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).

Ciliated protists are a large group of single-cell eukaryotes, leading to the resting cysts in unfavorable environmental condition. However, the underlying molecular mechanism of encystment in the free-living ciliates is poorly understood. Here we show that the resting cysts are better than the vegetative cells of Euplotes encysticus in adverse survivor with respect to energy metabolism. Therefore scale identification of encystment-related proteins in Euplotes encysticus was investigated by iTRAQ analysis. We analyzed a total of 130 proteins, in which 19 proteins involving 12 upregulated and 7 downregulated proteins were associated with encystment in the resting cysts in comparison with the vegetative cells. Moreover, direct fluorescent labeling analysis showed that the vegetative cells treated with shRNA-β-tubulin recombinant E. coli accumulated a large number of granular materials, and dramatic cell morphology changes. Importantly, the cell membrane rupture phenomenon was observed after three weeks of shRNA-β-tubulin interference as compared to the control group. These results revealed that different proteins might play an important role in the process of the vegetative cells into the resting cysts. These results will help to reveal the morphological changes and molecular mechanism of resting cyst formation of ciliates.

Ciliated protozoans form dormant cysts for survival under adverse conditions. The molecular mechanisms regulating this process are critical for understanding how single-celled eukaryotes adapt to the environment. Despite the accumulated data on morphology and gene coding sequences, the molecular mechanism by which lncRNAs regulate ciliate encystment remains unknown. Here, we first detected and analyzed the lncRNA expression profile and coexpressed mRNAs in dormant cysts versus vegetative cells in the hypotrich ciliate Pseudourostyla cristata by high-throughput sequencing and qRT-PCR. A total of 853 differentially expressed lncRNAs were identified. Compared to vegetative cells, 439 and 414 lncRNAs were upregulated and downregulated, respectively, while 47 lncRNAs were specifically expressed in dormant cysts. A lncRNA-mRNA coexpression network was constructed, and the possible roles of lncRNAs were screened. Three of the identified lncRNAs, DN12058, DN20924 and DN30855, were found to play roles in fostering encystment via their coexpressed mRNAs. These lncRNAs can regulate a variety of physiological activities that are essential for encystment, including autophagy, protein degradation, the intracellular calcium concentration, microtubule-associated dynein and microtubule interactions, and cell proliferation inhibition. These findings provide the first insight into the potentially functional lncRNAs and their coexpressed mRNAs involved in the dormancy of ciliated protozoa and contribute new evidence for understanding the molecular mechanisms regulating encystment.

MicroRNAs (miRNAs) regulate the expression of target genes in diverse cellular processes and play important roles in different physiological processes. However, little is known about the microRNAome (miRNAome) during encystment of ciliated protozoa. In the current study, we first investigated the differentially expressed miRNAs and relative signaling pathways participating in the transformation of vegetative cells into dormant cysts of Pseudourostyla cristata (P. cristata). A total of 1608 known miRNAs were found in the two libraries. There were 165 miRNAs with 1217 target miRNAs. The total number of differential miRNAs screened between vegetative cells and dormant cysts databases were 449 with p < 0.05 and |log2 fold changes| > 1. Among them, the upregulated and downregulated miRNAs were 243 and 206, respectively. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that some of the differentially expressed miRNAs were mainly associated with oxidative phosphorylation, two-component system, and biosynthesis of amino acids. Combining with our bioinformatics analyzes, some differentially expressed miRNAs including miR-143, miR-23b-3p, miR-28, and miR-744-5p participates in the encystment of P. cristata. Based on these findings, we propose a hypothetical signaling network of miRNAs regulating or promoting P. cristata encystment. This study shed new lights on the regulatory mechanisms of miRNAs in encystment of ciliated protozoa.