Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Invasion by exogenous RNA: cellular defense strategies and implications for RNA inference.

Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms. Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA, including endo-lysosomal degradation, RNA-dependent repression and innate immune clearance. Across a variety of mechanisms, the natural anti-sense RNA-dependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference (RNAi). To optimize the efficiency of RNAi silencing, a comprehensive understanding of the whole life cycle of exogenous RNA, from cellular entry to its decay, is vital. In this paper, we review recent progress in comprehending the recognition and elimination of foreign RNA by cells, focusing on cellular entrance, intracellular transportation, and immune-inflammatory responses. By leveraging these insights, we highlight the potential implications of these insights for advancing RNA interference efficiency, underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms, and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.

The pooled prevalence and influential factors of non-suicidal self-injury in non-clinical samples during the COVID-19 outbreak: A meta-analysis.

BACKGROUND: COVID-19 has had an enormous impact on the mental health of people around the world, particularly adolescents. Non-suicidal self-injury (NSSI) is one of the most prominent and dangerous behaviors associated with suicide. However, few meta-analyses of the NSSI prevalence have ever been conducted since the COVID-19 outbreak. Here, we conducted a meta-analysis to estimate the pooled prevalence and elucidate the influencing factors for NSSI. METHODS: We searched PubMed, Web of Science, Embase, APA PsycINFO, CNKI and Wanfang Database for relevant literature published before April 2022. Pooled prevalence and 95 % confidence interval (CI) were used to assess NSSI prevalence. Subgroup and meta-regression analyses were performed to clarify the potential influencing factors. RESULTS: A total of 15 studies with 24,055 participants were eventually included. The results showed that the pooled overall prevalence of NSSI among overall samples during the COVID-19 pandemic was 22.5 % (95 % CI: 17.2 % to 28.9 %). Subgroup and meta-regression analyses revealed that the crucial influencing factors for NSSI included gender, age, regional distribution, and suicidal ideation. Specifically, the NSSI prevalence among adolescents and adults during the pandemic was 32.40 % and 15.70 %, respectively. Most importantly, gender is a significant influencing factor for NSSI among adolescents. CONCLUSIONS: The pooled prevalence of NSSI during the COVID-19 outbreak has surged to alarming heights, especially among adolescents. The prevalence of NSSI may be influenced by complex factors such as gender and age. Therefore, it is critical to pay attention to NSSI behaviors in the adolescent population, particularly male adolescents who appear to be susceptible.

Single-cell transcriptome reveals cell division-regulated hub genes in the unicellular eukaryote Paramecium.

The transition from growth to division during the cell cycle encompasses numerous conserved processes such as large-scale DNA replication and protein synthesis. In ciliate cells, asexual cell division is accompanied by additional cellular changes including amitotic nuclear division, extensive ciliogenesis, and trichocyst replication. However, the molecular mechanisms underlying these processes remain elusive. In this study, we present single-cell gene expression profiles of Paramecium cf. multimicronucleatum cells undergoing cell division. Our results reveal that the most up-regulated genes in dividing cells compared to growing cells are associated with 1) cell cycle signaling pathways including transcription, DNA replication, chromosome segregation and protein degradation; 2) microtubule proteins and tubulin glycylases which are essential for ciliogenesis, nuclei separation and structural differentiation signaling; and 3) trichocyst matrix proteins involved in trichocyst synthesis and reproduction. Furthermore, weighted gene co-expression network analysis identified hub genes that may play crucial roles during cell division. Our findings provide insights into cell cycle regulators, microtubules and trichocyst matrix proteins that may exert influence on this process in ciliates.

Developmental mRNA clearance by PIWI-bound endo-siRNAs in Paramecium.

The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether similar processes exist in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of PIWI-clade Argonautes involved in various small RNA (sRNA) pathways, many of which have not yet been investigated. Here, we investigate the function of a PIWI protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endogenous small interfering RNA (endo-siRNA) pathway involved in the clearance of untranslated mRNAs. These endo-siRNAs are found in clusters that are strictly antisense to their target mRNAs and are a subset of siRNA-producing clusters (SRCs). Furthermore, the endo-siRNAs are 2'-O-methylated by Hen1 and require Dcr1 for their biogenesis. Our findings suggest that sRNA-mediated developmental mRNA clearance extends beyond metazoans and may be a more widespread mechanism than previously anticipated.

Small RNA-mediated genome rearrangement pathways in ciliates.

Most eukaryotes employ a combination of transcriptional and post-transcriptional silencing mechanisms to suppress transposons, yet ciliates employ a more extreme approach. They separate germline and somatic functions into distinct nuclei, enabling the elimination of transposons from the active somatic genome through diverse small RNA-mediated genome rearrangement pathways during sexual processes.

An in vitro assay for enzymatic studies on human ALG13/14 heterodimeric UDP-N-acetylglucosamine transferase.

The second step of eukaryotic lipid-linked oligosaccharide (LLO) biosynthesis is catalyzed by the conserved ALG13/ALG14 heterodimeric UDP-N-acetylglucosamine transferase (GnTase). In humans, mutations in ALG13 or ALG14 lead to severe neurological disorders with a multisystem phenotype, known as ALG13/14-CDG (congenital disorders of glycosylation). How these mutations relate to disease is unknown because to date, a reliable GnTase assay for studying the ALG13/14 complex is lacking. Here we describe the development of a liquid chromatography/mass spectrometry-based quantitative GnTase assay using chemically synthesized GlcNAc-pyrophosphate-dolichol as the acceptor and purified human ALG13/14 dimeric enzyme. This assay enabled us to demonstrate that in contrast to the literature, only the shorter human ALG13 isoform 2, but not the longer isoform 1 forms a functional complex with ALG14 that participates in LLO synthesis. The longer ALG13 isoform 1 does not form a complex with ALG14 and therefore lacks GnTase activity. Importantly, we further established a quantitative assay for GnTase activities of ALG13- and ALG14-CDG variant alleles, demonstrating that GnTase deficiency is the cause of ALG13/14-CDG phenotypes.

The taxonomy and molecular phylogeny of two epibiotic colonial peritrich ciliates (Ciliophora, Peritrichia).

Two colonial sessilid peritrichs, Epistylis qingdaoensis n. sp. and Carchesium cyclopidarum Nenninger, 1948, were isolated from a marine crustacean and a freshwater mayfly, respectively. Morphological characters for each species were revealed by in vivo observations and silver staining methods. Epistylis qingdaoensis n. sp. is characterized by the apperance of the colony which is up to 250 µm high and usually contains fewer than eight zooids, the single-layered peristomial lip, the conspicuously conical peristomial disc, and the structure of infundibular polykinety 3 which comprises three isometric ciliary rows. Carchesium cyclopidarum is recognized by the colony height of about 600 µm, the small zooid size of 35-50 × 20-30 µm, and the low number of silverlines. Phylogenetic analyses based on small subunit ribosomal DNA sequences were performed to reveal their evolutionary relationships. Surprisingly, neither species clustered with its congeners. Epistylis qingdaoensis n. sp. nested within a clade of Zoothamnium species that was sister to a clade comprising core Epistylis species and several Zoothamnium species. Carchesium cyclopidarum nested within a clade comprising solitary peritrichs and was distantly related to the type species of Carchesium, C. polypinum. These findings imply for the first time that Carchesium is non-monophyletic.

A small RNA-guided PRC2 complex eliminates DNA as an extreme form of transposon silencing.

In animal germlines, transposons are silenced at the transcriptional or post-transcriptional level to prevent deleterious expression. Ciliates employ a more direct approach by physically eliminating transposons from their soma, utilizing piRNAs to recognize transposons and imprecisely excise them. Ancient, mutated transposons often do not require piRNAs and are precisely eliminated. Here, we characterize the Polycomb Repressive Complex 2 (PRC2) in Paramecium and demonstrate its involvement in the removal of transposons and transposon-derived DNA. Our results reveal a striking difference between the elimination of new and ancient transposons at the chromatin level and show that the complex may be guided by Piwi-bound small RNAs (sRNAs). We propose that imprecise elimination in ciliates originates from an ancient transposon silencing mechanism, much like in plants and metazoans, through sRNAs, repressive methylation marks, and heterochromatin formation. However, it is taken a step further by eliminating DNA as an extreme form of transposon silencing.

Investigation of Parental Acceptance of General Anesthesia With Mask Induction in Children Undergoing Frenulectomy.

PURPOSE: To investigate parental acceptance of the use of general anesthesia with mask inhalation (GAMI) in the treatment of ankyloglossia. DESIGN: Parents of children with ankyloglossia received questionnaires to analyze the related factors of their acceptance of GAMI. METHODS: From July 2017 to November 2020, 131 parents of children with ankyloglossia in our hospital were enrolled and received investigation questionnaires. A total of 129 valid questionnaires were returned. The level of acceptance was evaluated using the visual analogue scale (VAS). We described the parental acceptance in a statistical method and performed univariant and multivariate analyses to identify related factors using SPSS 20.0. FINDINGS: A total of 129 (98.5%) parents completed the questionnaires. Only one patient (0.8%) experienced short-term (4 hours) abdominal bloating after surgery with GAMI. The average VAS regarding parental acceptance of the use of GAMI in the treatment was 43.80 mm (± 29.49), with only 17.8% of parents exhibiting a high level of acceptance of the anesthesia technique, while they had a relatively high level of satisfaction after surgery. CONCLUSIONS: Parents had a low level of acceptance of using GAMI in the treatment of ankyloglossia before surgery due to various factors.

Regulation of Semaphorin3A in the process of cutaneous wound healing.

Semaphorin 3A (Sema3A) has been recognized as a crucial regulator of morphogenesis and homeostasis over a wide range of organ systems. However, its function in cutaneous wound healing is poorly understood. In our study, we demonstrated that Sema3A adenovirus plasmids transfection limited keratinocyte proliferation and decreased migrative capacity as assessed by in vitro wound healing assay. Sema3A transduction inhibited TGF-ß1-mediated keratinocyte migration and EMT process. Besides, we applied mice with K14-Cre-mediated deletion of Sema3A and found that Sema3A depletion postponed wound closure with decreased re-epithelialization and matrix growth. Contrary to the results obtained with full-length Sema3A plasmids transfection, increased keratinocyte migration with recombinant Sema3A proteins resulted in quicker closure of the wounding area after a scratch. Further, exogenously applied recombinant Sema3A worked with EGF to maintain the activation of EGFR by interacting with NRP1 and thereby regulated the internalization of the EGFR-NRP1 complex. Taken together, these results indicated a paradoxical role of autonomous and non-autonomous Sema3A expression during wound healing. Combined administration of recombinant EGF and Sema3A proteins could accelerate the process of wound repair, thus providing promising treatment prospects in the future.

Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

The pooled prevalence of the mental problems of Chinese medical staff during the COVID-19 outbreak: A meta-analysis.

BACKGROUND: The COVID-19 pandemic has had a great impact on the mental health of the medical staff in China, especially those on the first-line (frontline) of the pandemic. But the profile of the mental problem of nationwide Chinese medical staff is still unclear, especially about the sleep problems. METHODS: There are five databases (PubMed, Embase, CNKI, Wanfang Database and Web of Science) searched to identify the published studies on the mental health of the medical staff in China during the COVID-19 outbreak. The pooled prevalence of mental problems of Chinese medical staff during the pandemic were calculated, especially for the first-line medical staff. Subgroup analysis and meta-regression analysis were performed to identify the potential impact factors. RESULTS: A total of 71 articles including 98,533 participants are included in this meta-analysis. The results showed that the pooled prevalence of the mental problems was as follows: anxiety problem 27%, depression problem 29%, sleep problem 40%. Subgroup analysis showed that there were significant differences in the prevalence of anxiety and depression problems between first-line and non-first-line medical staff (p < 0.01). Sex had a significant impact on the sleep of first-line medical staff (p < 0.01). LIMITATIONS: There may be heterogeneity among the included studies. The analysis of potential influencing factors remains limited. CONCLUSIONS: The prevalence of adverse mental problems among medical staff is high during the COVID-19 outbreak. We need to pay special attention to the mental health of first-line medical staff, especially the sleep problems of female first-line workers.

Timing and characteristics of nuclear events during conjugation and genomic exclusion in Paramecium multimicronucleatum.

Ciliated protists are ideal material for studying the origin and evolution of sex, because of their nuclear dimorphism (containing both germline micronucleus and somatic macronucleus in the same cytoplasm), special sexual processes (conjugation and autogamy), and high diversity of mating-type systems. However, the study of sexual process is limited to only a few species, due to the difficulties in inducing or observing conjugation. In the present study, we investigate the conjugation process in Paramecium multimicronucleatum: (1) of the three prezygotic divisions, all micronuclei undergo the first two divisions (meiosis I, II), while a variable number of nuclei undergo the third division (mitosis); (2) the synkaryon divides three times after fertilization, giving rise to eight products that differentiate into four macronuclear anlagen and four micronuclei; (3) cells restore the vegetative stage after two successive cell fissions during which the macronuclear anlagen are distributed into daughter cells without division, while micronuclei divide mitotically; (4) the parental macronucleus begins to fragment following the first meiotic division and finally degenerates completely; (5) the entire process takes about 110 h, of which about 85 h are required for macronuclear development. In addition, we describe for the first time the process of genomic exclusion occurring between amicronucleate and micronucleate cells of P. multimicronucleatum, during which the micronucleate cell contributes a pronucleus to the amicronucleate cell, resulting in both exconjugants being homozygotes. These results provide new insights into the diversity of sexual processes and lay an important cytological basis for future in-depth studies of mating systems in ciliates.

New considerations on the phylogeny of Sessilida (Protista: Ciliophora: Peritrichia) based on multiple-gene information, with emphasis on colonial taxa.

The subclass Peritrichia, containing two orders Sessilida and Mobilida, is a major group of ciliates with worldwide distribution and high species diversity. Several studies have investigated the phylogeny of peritrichs; however, the evolutionary relationships and classification of some families and genera within the Sessilida remain unclear. In the present study, we isolated and identified 22 peritrich populations representing four families and six genera and obtained 64 rDNA sequences to perform phylogenetic analyses and assess their systematic relationships. Ancestral character reconstruction was also carried out to infer evolutionary routes within the Sessilida. The results indicate: (1) family Vaginicolidae is monophyletic and acquisition of the typical peritrich lorica represents a single evolutionary divergence; (2) core epistylidids evolved from a Zoothamnium-like ancestor and experienced spasmoneme loss during evolution; (3) Campanella clusters with species in the basal clade and shows stable morphological differences with other epistylidids, supporting its assignment to a separate family; (4) the structure of the peristomial lip may be a genus-level character rather than a diagnostic character for discriminating Epistylididae and Operculariidae, thus a redefinition of Operculariidae should be carried out when more species have been investigated; (5) some characters, such as lifestyle (solitary or colonial), spasmoneme and living habit (sessile or free-swimming), evolved repeatedly among sessilids indicating that species with non-contractile stalks or that are free-swimming have multiple evolutionary routes and might derive from any sessilid lineage without a lorica. The close phylogenetic relationships of some morphologically distinct sessilids imply that the diagnoses of some genera and families should be improved.

How do humans group non-rigid objects in multiple object tracking?: Evidence from grouping by self-rotation.

Previous studies on perceptual grouping found that people can use spatiotemporal and featural information to group spatially separated rigid objects into a unit while tracking moving objects. However, few studies have tested the role of objects' self-motion information in perceptual grouping, although it is of great significance to the motion perception in the three-dimensional space. In natural environments, objects always move in translation and rotation at the same time. The self-rotation of the objects seriously destroys objects' rigidity and topology, creates conflicting movement signals and results in crowding effects. Thus, this study sought to examine the specific role played by self-rotation information on grouping spatially separated non-rigid objects through a modified multiple object tracking (MOT) paradigm with self-rotating objects. Experiment 1 found that people could use self-rotation information to group spatially separated non-rigid objects, even though this information was deleterious for attentive tracking and irrelevant to the task requirements, and people seemed to use it strategically rather than automatically. Experiment 2 provided stronger evidence that this grouping advantage did come from the self-rotation per se rather than surface-level cues arising from self-rotation (e.g. similar 2D motion signals and common shapes). Experiment 3 changed the stimuli to more natural 3D cubes to strengthen the impression of self-rotation and again found that self-rotation improved grouping. Finally, Experiment 4 demonstrated that grouping by self-rotation and grouping by changing shape were statistically comparable but additive, suggesting that they were two different sources of the object information. Thus, grouping by self-rotation mainly benefited from the perceptual differences in motion flow fields rather than in deformation. Overall, this study is the first attempt to identify self-motion as a new feature that people can use to group objects in dynamic scenes and shed light on debates about what entities/units we group and what kinds of information about a target we process while tracking objects.

The hidden genomic diversity of ciliated protists revealed by single-cell genome sequencing.

BACKGROUND: Ciliated protists are a widely distributed, morphologically diverse, and genetically heterogeneous group of unicellular organisms, usually known for containing two types of nuclei: a transcribed polyploid macronucleus involved in gene expression and a silent diploid micronucleus responsible for transmission of genetic material during sexual reproduction and generation of the macronucleus. Although studies in a few species of culturable ciliated protists have revealed the highly dynamic nature of replicative and recombination events relating the micronucleus to the macronucleus, the broader understanding of the genomic diversity of ciliated protists, as well as their phylogenetic relationships and metabolic potential, has been hampered by the inability to culture numerous other species under laboratory conditions, as well as the presence of symbiotic bacteria and microalgae which provide a challenge for current sequencing technologies. Here, we optimized single-cell sequencing methods and associated data analyses, to effectively remove contamination by commensal bacteria, and generated high-quality genomes for a number of Euplotia species. RESULTS: We obtained eight high-quality Euplotia genomes by using single-cell genome sequencing techniques. The genomes have high genomic completeness, with sizes between 68 and 125 M and gene numbers between 14K and 25K. Through comparative genomic analysis, we found that there are a large number of gene expansion events in Euplotia genomes, and these expansions are closely related to the phenotypic evolution and specific environmental adaptations of individual species. We further found four distinct subgroups in the genus Euplotes, which exhibited considerable genetic distance and relative lack of conserved genomic syntenies. Comparative genomic analyses of Uronychia and its relatives revealed significant gene expansion associated with the ciliary movement machinery, which may be related to the unique and strong swimming ability. CONCLUSIONS: We employed single-cell genomics to obtain eight ciliate genomes, characterized the underestimated genomic diversity of Euplotia, and determined the divergence time of representative species in this subclass for the first time. We also further investigated the extensive duplication events associated with speciation and environmental adaptation. This study provides a unique and valuable resource for understanding the evolutionary history and genetic diversity of ciliates.

Notes on Homalogastra similis nom. nov. (Ciliophora, Scuticociliatia), a replacement name for the junior homonym Homalogastra binucleata Liu et al., 2020.

We recently established a new species, Homalogastra binucleata, with a morphological description and phylogenetic analyses based on SSU rRNA gene sequence data (Liu et al., 2020). Helmut Berger (Salzburg, Austria) commented that Homalogastra binucleata Liu et al., 2020 is a junior primary homonym of H. binucleata Song, 1993, the latter having been transferred to the genus Uronemita by Liu et al. (2016). According to article 57.2 of the ICZN (1999), the junior primary homonym, Homalogastra binucleata Liu et al., 2020, is permanently invalid. It is replaced by Homalogastra similis nom. nov.

The Dental Health of Orphan and Non-orphan Children in Fuyang City, China.

PURPOSE: To assess the dental health of orphan and non-orphan children in Fuyang City, China. MATERIALS AND METHODS: A total of 332 orphan children were selected from social child welfare institutes and 590 non-orphan children were selected from the Experimental Primary and Middle Schools through random sampling in Fuyang City, China. The indices for Decayed, Missing, Filling (DMF) in orphan and non-orphan children were determined by dental inspections to assess their dental health. The periodontal status of orphan and non-orphan children aged 12-15 years was determined using the percentages of bleeding gingiva and dental calculus. RESULTS: In the age range of 3 to 5 years, the percentage of caries (81%) and the mean DMF (4.41; SD: 2.06; 95% CI: 3.82 to 5.00) of orphan children were statistically significantly higher than the percentage of caries (64%) and the mean DMF of the non-orphan control cohort (3.29; SD: 2.05; 95% CI: 2.87 to 3.71; p < 0.05). In the age range of 12 to 15 years, the percentage of caries (50.8%) and the mean DMF (1.28; SD: 1.26; 95% CI: 1.12 to 1.43) of orphan children were statistically significantly higher than the percentage of caries (34.5%) and the mean DMF (1.11; SD: 1.24; 95% CI: 1.01 to 1.23; p < 0.05) of non-orphan children. For orphan children ages 3 to 5 years, the percentage of restorations was statistically significantly lower (p < 0.05) than that of non-orphan children (30%). For orphan children ages 12 to 15 years, the percentage of restorations was 3.9%, statistically significantly lower (p < 0.05) than that of non-orphan children (19.4%). In orphan children ages 12 to 15 years, the percentages of bleeding gingiva (76.0%) and dental calculus (69.3%) were statistically significantly higher (p < 0.05) than those of the non-orphan children (46.2% and 39.1%, respectively). There was no statistically significant difference in the evaluation indicators above between different genders within the groups studied, except the percentage of restorations. CONCLUSION: The dental health of orphan children in Fuyang City is worse than that of non-orphan children of the same age ranges. The values determined in this study can be used as a starting metric to measure the effectiveness of dental health care programs in improving the dental health of both orphan and non-orphan children.

The Morphology, Taxonomy, and Phylogenetic Analyses of Five Freshwater Colonial Peritrich Ciliates (Alveolata, Ciliophora), Including the Descriptions of Two New Species.

The morphology and phylogeny of two new sessilid species, Zoothamnium weishanicum n. sp. and Epicarchesium sinense n. sp., two insufficiently known species, Zoothamnium arbuscula Ehrenberg, 1831 and Zoothamnium hentscheli Kahl, 1935, and a well-known species, Carchesium polypinum (Linnaeus, 1767) Ehrenberg, 1838, collected from freshwater habitats of China, were investigated. Zoothamnium weishanicum n. sp. is characterized by its inverted bell-shaped zooids, double-layered peristomial lip, alternately branched stalk, and two different-length rows in infundibular polykinety 3 (P3). Epicarchesium sinense n. sp. is recognized by its asymmetric-pyriform zooids, single-layered peristomial lip, conspicuous cortical blisters on the pellicle, dichotomously branched stalk, and P3 containing one short inner row and two long outer rows. Based on previous and newly obtained data of the three known species, improved diagnoses and redescriptions are provided including, for the first time, data on the infraciliature of Z. arbuscula and Z. hentscheli. In addition, we analyzed the phylogeny of each species based on SSU rDNA sequence data.