and signaling pathways involved in the formation of ciliate cysts at the molecular level are rare 8 . In this study, we have investigated the molecular mechanism underlying the cyst formation in ciliates using comparative transcriptomic analysis, quantitative real-time PCR (qRT-PCR) and bioinformatic techniques. Furthermore, we have focused on the analysis of several important signaling pathways related to the encystment. Therefore, we have proposed a hypothetical network that regulates Pseudourostyla cristata encystment. Our study generates novel insights into the molecular mechanisms of the cyst formation in free-living ciliates.www.nature.com/scientificreports www.nature.com/scientificreports/ dormant cysts of P. cristata (Fig. 4). Validation of results by transcriptome sequencing showed that mRNA outcomes were consistent with the transcriptomic data of trophonts and dormant cysts in P. cristata (Fig. 4).

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.A wide variety of protists, including hymenostome ciliates, have relatively simple body structures with few morphological characters for species circumscription, which makes them difficult to separate from each other (Borden et al. 1977;Simon et al. 2008). Early taxonomists identified and classified such organisms based only on morphological and cell cycle characters. As a consequence, independent species were sometimes misidentified as morphologically similar species within a morphospecies complex (for instance, Paramecium aurelia). The development of modern methods, especially gene sequencing and phylogenetic analyses, has improved this situation and the taxonomic positions of several such species have been clarified in recent years (Quintela-Alonso et al. 2013;Zhao et al. 2016).Cryptic species of ciliates were first discovered by Sonneborn (1939) for the model organism P. aurelia Ehrenberg, 1838. Cryptic species have since been found in a variety of ciliate genera including Aspidisca, Colpidium, Euplotes, Glaucoma, Oxytricha, Paramecium, Pseudourostyla, Stylonychia, Tetrahymena, Tokophrya, and Uronychia (Dini and Nyberg 1993;Przybos 1986;Simon et al. 2008). The probable wide occurrence of cryptic speciation has been cited as evidence for the underestimation of ciliate species diversity (Foissner et al. 2007).Tetrahymena pyriformis (Ehrenberg, 1830) Lwoff, 1947 is another example now known to be a species complex (Nanney and McCoy 1976). Species of Tetrahymena were previously assigned to the genera Leucophyrs or Glaucoma until the establishment of the genus Tetrahymena by Furgason (1940). Using mating type tests, Gruchy (1955) first discovered the heterogeneity of T. pyriformis by describin...

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