Skip to main content
NCBI home page
Marine Life Science & Technology logoLink to Marine Life Science & Technology
. 2022 Nov 21;4(4):609–623. doi: 10.1007/s42995-022-00147-w

Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists

Zhicheng Chen 1,#, Jia Li 1,#, Dayana E Salas-Leiva 2, Miaoying Chen 1, Shilong Chen 1, Senru Li 1, Yanyan Wu 1, Zhenzhen Yi 1,✉
PMCID: PMC10077286  PMID: 37078085

Abstract

Adaptations of ciliates to hypoxic environments have arisen independently several times. Studies on mitochondrion-related organelle (MRO) metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes. To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis, mass-culture and single-cell transcriptomes of two anaerobic species, Metopus laminarius (class Armophorea) and Plagiopyla cf. narasimhamurtii (class Plagiopylea), were sequenced and their MRO metabolic maps were compared. In addition, we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes (i.e., Armophorea, Litostomatea, Muranotrichea, Oligohymenophorea, Parablepharismea and Plagiopylea). We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates. The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates, even among closely related species. Notably, our findings indicate the existence of group-specific functional relics of electron transport chains (ETCs). Detailed group-specific ETC functional patterns are as follows: full oxidative phosphorylation in Oligohymenophorea and Muranotrichea; only electron-transfer machinery in Armophorea; either of these functional types in Parablepharismea; and ETC functional absence in Litostomatea and Plagiopylea. These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times. Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42995-022-00147-w.

Keywords: Anaerobic metabolism, Mitochondrion-related organelle, Single-cell transcriptome, Evolutionary transition

Introduction

Oxygen plays a key role in typical mitochondrial ATP production. Nevertheless, numerous eukaryotes, especially single-celled forms (protists), thrive in a variety of hypoxic and anoxic habitats, such as deep seas, marine sediments, and sewage (Hackstein 2018; Hu 2014; Zhu et al. 2021). These protists possess reduced mitochondrion-related organelles (MROs) as an adaptation to low-oxygen conditions (Leger et al. 2016; Müller et al. 2012). Among obligate and facultative anaerobic protists, ciliates contain numerous free-living and endosymbiotic species that have been classified in most ciliate classes (Fig. 1) (Gao et al. 2016; Rotterová et al. 2020). Studies have revealed that the adaptation of ciliates to anaerobic conditions has occurred multiple times, indicating that MRO metabolisms of anaerobic ciliates might have diverse patterns (Embley et al. 1995; Lewis et al. 2019; Rotterová et al. 2020, 2022).

Fig. 1.

Fig. 1

Open in a new tab

Schematic depiction of ciliate anaerobes and their four group-specific functional patterns for MRO energy pathways. The figure is drawn following the systems of Gao et al. (2016) and Rotterová et al. (2020). Species are color coded for their niche environment aerobic (yellow), anaerobic (brown), and facultative (purple). In the rightmost column, classes within the same green box share conserved MRO functionality. The MRO of class Parablepharismea, placed in the middle of the first and the second green box, seems to be a functional and evolutionary continuum

In recent years, the study of MRO metabolisms in various protist groups inferred from both genomic and transcriptomic data has revealed a series of important adaptive evolutionary events (Gawryluk and Stairs 2021; Stairs et al. 2015). However, MRO metabolisms of anaerobic ciliates remain comparatively understudied, although the morphology and phylogeny of such ciliates have long been investigated (Fenchel and Finlay 1991; Fenchel et al. 1977; Jiang et al. 2021a, b; Kofoid 1943; Li et al. 2020, 2021a, b; Vďačný et al. 2011, 2019; Zhuang et al. 2021). In a previous study, the prediction of MRO metabolisms inferred from transcriptomes and genomes revealed that the anaerobic ciliate Nyctotherus ovalis, which lives in the hindgut of cockroaches, possesses hydrogen-producing mitochondria with an incomplete electron transport chain (ETC) and tricarboxylic acid (TCA) cycle (de Graaf et al. 2011). No additional or related research was published for several years, possibly due to the difficulties both of culturing anaerobic ciliates and of sequencing and assembling their genomes. Recently, MRO metabolisms of 11 anerobic species belonging to six ciliate classes were predicted from single-cell transcriptomic and genomic/metagenomic assemblies (Feng et al. 2020; Lewis et al. 2019; Park et al. 2021; Rotterová et al. 2020). Among these species, only the MRO of Isotricha intestinalis seems not to produce hydrogen, while [FeFe]-hydrogenase with a mitochondrial targeting signal (MTS) was detected in each of the other ten species. Components of ETCs seem to be completely lost in the MROs of Entodinium furca, E. caudatum, Diplodinium dentatum, and Plagiopyla frontata, whereas the incomplete ETCs of the remaining seven species have undergone a variable reduction in their components. These 11 species are from the classes Armophorea (Metopus contortus, Heterometopus sp. CSS, and metopid sp. SK), Parablepharismea (Parablepharisma sp.), Muranotrichea (Muranothrix gubernata), Oligohymenophorea (Cyclidium porcatum), Plagiopylea (Plagiopyla frontata), and Litostomatea (E. furca, E. caudatum, D. dentatum, and I. intestinalis). Based on these data, different degrees of independent MRO proteome reductive evolution among three species from Armophorea, Plagiopylea, and Oligohymenophorea were demonstrated (Lewis et al. 2019), and evolutionary adaptations of APM (Armophorea, Parablepharismea, Muranotrichea) ciliates to anaerobiosis were revealed (Rotterová et al. 2020). To date, the MRO metabolisms of most ciliate classes containing anaerobic species have not been explored (Fig. 1), and only two classes (Armophorea and Litostomatea) include MRO metabolic maps of more than one anaerobic species. Hence, evolutionary patterns of anaerobiosis remain unclear both among and within various ciliate groups due to limited numbers of predicted MRO metabolic maps (Embley et al. 1995; Lewis et al. 2019; Rotterová et al. 2020).

Comparison of MRO metabolisms from various groups/species depicting patterns of protein presence and absence is essential for outlining the adaptive evolution of MROs strategies in ciliates. Furthermore, inaccurate evolutionary reconstructions might be inferred when MRO metabolisms are predicted using data derived from techniques known to have biases that lead to incomplete data acquisition. For example, compared to mass-culture transcriptomes, single-cell transcriptomes usually have low efficiency of cDNA synthesis, which results in the absence of transcripts and limits the detection of target genes (Islam et al. 2014). To date, MRO metabolisms have been predicted for 12 anaerobic ciliate species, six of which were based on single-cell transcriptomics due to the difficulty of culturing anaerobes. To the best of our knowledge, no study has focused on the potential for or limitations of MRO metabolism prediction using single-cell transcriptomes (Feng et al. 2020; Lewis et al. 2019). We hypothesized that compared to mass-culture transcriptomes, single-cell transcriptomes are relatively effective for the discovery of MRO proteins, considering that single-cell transcriptomics applied to five ciliate species allowed the recovery of 90–100% aminoacyl-tRNA synthetases and 66–94% of core eukaryotic proteins of mass-culture transcriptomes (Kolisko et al. 2014).

Here, we evaluated the success of MRO protein discovery using single-cell transcriptomes and mass-culture transcriptomes in the anaerobic ciliates M. laminarius and P. cf. narasimhamurtii and predicted their MRO metabolisms. We compared the MRO metabolic patterns of congeners within the genera Metopus and Plagiopyla, respectively, to test whether MRO metabolic patterns of closely related species are similar. Finally, to deepen our understanding of the evolutionary patterns of anaerobiosis in ciliates, all available MRO metabolic maps of anaerobic ciliates were compared.

Results

Species identification of Metopus laminarius and Plagiopyla cf. narasimhamurtii

According to the original description of Metopus laminarius (Kahl 1927), its typical morphological characters include: preoral dome overhangs the adoral zone of membranelle and twisted towards the left of the cell; adoral zone of membranelle contains around 20–24 membranelles; oblong macronucleus located in the cell center with a small micronucleus closely nearby; the posterior contractile vacuole is present (Bourland et al. 2014; Kahl 1927). Our population matches these features well (Fig. 2, Supplementary Fig. S1). The 18S rDNA sequence similarity between our population and a known classified specimen is 99.87%. So, we identified it as M. laminarius.

Fig. 2.

Fig. 2

Open in a new tab

Micrograph and predicted MRO metabolic map of Metopus laminarius. Rounded rectangles depicted by solid outline indicate proteins detected in both mass-culture transcriptomes (MT) and single-cell transcriptomes (ST). Predicted proteins are colored according to pathways (dark blue: amino acid metabolism; light blue: pyruvate and propionate metabolism; yellow: ETC; dark green: fatty acid, lipid and lipoate metabolisms; light green: TCA cycle; purple: Fe-S cluster biosynthesis; light purple: glycolysis; brown: carriers; orange: protein import system). Subunits of ETC complexes are listed in grey boxes on the bottom right of the figure. Detailed information of predicted proteins in all pathways is listed in Supplementary Tables S1, S2. Scale bar = 50 μm

According to the original description of Plagiopyla narasimhamurtii (Nitla et al. 2019), its typical morphological characters include: around 60 rows of somatic monokinetids; striated band; three dense cytoproct-related ciliary rows on the left; two types of extrusive organelles (Nitla et al. 2019). Our population matches these features well (Fig. 3, Supplementary Fig. S1). However, the 18S rDNA sequence similarity between our population and the original species is 97.52%. For this reason, we identified it comparable to P. cf. narasimhamurtii.

Fig. 3.

Fig. 3

Open in a new tab

Micrograph and predicted MRO metabolic map of Plagiopyla cf. narasimhamurtii. Rounded rectangles enclosed by a solid outline indicate proteins detected in both mass-culture transcriptomes (MT) and single-cell transcriptomes (ST). Dashed lines indicate that the protein was identified in MT but not in ST. Predicted proteins are colored according to pathways (dark blue: amino acid metabolism; light blue: pyruvate and propionate metabolism; yellow: ETC; dark green: fatty acid, lipid and lipoate metabolisms; light green: TCA cycle; purple: Fe-S cluster biosynthesis; light purple: glycolysis; brown: carriers; orange: protein import system). Subunits of ETC complexes are listed in the grey box on the bottom right of the figure. Detailed information of the predicted proteins in all pathways is listed in Supplementary Tables S3, S4. Scale bar = 50 μm

Predicted MRO proteins from single-cell and mass-culture transcriptomes

Single-cell and mass-culture transcriptomes of M. laminarius (Armophorea, cell length of 125–175 μm) and of P. cf. narasimhamurtii (Plagiopylea, cell length of 65–90 μm) were sequenced. The cDNA of a single cell of P. cf. narasimhamurtii did not pass quality control (i.e., low concentration) for sequencing even though we tried seven times. Thus, ten P. cf. narasimhamurtii cells containing the equivalent amount of cDNA (42.48 ng) and one M. laminarius cell (46.35 ng) were sequenced using the single-cell RNA-seq method. For M. laminarius, the N50 and GC contents were 1284 and 50.26% in the single-cell transcriptomes, and 1512 and 48.04% in mass-culture transcriptomes, respectively. For P. cf. narasimhamurtii, the N50 and GC contents were 1025 and 24.18% in the single-cell transcriptomes and 939 and 31.35% in the mass-culture transcriptomes, respectively.

To characterize the MRO metabolic capabilities of M. laminarius and P. cf. narasimhamurtii, we looked for proteins involved in pathways including pyruvate and propionate metabolism, ETC, TCA cycle, Fe-S cluster biosynthesis, protein import system, carriers, oxidative stress, and amino acid, fatty acid, and lipid and lipoate metabolisms. In addition, all ten proteins involved in glycolysis, the cytoplasmic pathway associated with MRO metabolisms, were searched (Supplementary Tables S1–S4). Within these pathways, we did not find any MRO-encoded protein. In total, 70 predicted nuclear-encoded MRO proteins were detected both in the single-cell and in the mass-culture transcriptomes of M. laminarius. In contrast, 36 and 42 predicted nuclear-encoded MRO proteins were detected in single-cell and mass-culture transcriptomes of P. cf. narasimhamurtii, respectively (Table 1). Frataxin (Yfh1), monothiol glutaredoxin-5 (Grx5), phosphoenolpyruvate mutase (PEPM), translocases of the inner mitochondrial membrane 22, 44 (Tim22, Tim44) and mitochondrial-processing peptidase subunit beta (MPPβ) were detected in mass-culture transcriptomes but not in single-cell transcriptomes of P. cf. narasimhamurtii. The gene expression levels of these six MRO proteins (as well as others in the same pathways) were estimated in mass-culture transcriptomes of P. cf. narasimhamurtii to determine whether their non-detection in single-cell transcriptomes was due to low-gene expression (Table 2). For the Fe-S cluster biosynthesis pathway, the gene encoding Iron-sulfur cluster biogenesis chaperone (Ssq1), which was detected in both single-cell and mass-culture transcriptomes, was most abundant (Fragments Per Kilobase of transcript per Million mapped reads or FPKM > 1000). Compared to Ssq1, FPKM values of Yfh1 (1.70) and Grx5 (34.21), which were not detected in single-cell transcriptomes, were much lower. For the lipid and lipoate metabolism pathway, PEPM, which was not detected in single-cell transcriptomes, had the highest expression level (FPKM = 56.35). In the case of the protein import system pathway, the mitochondrial import receptor subunit TOM40 (Tom40), which was detected in both single-cell and mass-culture transcriptomes, was most abundant (FPKM = 21.31). Compared to Tom40, low expression levels were observed for the genes encoding Tim22 (FPKM = 4.40), Tim44 (FPKM = 8.87), and MPPβ (FPKM = 2.77), which were not detected in single-cell transcriptomes.

Table 1.

Numbers of predicted MRO proteins in Metopus laminarius and Plagiopyla cf. narasimhamurtii

Pathway M. laminarius P. cf. narasimhamurtii
(Single-cell transcriptomes) (Mass-culture transcriptomes) (Single-cell transcriptomes) (Mass-culture transcriptomes)
Fe-S cluster biosynthesis 8 8 5a 7
Pyruvate and propionate metabolism 8 8 6 6
ETC & TCA cycle 28 28 8 8
Amino acid metabolism 7 7 8 8
Fatty acid metabolism 1 1 0 0
Lipid and lipoate metabolism 5 5 3a 4
Carriers 3 3 3 3
Protein import system 8 8 3a 6
Oxidative stress 2 2 0 0
Total 70 70 36 42
Open in a new tab

aSome proteins in this pathway were not detected in single-cell transcriptomes but in mass-culture transcriptomes

Table 2.

Genetic expression levels in mass-culture transcriptomes of Plagiopyla cf. narasimhamurtii. FPKM: Fragments Per Kilobase of transcript per Million mapped reads or FPKM > 1000

Pathway Protein P. cf. narasimhamurtii
FPKM
Fe-S cluster biosynthesis Atm1 80.98
Yfh1 1.70a
Ssq1 9961.43
Erv1 4.42
Grx5 34.21a
Nfs1 17.12
Isu1 80.98
Lipid and lipoate metabolism PEPM 56.35a
PTPMT 1.03
LPLA 11.55
PGPS 25.12
Protein import system Tim16 2.02
Tim17 20.26
Tim22 4.40a
Tim44 8.87a
Tom40 21.31
MPPβ 2.77a
Open in a new tab

aMeans proteins not detected in single-cell transcriptomes

Prediction of MRO proteins and their metabolic potential

Pyruvate and energy metabolisms

Four subunits (E1α, E1β, E2, E3) of the pyruvate dehydrogenase (PDH) from canonical mitochondrial pyruvate metabolism producing acetyl-CoA were identified in M. laminarius and P. cf. narasimhamurtii. No evidence was detected for pyruvate: ferredoxin oxidoreductase (PFO) and pyruvate: NADP oxidoreductase (PNO). To catalyze acetyl-CoA, both species are enabled for using acetate: succinate CoA transferase (ASCT) and succinyl CoA synthetase (SCS) to produce ATP by substrate-level phosphorylation. Additionally, methyl CoA mutase (MMM) and two subunits of propionyl-CoA carboxylase (PCCα and PCCβ), which catalyze propionyl CoA to succinyl-CoA, were identified in M. laminarius. Malic enzyme was present in P. cf. narasimhamurtii but not found in M. laminarius which can catalyze malate to pyruvate. And [FeFe]-hydrogenase with C-terminal NuoE-like and NuoF-like domains was detected in both species (Figs. 2, 3).

The ETC systems differed in their degrees of reduction in M. laminarius and P. cf. narasimhamurtii. For example, M. laminarius retains some components of complexes I to III but has lost complex IV and complex V (F1F0ATP-synthase) (Fig. 2). In complex I, NADH dehydrogenase subunit 8 (Nad8) of the ubiquinone-reducing Q-module was detected, but Nad7, Nad9, and Nad10 were not detected. Nad11, NADH-quinone oxidoreductase subunits E (NuoE) and F (NuoF) of the NADH-dehydrogenase N-module of complex I were also identified. However, subunits of the proton-pumping P-module (Nad1-Nad6) of complex I appear to have been lost. Several accessory proteins in complex I, including NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunits 2, 5, and 9 (NDUFA2, NDUFA5, and NDUFA9), NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, 5, and 6 (NDUFS4, NDUFS5, and NDUFS6) were detected. In complex II, only succinate dehydrogenase subunit alpha (SdhA) and succinate dehydrogenase subunit beta (SdhB) were detected. In complex III, the electron transfer proteins rieske iron-sulfur protein (Rieske) and cytochrome C1 (CytC1) were identified. Furthermore, quinone-utilizing enzymes including alternative oxidase (AOX), glycerol-3-phosphate dehydrogenase (G3PDH), and electron transferring flavoprotein system (ETF) (i.e., ETFβ and ETFDH) were also detected. All these findings suggest that the ETC of M. laminarius appears to maintain its function of transferring electrons but has lost its ATP synthesizing function. Compared to M. laminarius, P. cf. narasimhamurtii has lost more ETC components, as only SdhA in complex II was detected. This suggests that SdhA could work in other pathways instead of the ETC and that the ETC of P. cf. narasimhamurtii might have lost its oxidative phosphorylation function.

A nearly complete TCA cycle was predicted in M. laminarius (i.e., only citrate synthase (CS) was not detected) (Fig. 2). In the case of P. cf. narasimhamurtii, only malate dehydrogenase (MDH), fumarate hydratase (FH), E1 component family protein of 2-oxoglutarate dehydrogenase (OGDH), SCS, and SdhA from the TCA cycle were detected (Fig. 3). These proteins might be functional in other pathways instead of TCA cycle. For instance, SdhA could act in reverse as a fumarate reductase (FRD) to convert fumarate into succinate as in other anaerobic eukaryotes, and then succinate can be used as CoA acceptor in the ASCT/SCS cycle.

Fe-S cluster synthesis and transporters

The machineries for Iron-Sulfur cluster assembly (ISC), sulfur mobilization (SUF), nitrogen fixation (NIF), and cysteine sulfinate desulfinate (CSD) were searched. Fe-S cluster biogenesis in MROs of both M. laminarius and P. cf. narasimhamurtii could only be carried out via the ISC pathway. The ISC pathway of M. laminarius is nearly complete. For this species, Fe-S cluster components could firstly be synthesized on Iron-Sulfur cluster assembly protein 1 (Isu1), with cysteine desulfurase (Nfs1) acting as the sulfur donor. Unfortunately, no Fe2+ donor was detected in this species. [2Fe-2S] clusters could be transferred to recipient apoproteins with the assistance of Grx5, Ssq1, and GrpE protein (Mge1) detected in this species. We were unable to detect the Iron-Sulfur cluster assemblies 1 (Isa1) and 2 (Isa2) or the Iron-Sulfur cluster assembly factor IBA57 (Iba57), which play important roles in the maturation of proteins with [4Fe-4S] clusters. By contrast, both the aconitase (Aco) containing [4Fe-4S] cluster and Iron-Sulfur cluster scaffold protein (Nfu1) transferring [4Fe-4S] clusters were found. We think that Isa1, Isa2, and Iba57 might not have been detected because this work relies on transcriptome sequencing, otherwise alternative proteins with the same functions should have been found in M. laminarius. The ISC pathway of P. cf. narasimhamurtii is similar to that of M. laminarius, with the addition of Fe2+ donor Yfh1 and loss of Nfu1 for [4Fe-4S] cluster transport.

Two proteins which export Fe-S cluster assembled by ISC system were detected in both M. laminarius and P. cf. narasimhamurtii. These were an ATP-binding cassette transporter subfamily B member 7 (Atm1) located in the mitochondrial inner membrane and a mitochondrial FAD-linked sulfhydryl oxidase (Erv1) located in the intermembrane space.

Other MRO pathways

Several enzymes involved in the metabolism of amino acids were found in both M. laminarius and P. cf. narasimhamurtii. These were alanine aminotransferase (ALAT), branched-chain amino acid aminotransferase (BCAT), 2-amino-3ketobutyrateCoA ligase (KBL), serine hydroxymethyl transferase (SHMT), aspartate aminotransferase (AspAT), and three subunits (GCSP, GCST, and GCSL) of glycine cleavage system (GCS), which are related to degradation of alanine, leucine, glycine, aspartic acid, and glutamic acid, respectively. Tryptophanase (TRN) was detected only in P. cf. narasimhamurtii.

In the fatty acid activation pathway, only 3-oxoacyl-[acyl-carrier-protein] reductase FabG (FabG), which can reduce beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, was identified in M. laminarius. No similar protein was detected in P. cf. narasimhamurtii.

In the lipid and lipoate metabolism, PEPM, phosphatidyl glycerophosphate phosphatase (PTPMT), CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase (PGPS), and lipoate-protein ligase A (LPLA), were present in both species. Phosphatidylserine decarboxylase proenzyme 1 (PSD1) was detected only in M. laminarius.

The protein import system included translocase of the outer mitochondrial membrane (Tom complex), mitochondrial sorting and assembly machinery (Sam complex), and translocase of the inner mitochondrial membrane (Tim complex). One Tom protein (i.e., Tom40) and no Sam complex were detected in MROs of both species. Tim22 complex, which is the core translocase, was detected in both species. Four subunits of the Tim23 complex (i.e., Tim16, Tim17, Tim44, heat shock 70 kDa protein (Hsp70)), were detected in M. laminarius and P. cf. narasimhamurtii, while Tim23 was only detected in M. laminarius. Oxidase assembly proteins 1 (Oxa) in the inner mitochondrial membrane, and mitochondrial processing peptidase (MPP) complex (i.e., MPPα and MPPβ) cleaving mitochondrial MTS, were present in M. laminarius.

In the oxidative stress pathway, we detected superoxide dismutase (SOD) and peroredoxin (Prx) in M. laminarius. No proteins of the oxidative stress pathway were detected in P. cf. narasimhamurtii. Finally, ADP/ATP translocase (AAC), pyridine nucleotide transhydrogenase (PNT), and mitochondrial carrier protein (MCP), carriers which could transport ADP/ATP, NAD (P), and other substances, respectively, were also found in both species.

Discussion

Discovery effectiveness of MRO proteins using single-cell transcriptomes

Previous studies have made silico-predictions of MRO metabolisms based on mass-culture/single-cell transcriptomes and genomes/metagenomes of six and four anaerobic ciliate species, respectively, as well as two that were inferred from a combination of transcriptomes and genomes/metagenomes (Fig. 4) (de Graaf et al. 2011; Feng et al. 2020; Lewis et al. 2019; Park et al. 2021; Rotterová et al. 2020). Transcriptomes are the mostly widely used resource in these studies. Inferring gene loss from transcriptomic data can be problematic because some genes may only be expressed under certain conditions (Liu et al. 2017; Xu et al. 2020). Nevertheless, predicting the absence or presence of MRO proteins of anaerobic ciliates based on transcriptomes might be the best choice at present. It is difficult to obtain full genomes of ciliates as they have two kinds of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), in a single cell. Complete MAC genomes are difficult to sequence and assemble due to extensive fragmentation and duplication of chromosomes (Sheng et al. 2018; Wang et al. 2021a, b; Yan et al. 2019). To make things more complicated, there is a high similarity between the MIC and MAC genomes, and MIC purification is still elusive (Chen et al. 2016; Duan et al. 2021). By contrast, ciliate transcriptomes are easily obtained, especially with recent advances in single-cell transcriptome sequencing which have opened the door for sequencing unculturable species (Chen et al. 2020; Xu et al. 2020; Zhang et al. 2021). Additionally, since most anaerobic ciliates are sensitive to changes in dissolved oxygen (DO) concentrations, cells or cell cultures are collected under fixed, low DO concentrations prior to sequencing. This means that these samples are enriched in transcripts for MRO metabolism and that the absence of some of these proteins inferred from transcriptomes is characteristic of the target species rather than being the result of metabolic fluxes caused by DO changes in the medium. It has also been demonstrated in the flagellate Euglena gracilis that the transcriptome is a reliable alternative to reconstruct metabolic pathways when the complete genome is difficult to assemble (Ebenezer et al. 2019).

Fig. 4.

Fig. 4

Open in a new tab

Presence and absence of proteins in ETC and pyruvate metabolisms from 14 anaerobic ciliate species and aerobic Tetrahymena thermophila (phylogenetic tree constructed inferred from 18S rDNA sequences is on the left). Data in the Coulson plots are derived from the proteome (P), single-cell transcriptomes (ST), mass-culture transcriptomes (MT), genome (G), and metagenomic (MG). Metopus laminarius and Plagiopyla cf. narasimhamurtii, species newly investigated in this study are in green. Presence and absence of MRO proteins for other species are from references: T. thermophila (Smith et al. 2007), Cyclidium porcatum, P. frontata, M. contortus (Lewis et al. 2019), Nyctotherus ovalis (de Graaf et al. 2011), metopid sp. SK, Heterometopus sp. CSS, Muranothrix gubernata, Parablepharisma sp. (Rotterová et al. 2020), Isotricha intestinalis, Diplodinium dentatum, Entodinium furca (Feng et al. 2020), and E. caudatum (Park et al. 2021). The pie charts in the most bottom column with dark brown background represent the legends of the upper pie charts

In our investigation, 100% of MRO proteins in Metopus laminarius (70 proteins out of 70, cell length of 125–175 μm) and 85% of MRO proteins in Plagiopyla cf. narasimhamurtii (36 proteins out of 42, cell length of 65–90 μm) were detected in single-cell transcriptomes compared to mass-culture transcriptomes (Table 1), showing that single-cell transcriptomes are not perfect but relatively effective for the discovery of MRO proteins. This is consistent with the findings of Kolisko et al. (2014) using single-cell transcriptomes, which recovered 66–94% of the core eukaryotic proteins that were obtained using mass-culture transcriptomes in five ciliate species (cell length of 50–500 μm). Conversely, many housekeeping genes could not be detected using single-cell transcriptomes of the dinoflagellate Karlodium veneficum or of the haptophyte Prymnesium parvum (Liu et al. 2017). Liu et al. (2017) suggested that the observed differences in single-cell transcriptomes were due to elevated stochasticity on the level of individual genes instead of physiological differences among cells because collective expression levels of major pathways and function were similar across different single-cell transcriptomes of the same protist species. In our study, the failure to detect some MRO proteins using single-cell transcriptomes likewise is unlikely to be caused by physiological differences, since a single-cell transcriptome from cDNA of one M. laminarius cell performs better than that from ten Plagiopyla cf. narasimhamurtii cells. It has been demonstrated that single-cell transcriptomes of organisms with low RNA contents per cell usually have higher stochasticity in gene expression levels and low transcript recovery rate (Liu et al. 2017; Marinov et al. 2014; Taniguchi et al. 2010). Previous study suggests that compared to protist that are large in size, small taxa, e.g., K. veneficum (cell length of ~ 15 μm) and Prymnesium parvum (cell length of ~ 8 μm), probably have much lower mRNA copy numbers, and hence their single-cell transcriptomes recover fewer genes (Liu et al. 2017). It is noteworthy that the two ciliate species studied here, and the five ciliate species studied by Kolisko et al. (2014), are much larger than K. veneficum or P. parvum. Additionally, ciliates possess very high gene copy numbers in their MAC which are produced from duplications (Wang et al. 2021a, b; Yan et al. 2019). Thus, it is reasonable to predict that single-cell transcriptomes are probably more effective in gene discovery in large (e.g., ciliates) rather than smaller (e.g., flagellate) groups. In short, this technology is not perfect, but it enables broad evolutionary exploration of MROs by offering access to many unculturable anaerobic large protists.

Reduction levels of MROs might vary among closely related anaerobic ciliate species

As revealed in all 12 reported anaerobic ciliate species (de Graaf et al. 2011; Lewis et al. 2019; Park et al. 2021; Ricard et al. 2006; Rotterová et al. 2020), the composition and function of the MROs in the newly sequenced M. laminarius and P. cf. narasimhamurtii indicate that they have undergone significant reductive changes while adapting to their anaerobic niche (Figs. 2, 3). Retention of the subsurface cristae or their remnants of obligate anaerobic ciliates in the APM clade studied to date (Clarke et al. 1993; Lewis et al. 2019; Rotterová et al. 2020), suggested that their MRO metabolisms are not as reduced as other obligate anaerobes (Rotterová et al. 2022). However, the MRO metabolic pathways of the M. laminarius show that, compared to other armophorean species, subunits of the proton-pumping P-module (Nad1–Nad6) of complex I appear to have been lost, as well as other MRO-encoded related proteins (Fig. 5).

Fig. 5.

Fig. 5

Open in a new tab

Presence and absence of proteins in several MRO metabolic pathways associated with congeners of Metopus (A), Plagiopyla (B), and Entodinium (C). Colored dots indicate proteins that were detected, and gray dots indicate non-detected proteins

The MRO components among species within class Armophorea, even congeners differ (Fig. 5, Supplementary Table S5). For example, some MRO proteins are detected in one Metopus species but not in another. There are twelve of these proteins in the ETC pathway, two in the TCA cycle pathway, three in pyruvate and propionate metabolism, six in Fe-S Cluster Biogenesis, one in the GCS system, and eight in the protein import system (Fig. 5A). Our 18S rDNA tree (Supplementary Fig. S1) indicated that these two Metopus species do not group together. Hence, we also compared MRO metabolic pathways of all available armophorean species and found 64 proteins involved in seven pathways that are only detected in some species but not in others (Supplementary Table S5). Similarly, some MRO proteins are detected in one Plagiopyla species but not in another. They are one in the ETC pathway, one in the TCA cycle pathway, five in Fe-S cluster biogenesis, one in the GCS system, and nine in the import system (Fig. 5B). By contrast, MRO components of two Entodinium species within class Litosomatea are very similar to each other. Only one MRO protein in the pyruvate metabolism pathway, one in the amino acid metabolism pathway, and one in Fe-S cluster biogenesis pathway were detected in E. furca but not E. caudatum (Fig. 5C). In the future, more taxa are needed to test whether MRO metabolic patterns of closely related species are similar in some classes but not in others.

Divergent MRO patterns with conserved functionality within groups of anaerobic ciliates

Up to date, anaerobic species have been reported in at least 14 ciliate classes (Fig. 1). Classes Armophorea, Plagiopylea, Odontostomatea, Cariacotrichea, Parablepharismea, Muranotrichea, include only anaerobes or facultative anaerobes (Fernandes et al. 2018; Lewis et al. 2019; Li et al. 2020; Lynn 2008; Orsi et al. 2012; Rotterová et al. 2020; Yan et al. 2019), whereas the predominantly aerobic classes Oligohymenophorea Prostomatea, Phyllopharyngea, Colpodea, Spirotrichea, Litostomatea, Heterotrichea, and Karyorelictea also include obligate or facultative anaerobic taxa (Fig. 1) (Arregui et al. 2010; Beinart et al. 2018a; Berger and Lynn 1992; Bernard and Fenchel 1996; Clarke et al. 1993; Edgcomb and Pachiadaki 2014; Embley et al. 1995; Feng et al. 2020; Finlay et al. 1986; Foissner and Foissner 1995; Hines et al. 2018; Pan et al. 2019). Notably, Zosterodasys sp. was reported to be the only anaerobic species of class Nassophorea (Finlay et al. 1993; Rotterová et al. 2022), but it has been assigned to class Phyllopharyngea (Gong et al. 2009). A recent study revealed that Copemetopus verae might represent an anaerobic lineage sister to class Protocruziea (Campello-Nunes et al. 2022; Rotterová et al. 2022), but its assignment varied in different trees (Supplementary Fig. S1 in this study) (Campello-Nunes et al. 2022).

In an early study, MROs were classified into five types: aerobic mitochondria, anaerobic mitochondria, hydrogen-producing mitochondria, hydrogenosomes, and mitosomes (Müller et al. 2012). Subsequently, a series of studies has revealed functionally intermediate organelles (Gawryluk et al. 2016; Stairs et al. 2014). In this study, we compared components and functions of several main MRO pathways instead of classifying types of MROs for all 14 anaerobic ciliate species studied to date (Fig. 4). It is not possible to outline consistent evolutionary patterns of MRO metabolic pathways among anaerobic ciliates using the remaining components as characters (Figs. 2, 3). However, conserved functional patterns of MRO pathways could be revealed for phylogenetically closely related species (Rotterová et al. 2022). Only proteins related to pyruvate and energy metabolisms have been carefully considered in all 12 previous published anaerobic ciliates, so evolutionary patterns of MRO metabolic functions of these pathways among these anaerobic ciliates including the two we studied here are discussed in detail below.

Pyruvate metabolism converts pyruvate and coenzyme A into acetyl-CoA while reducing NAD+ to NADH. The PDH complex functions in the classical mitochondrial pyruvate metabolism, while PFO and PNO are pyruvate oxidation enzymes in MROs of anaerobic protists (de Graaf et al. 2011; Gawryluk et al. 2016; Hug et al. 2010; Stairs et al. 2014). No pyruvate oxidation enzyme was detected in Parablepharisma sp., Isotricha intestinalis, Entodinium furca, E. caudatum, or Diplodinium dentatum, probably due to incomplete metagenomic or single-cell transcriptomic datasets (Feng et al. 2020; Park et al. 2021; Rotterová et al. 2020). Interestingly, all anaerobic ciliates with pyruvate oxidation enzymes, as well as the aerobic ciliate Tetrahymena thermophila, contain at least one subunit of PDH (de Graaf et al. 2011; Feng et al. 2020; Lewis et al. 2019; Park et al. 2021; Rotterová et al. 2020; Smith et al. 2007). Furthermore, a complete PDH complex has been predicted in Nyctotherus ovalis, two Plagiopyla species, two Metopus species and metopid sp. SK (Fig. 4). Mitochondrial type PNO protein is only detected in Cyclidium porcatum, and it has been suggested that PDHE3 of this species is part of another pathway (Lewis et al. 2019). We suggest that sequencing of additional anaerobic ciliates, especially species phylogenetically close to C. porcatum, is needed to assess the evolutionary patterns of mitochondrial PNO within ciliates.

MROs of all 14 anaerobic ciliate species studied to date, except for I. intestinalis, can produce hydrogen (Fig. 4). Surprisingly, [FeFe]-hydrogenase in typical hydrogenosomes of other protist groups, as well as their maturases (HydE, HydF, and HydG), are absent in MROs of hydrogen-producing anaerobic ciliates (Figs. 2, 3), as revealed by previous studies (de Graaf et al. 2011; Feng et al. 2020; Lewis et al. 2019; Rotterová et al. 2020, 2022). Instead, except for E. furca, E. caudatum, and D. dentatum with partial sequences (Feng et al. 2020; Park et al. 2021), [FeFe]-hydrogenases in all other anaerobic ciliates are fusion proteins containing not only hydrogenase but also NuoE and NuoF (present study; de Graaf et al. 2011; Lewis et al. 2019; Rotterová et al. 2020). These NuoE and NuoF domains are similar to NADH dehydrogenase of prokaryotes. A previous investigation suggested that NuoE and NuoF domains are similar to NADH dehydrogenase of prokaryotes, and produce hydrogen with NADH oxidation (Losey et al. 2020; Rotterová et al. 2022). These complex [FeFe]-hydrogenases seem to be acquired by horizontal gene transfer (HGT) from prokaryotes and are important for ciliate early transitions to anaerobic environments (de Graaf et al. 2011; Lewis et al. 2019; Losey et al. 2020; Rotterová et al. 2020, 2022).

Energy metabolisms are variable among the 14 anaerobic ciliate species for which data are available. Neither oxidative phosphorylation nor substrate-level phosphorylation for ATP production seems to be carried out by MROs of four species in class Litostomatea (I. intestinalis, E. furca, E. caudatum, and D. dentatum) (Fig. 4) (Feng et al. 2020; Park et al. 2021). MROs of C. porcatum and Muranothrix gubernata produce ATP by both oxidative phosphorylation and substrate-level phosphorylation (Lewis et al. 2019; Rotterová et al. 2020), while MROs of all other eight anaerobic species only produce ATP by substrate-level phosphorylation (Figs. 2, 3, 4) (de Graaf et al. 2011; Feng et al. 2020; Lewis et al. 2019; Rotterová et al. 2020). Some ETC components of C. porcatum and M. gubernata are missing, but their ETC retains functions necessary for oxidative phosphorylation. MRO proteins which are needed for proton-production (Nad11, NuoE, NuoF of complex I and SdhA of complex II in both species), proton-pumping (Nad1–Nad6 of complex I in M. gubernata), electron-transferring (i. Nad7–Nad10 of complex I, Rieske of complex III, AOX in C. porcatum; ii. Nad4L, Nad5, Nad6, Nad7, Nad9, and Nad10 of complex I, SdhB in complex II, Rieske and CytC1 in complex III, AOX in M. gubernata), and F1F0ATP-synthase functions (complex V in two species) have been detected (Lewis et al. 2019; Rotterová et al. 2020). The capability of generating ATP by ETC in P. sp. of class Parablepharismea should be further supported since alpha and beta subunits (ATP1, ATP2) of complex V have not been detected, although the gamma subunit (ATP3) is present. Anaerobic species in class Plagiopylea (two Plagiopyla species) and Litostomatea (I. intestinalis, E. furca, E caudatum, and D. dentatum) seem to have completely lost all functions of ETC (Figs. 3, 4) (Feng et al. 2020; Lewis et al. 2019; Park et al. 2021; Rotterová et al. 2020). Five anaerobic ciliates in the class Armophorea (two Metopus species, N. ovalis, metopid sp. SK, Heterometopus sp. CSS) cannot synthetize ATP by ETC due to the absence of complex V, but they can transfer electrons in their partial ETC due to the presence of the remaining components of several complexes and AOX (Figs. 2, 4) (Lewis et al. 2019; Rotterová et al. 2020). All these predicted results are consistent with transmission electron microscopy (TEM) images showing presence of cristae in C. porcatum, M. gubernata, M. contortus, H. sp. CSS, and N. ovalis, as well as absence of cristae in P. frontata (Beinart et al. 2018b; Cuthill et al. 2005; Esteban et al. 1995; Lewis et al. 2019; Rotterová et al. 2020). The presence or absence of cristae should be checked by TEM in the other seven anaerobic ciliate species. Similarly, as reported in other anaerobic protist groups, e.g. Trichomonas vaginalis and Pygsuia biforma (Dolezal et al. 2007; Stairs et al. 2014), all 14 anaerobic ciliates studied to date (except for three I. intestinalis, E. furca, E caudatum, and D. dentatum) can acquire ATP by substrate-level phosphorylation using ASCT and SCS (Figs. 2, 3, 4) (Lewis et al. 2019; Rotterová et al. 2020).

To summarize, except for I. intestinalis, MROs of all other 13 anaerobic ciliate species studied have [FeFe]-hydrogenase-producing hydrogen. Furthermore, group-specific functional patterns for MRO energy pathways are present in anaerobic ciliates (Fig. 1), although broader taxon sampling from more groups and within groups is needed to test this hypothesis. Additionally, group-specific functional patterns for MRO energy pathways do not evolve following the phylogeny of anaerobic ciliates. These findings indicate that adaptation of ciliates to anaerobic niches is group-specific and may have occurred multiple times.

Materials and methods

Sampling, culture and identification of anaerobic ciliates

Metopus laminarius was isolated from sediment in the Pearl River in Guangzhou, China (23° 11′ N; 113° 38′ E), where the dissolved oxygen was 1.99 mg/L, and the salinity was 0. Plagiopyla cf. narasimhamurtii was isolated from a mangrove forest in Zhanjiang, China (21° 12′ N; 110° 25′ E), where the dissolved oxygen was 0.23 mg/L, and the salinity was 18. M. laminarius and P. cf. narasimhamurtii were cultured in CMV medium (Narayanan et al. 2007) and artificial sea water, respectively. Both culture mediums were flushed with N2 for 20 min to remove O2, and then filled into air-tight culture flasks without headspace. Cultures of single ciliate species were obtained by transferring about 20 ciliate cells to culture flasks containing an anaerobic medium using a micropipette.

Live isolated ciliate cells were observed by bright field under microscopy, and the protargol staining method was used to reveal ciliary pattern (Wang et al. 2021a, b; Wu et al. 2020). To verify the taxonomic identification of the studied cells, we also sequenced their 18S rDNA. The protocol was as follows: total DNA was extracted using REDExtract-N-AmpTMTissue PCR Kit (SIGMA, USA) according to the manufacturer’s instructions. The universal eukaryotic primers (82F: 5′-GAA ACT GCG AAT GGCTC-3′;18S-R: 5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′) (Medlin et al.1988) were used to amplify the 18S rDNA of ciliates. PCR reactions were carried out using 2 × Taq plus Master Mix (Biosharp, China) with the following conditions: initial denaturation step of 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 90 s, and a final extension step at 72 °C for 5 min. The PCR products were sequenced in Guangzhou Tianyi huiyuan Co., Ltd. Finally, two new 18S rDNA sequences were searched against the NT database.

RNA extraction, cDNA synthesis, and transcriptome sequencing

For single-cell transcriptomics, one M. laminarius cell was picked using a micropipette, and rapidly washed five times in sterile phosphate-buffered saline (PBS). After being starved for 24 h, it was transferred to 0.2 ml RNA-free PCR tube with 0.95 μl Lysis Buffer in SMART-Seq v4 kit (TaKaRa, Japan) and immediately stored at − 80 °C. Subsequently, cDNA was synthesized and purified using the SMART-Seq v4 kit (TaKaRa, Japan) and the Agencourt AMPure XP kit (Beckman Coulter Inc, USA), respectively. The cDNA library was sequenced using Illumina Novaseq 150-bp paired-end reads in Beijing Novozhiyuan Technology Co., Ltd. Unfortunately, cDNA of a single cell of P. cf. narasimhamurtii did not pass quality control (i.e., sufficient concentration) for sequencing. Hence, cDNA of 10 P. cf. narasimhamurtii cells was sequenced using the same protocol described for M. laminarius cell but using sterile artificial seawater rather than PBS for washing.

For mass-culture transcriptome experiments, approximately 105 cells of M. laminarius and P. cf. narasimhamurtii were harvested, respectively, by centrifugation at 4500 g for 5 min. After being starved for 24 h, total RNA was extracted using RNeasy Micro Kit (Qiagen, Hilden, Germany). After a quality check, a library was prepared using NebNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, USA) and sequenced using Illumina Novaseq 150-bp paired-end reads in Zhejiang Annuouda Biological Technology Co., Ltd.

In silico predictions of MRO metabolism and estimation of transcript expression abundance

Trimmomatic v0.33 (Bolger et al. 2014) was used to filter raw reads and trim adapters. Then, reads with half bases with a quality value of Q < 19 were filtered out. The transcriptome was de novo assembled using Trinity v2.4.0 (Grabherr et al. 2011). Gene prediction was done with Prodigal V2.6.3 (Hyatt et al. 2010), and then decontamination was assisted by eliminating identifying contaminant taxa using Plast V2.3.2 (Nguyen and Lavenier 2009). Coding sequences from cleanly assembled transcriptomes were predicted using TransDecoder (Haas et al. 2013) with the Tetrahymena genetic code (NCBI translation Table 6) and the protozoan mitochondrial genetic code (NCBI translation Table 4).

Putative MRO proteins were detected from translated transcriptomes by BlastP (Camacho et al. 2009) searches, using reference proteins from model ciliate species Tetrahymena thermophila (Smith et al. 2007), and three published anerobic ciliate species, i.e., M. contortus, P. frontata, and Cyclidium porcatum (Lewis et al. 2019). Candidate sequences were annotated with the NR database for preliminary identification of target proteins. For each target protein, candidate sequences and hundreds of their best hit sequences in GenBank, as well as sequences from reference ciliates, were aligned using AliView v1.23 (Larsson 2014). Phylogenetic trees, constructed using IQTree2.1 online server (Trifinopoulos et al. 2016), were used to confirm orthology. Mitochondrial-targeting signals (MTS) predictions were applied using Mitoprot (Claros and Vincens 1996), MitoFates (Fukasawa et al. 2015), and TargetP (Emanuelsson et al. 2007).

The gene/transcript expression abundances were estimated by RSEM (Li and Dewey 2011) and Bowtie2 (Langmead and Salzberg 2012). Cleaned paired-end reads were mapped back onto the assembled transcriptome, and the read count for each predicted gene was obtained from the mapping results. The FPKM method was used to compare for the gene expression levels in mass-culture transcriptomes (Mortazavi et al. 2008).

Construction of 18S rDNA phylogenetic trees

A dataset containing 98 18S rDNA sequences was used to construct a phylogenetic tree, which encompassed two new sequences of M. laminarius and P. cf. narasimhamurtii generated in this study. Also, 44, 18, and 34 sequences of class Armophorea, Plagiopylea, and other ciliate taxa were downloaded from GenBank (Supplementary Fig. S1). To facilitate the interpretation of the results shown in the figures, we also reconstructed the phylogenetic relationships among the taxa used for the comparison among MRO metabolic pathways. For this purpose, we selected 14 anaerobic ciliate species: Nyctotherus ovalis, Heterometopus sp. CSS, metopid sp. SK, Muranothrix gubernata, Parablepharisma sp., C. porcatum, M. contortus, M. laminarius, P. frontata, P. cf. narasimhamurtii, Entodinium furca, E. caudatum, Diplodinium dentatum, and Isotricha intestinalis, and the aerobic ciliate Tetrahymena thermophila from above 98 18S rDNA sequences. Sequences of two datasets were aligned using MAFFT in GUIDANCE2 server (Sela et al. 2015) with default settings, and then aligned sequences were manually trimmed using Seaview 4.7 (Gouy et al. 2010). Maximum likelihood (ML) analyses were performed in RAxML 8.2.12 via the CIPRES Science Gateway web server (Muller et al. 2010), using GTR GAMMA with 1000 bootstrap replicates.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1089 KB) (1.1MB, docx)
Supplementary file2 (XLSX 128 KB) (127.9KB, xlsx)
Supplementary file3 (XLSX 129 KB) (128.8KB, xlsx)
Supplementary file4 (XLSX 80 KB) (79.6KB, xlsx)
Supplementary file5 (XLSX 64 KB) (64.1KB, xlsx)
Supplementary file6 (XLSX 14 KB) (13.3KB, xlsx)

Acknowledgements

Many thanks to Dr. Alan Warren in Natural History Museum, UK for English improvement. Many thanks to Dr. Zhuo Shen in Sun Yat-sen University, China, for her help in species identification. Also, we thank Prof. Weibo Song in Ocean University of China, Dr. Lei Wu, Ms. Jiahui Xu, Ms. Zijing Quan, and Mr. Jian Wang in South China Normal University, for improving figures. This work is supported by the National Natural Science Foundation of China (Grant Number 32070406), Guangdong Basic and Applied Basic Research Foundation (Grant Number 2022A1515010773), and the Science and Technology Planning Project of Guangzhou (Grant Number 202102080168).

Author contributions

ZY designed the experiments. ZC, JL, and MC completed the experiments. ZY and DS directed data analysis. ZC, JL, MC, SC, SL, and YW analyzed the data. ZC and JL drafted the manuscript. DS and ZY revised the manuscript. All authors edited and approved the final manuscript.

Data availability

The 18S rDNA sequences were submitted into GenBank (accession numbers are SAMN29232525 for Metopus laminarius and SAMN29232526 for Plagiopyla cf. narasimhamurtii). And the single-cell transcriptomes and mass-culture transcriptomes of two species were submitted to GenBank under BioProject ID PRJNA851543.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Animal and human rights statement

This article does not contain human participants or animals.

Footnotes

Special topic: Ciliatology.

Zhicheng Chen and Jia Li contributed equally to this article.

References

  1. Arregui L, Pérez-Uz B, Zornoza A, Serrano S. A new species of the genus Metacystis (Ciliophora, Prostomatida, Metacystidae) from a wastewater treatment plant. J Eukaryot Microbiol. 2010;57:362–368. doi: 10.1111/j.1550-7408.2010.00484.x. [DOI] [PubMed] [Google Scholar]
  2. Beinart RA, Beaudoin DJ, Bernhard JM, Edgcomb VP. Insights into the metabolic functioning of a multipartner ciliate symbiosis from oxygen-depleted sediments. Mol Ecol. 2018;27:1794–1807. doi: 10.1111/mec.14465. [DOI] [PubMed] [Google Scholar]
  3. Beinart RA, Rotterová J, Čepička I, Gast RJ, Edgcomb VP. The genome of an endosymbiotic methanogen is very similar to those of its free-living relatives. Environ Microbiol. 2018;20:2538–2551. doi: 10.1111/1462-2920.14279. [DOI] [PubMed] [Google Scholar]
  4. Berger J, Lynn DH. Hydrogenosome-methanogen assemblages in the echinoid endocommensal plagiopylid ciliates, Lechriopyla mystax Lynch, 1930 and Plagiopyla minuta Powers, 1933. J Protozool. 1992;39:4–8. doi: 10.1111/j.1550-7408.1992.tb01277.x. [DOI] [Google Scholar]
  5. Bernard C, Fenchel T. Some microaerobic ciliates are facultative anaerobes. Eur J Protistol. 1996;32:293–297. doi: 10.1016/S0932-4739(96)80051-4. [DOI] [Google Scholar]
  6. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bourland WA, Wendell L, Hampikian G. Morphologic and molecular description of Metopus fuscus Kahl from North America and new rDNA sequences from seven metopids (Armophorea, Metopidae) Eur J Protistol. 2014;50:213–230. doi: 10.1016/j.ejop.2014.01.002. [DOI] [PubMed] [Google Scholar]
  8. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinform. 2009;10:421. doi: 10.1186/1471-2105-10-421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campello-Nunes PH, Silva-Neto ID, Sales MHO, Soares CAG, Paiva TS, Fernandes NM. Morphological and phylogenetic investigations shed light on evolutionary relationships of the enigmatic genus Copemetopus (Ciliophora, Alveolata), with the proposal of Copemetopus verae sp. Nov. Eur J Protistol. 2022;83:125878. doi: 10.1016/j.ejop.2022.125878. [DOI] [PubMed] [Google Scholar]
  10. Chen X, Gao S, Liu Y, Wang Y, Wang Y, Song W. Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macro-nuclei of the ciliated model organism, Tetrahymena thermophila. Sci China Life Sci. 2016;59:909–919. doi: 10.1007/s11427-016-5102-x. [DOI] [PubMed] [Google Scholar]
  11. Chen X, Wang C, Pan B, Lu B, Li C, Shen Z, Warren A, Li L. Single-cell genomic sequencing of three peritrichs (Protista, Ciliophora) reveals less biased stop codon usage and more prevalent programmed ribosomal frameshifting than in other ciliates. Front Mar Sci. 2020;7:602323. doi: 10.3389/fmars.2020.602323. [DOI] [Google Scholar]
  12. Clarke KJ, Finlay BJ, Esteban G, Guhl BE, Embley TM. Cyclidium porcatum n. sp.: a free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria. Eur J Protistol. 1993;29:262–270. doi: 10.1016/S0932-4739(11)80281-6. [DOI] [PubMed] [Google Scholar]
  13. Claros MG, Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem. 1996;241:779–786. doi: 10.1111/j.1432-1033.1996.00779.x. [DOI] [PubMed] [Google Scholar]
  14. Cuthill IC, Stevens M, Sheppard J, Maddocks T, Parraga CA, Troscianko TS. Disruptive coloration and background pattern matching. Nature. 2005;434:72–74. doi: 10.1038/nature03312. [DOI] [PubMed] [Google Scholar]
  15. de Graaf RM, Ricard G, van Alen TA, Duarte I, Dutilh BE, Burgtorf C, Kuiper JW, van der Staay GW, Tielens AG, Huynen MA, Hackstein JH. The organellar genome and metabolic potential of the hydrogen-producing mitochondrion of Nyctotherus ovalis. Mol Biol Evol. 2011;28:2379–2391. doi: 10.1093/molbev/msr059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dolezal P, Dancis A, Lesuisse E, Sutak R, Hrdy I, Embley TM, Tachezy J. Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis. Eukaryot Cell. 2007;6:1431–1438. doi: 10.1128/EC.00027-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Duan L, Cheng T, Wei F, Qiao Y, Wang C, Warren A, Niu J, Wang Y. New contribution to epigenetic studies: isolation of micronuclei with high purity and DNA integrity in the model ciliated protist, Tetrahymena thermophila. Eur J Protistol. 2021;80:125804. doi: 10.1016/j.ejop.2021.125804. [DOI] [PubMed] [Google Scholar]
  18. Ebenezer TE, Zoltner M, Burrell A, Nenarokova A, Novak Vanclova AMG, Prasad B, Soukal P, Santana-Molina C, O'Neill E, Nankissoor NN, Vadakedath N, Daiker V, Obado S, Silva-Pereira S, Jackson AP, Devos DP, Lukes J, Lebert M, Vaughan S, Hampl V, et al. Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol. 2019;17:11. doi: 10.1186/s12915-019-0626-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Edgcomb VP, Pachiadaki M. Ciliates along oxyclines of permanently stratified marine water columns. J Eukaryot Microbiol. 2014;61:434–445. doi: 10.1111/jeu.12122. [DOI] [PubMed] [Google Scholar]
  20. Emanuelsson O, Brunak S, von Heijne G, Nielsen H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc. 2007;2:953–971. doi: 10.1038/nprot.2007.131. [DOI] [PubMed] [Google Scholar]
  21. Embley TM, Finlay BJ, Dyal PL, Hirt RP, Wilkinson M, Williams AG. Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates. Proc Biol Sci. 1995;262:87–93. doi: 10.1098/rspb.1995.0180. [DOI] [PubMed] [Google Scholar]
  22. Esteban G, Fenchel T, Finlay B. Diversity of free-living morphospecies in the ciliate genus Metopus. Arch Protistenk. 1995;146:137–164. doi: 10.1016/S0003-9365(11)80106-5. [DOI] [Google Scholar]
  23. Fenchel T, Finlay BJ. The biology of free-living anaerobic ciliates. Eur J Protistol. 1991;26:201–215. doi: 10.1016/S0932-4739(11)80143-4. [DOI] [PubMed] [Google Scholar]
  24. Fenchel T, Perry T, Thane A. Anaerobiosis and symbiosis with bacteria in free-living ciliates. J Protozool. 1977;24:154–163. doi: 10.1111/j.1550-7408.1977.tb05294.x. [DOI] [PubMed] [Google Scholar]
  25. Feng JM, Jiang CQ, Sun ZY, Hua CJ, Wen JF, Miao W, Xiong J. Single-cell transcriptome sequencing of rumen ciliates provides insight into their molecular adaptations to the anaerobic and carbohydrate-rich rumen microenvironment. Mol Phylogenet Evol. 2020;143:106687. doi: 10.1016/j.ympev.2019.106687. [DOI] [PubMed] [Google Scholar]
  26. Fernandes NM, Vizzoni VF, Borges BDN, Soares CAG, da Silva-Neto ID, Paiva TDS. Molecular phylogeny and comparative morphology indicate that odontostomatids (Alveolata, Ciliophora) form a distinct class-level taxon related to Armophorea. Mol Phylogenet Evol. 2018;126:382–389. doi: 10.1016/j.ympev.2018.04.026. [DOI] [PubMed] [Google Scholar]
  27. Finlay BJ, Fenchel T, Gardener S. Oxygen perception and O2 toxicity in the freshwater ciliated protozoon Loxodes. J Protozool. 1986;33:157–165. doi: 10.1111/j.1550-7408.1986.tb05582.x. [DOI] [Google Scholar]
  28. Finlay BJ, Tellez C, Esteban GF. Diversity of free-living ciliates in the sandy sediment of a Spanish stream in winter. J Eukaryot Microbiol. 1993;139:2855–2863. [Google Scholar]
  29. Foissner W, Foissner I. Fine structure and systematic position of Enchelyomorpha vermicularis (Smith, 1899) Kahl, 1930, an anaerobic ciliate (Protozoa, Ciliophora) from from domestic sewage. Acta Protozool. 1995;34:21–34. [Google Scholar]
  30. Fukasawa Y, Tsuji J, Fu SC, Tomii K, Horton P, Imai K. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol Cell Proteomics. 2015;14:1113–1126. doi: 10.1074/mcp.M114.043083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Gao F, Warren A, Zhang Q, Gong J, Miao M, Sun P, Xu D, Huang J, Yi Z, Song W. The all-data-based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata) Sci Rep. 2016;6:24874. doi: 10.1038/srep24874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gawryluk RMR, Stairs CW. Diversity of electron transport chains in anaerobic protists. BBA-Bioenergetics. 2021;1862:148334. doi: 10.1016/j.bbabio.2020.148334. [DOI] [PubMed] [Google Scholar]
  33. Gawryluk RMR, Kamikawa R, Stairs CW, Silberman JD, Brown MW, Roger AJ. The earliest stages of mitochondrial adaptation to low oxygen revealed in a novel rhizarian. Curr Biol. 2016;26:2729–2738. doi: 10.1016/j.cub.2016.08.025. [DOI] [PubMed] [Google Scholar]
  34. Gong J, Stoeck T, Yi Z, Miao M, Zhang Q, Roberts DM, Warren A, Song W. Small subunit rRNA phylogenies show that the class Nassophorea is not monophyletic (phylum Ciliophora) J Eukaryot Microbiol. 2009;56:339–347. doi: 10.1111/j.1550-7408.2009.00413.x. [DOI] [PubMed] [Google Scholar]
  35. Gouy M, Guindon S, Gascuel O. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2010;27:221–224. doi: 10.1093/molbev/msp259. [DOI] [PubMed] [Google Scholar]
  36. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–652. doi: 10.1038/nbt.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013;8:1494–1512. doi: 10.1038/nprot.2013.084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Hackstein JHP. (Endo)symbiotic methanogenic archaea. Berlin: Springer; 2018. [Google Scholar]
  39. Hines HN, Onsbring H, Ettema TJG, Esteban GF. Molecular investigation of the ciliate Spirostomum semivirescens, with first transcriptome and new geographical records. Protist. 2018;169:875–886. doi: 10.1016/j.protis.2018.08.001. [DOI] [PubMed] [Google Scholar]
  40. Hu X. Ciliates in extreme environments. J Eukaryot Microbiol. 2014;61:410–418. doi: 10.1111/jeu.12120. [DOI] [PubMed] [Google Scholar]
  41. Hug LA, Stechmann A, Roger AJ. Phylogenetic distributions and histories of proteins involved in anaerobic pyruvate metabolism in eukaryotes. Mol Biol Evol. 2010;27:311–324. doi: 10.1093/molbev/msp237. [DOI] [PubMed] [Google Scholar]
  42. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 2010;11:119. doi: 10.1186/1471-2105-11-119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, Lonnerberg P, Linnarsson S. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods. 2014;11:163–166. doi: 10.1038/nmeth.2772. [DOI] [PubMed] [Google Scholar]
  44. Jiang L, Wang C, Zhuang W, Li S, Hu X. Taxonomy, phylogeny, and geographical distribution of the little-known Helicoprorodon multinucleatum Dragesco, 1960 (Ciliophora, Haptorida) and key to species within the genus. Eur J Protistol. 2021;78:125769. doi: 10.1016/j.ejop.2021.125769. [DOI] [PubMed] [Google Scholar]
  45. Jiang L, Zhuang W, El-Serehy HA, Al-Farraj SA, Warren A, Hu X. Taxonomy and molecular phylogeny of two new species of prostomatean ciliates with establishment of Foissnerophrys gen. n. (Alveolata, Ciliophora) Front Microbiol. 2021;12:686929. doi: 10.3389/fmicb.2021.686929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Kahl A. Neue und ergänzende Beobachtungen heterotricher Ciliaten. Arch Protistenk. 1927;57:121–203. [Google Scholar]
  47. Kofoid CA. On the type of the genus Diplodinium Schuberg, 1888 (class Ciliophora) Bull Zool Nomencl. 1943;1:167. [PubMed] [Google Scholar]
  48. Kolisko M, Boscaro V, Burki F, Lynn DH, Keeling PJ. Single-cell transcriptomics for microbial eukaryotes. Curr Biol. 2014;24:R1081–R1082. doi: 10.1016/j.cub.2014.10.026. [DOI] [PubMed] [Google Scholar]
  49. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Larsson A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. doi: 10.1093/bioinformatics/btu531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Leger MM, Eme L, Hug LA, Roger AJ. Novel hydrogenosomes in the microaerophilic jakobid Stygiella incarcerata. Mol Biol Evol. 2016;33:2318–2336. doi: 10.1093/molbev/msw103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Lewis WH, Lind AE, Sendra KM, Onsbring H, Williams TA, Esteban GF, Hirt RP, Ettema TJG, Embley TM. Convergent evolution of hydrogenosomes from mitochondria by gene transfer and loss. Mol Biol Evol. 2019;37:524–539. doi: 10.1093/molbev/msz239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 2011;12:323. doi: 10.1186/1471-2105-12-323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Li M, Hu G, Li C, Zhao WS, Zou H, Li WX, Wu SG, Wang GT, Ponce-Gordo F. Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs. Acta Trop. 2020;208:105531. doi: 10.1016/j.actatropica.2020.105531. [DOI] [PubMed] [Google Scholar]
  55. Li R, Zhuang W, Wang C, El-Serehy H, Al-Farraj SA, Warren A, Hu X. Redescription and SSU rRNA gene-based phylogeny of an anaerobic ciliate, Plagiopyla ovata Kahl, 1931 (Ciliophora, Plagiopylea) Int J Syst Evol Micr. 2021;71:004936. doi: 10.1099/ijsem.0.004936. [DOI] [PubMed] [Google Scholar]
  56. Li S, Zhuang W, Pérez-Uz B, Zhang Q, Hu X. Two anaerobic ciliates (Ciliophora, Armophorea) from China: morphology and SSU rDNA sequence, with report of a new species, Metopus paravestitus nov. spec. J Eukaryot Microbiol. 2021;68:e12822. doi: 10.1111/jeu.12822. [DOI] [PubMed] [Google Scholar]
  57. Liu Z, Hu SK, Campbell V, Tatters AO, Heidelberg KB, Caron DA. Single-cell transcriptomics of small microbial eukaryotes: limitations and potential. ISME J. 2017;11:1282–1285. doi: 10.1038/ismej.2016.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Losey NA, Poudel S, Boyd ES, McInerney MJ. The beta subunit of non-bifurcating NADH-dependent [FeFe]-Hydrogenases differs from those of multimeric electron-bifurcating [FeFe]-Hydrogenases. Front Microbiol. 2020;11:1109. doi: 10.3389/fmicb.2020.01109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Lynn DH. The ciliated protozoa: characterization, classification, and guide to the literature. Berlin: Springer; 2008. [Google Scholar]
  60. Marinov GK, Williams BA, McCue K, Schroth GP, Gertz J, Myers RM, Wold BJ. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing. Genome Res. 2014;24:496–510. doi: 10.1101/gr.161034.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5:621–628. doi: 10.1038/nmeth.1226. [DOI] [PubMed] [Google Scholar]
  62. Müller M, Mentel M, van Hellemond JJ, Henze K, Woehle C, Gould SB, Yu RY, van der Giezen M, Tielens AG, Martin WF. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol Mol Biol R. 2012;76:444–495. doi: 10.1128/MMBR.05024-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Narayanan N, Priya M, Haridas A, Manilal VB. Isolation and culturing of a most common anaerobic ciliate, Metopus sp. Anaerobe. 2007;13:14–20. doi: 10.1016/j.anaerobe.2006.10.003. [DOI] [PubMed] [Google Scholar]
  64. Nguyen VH, Lavenier D. PLAST: parallel local alignment search tool for database comparison. BMC Bioinform. 2009;10:329. doi: 10.1186/1471-2105-10-329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Nitla V, Serra V, Fokin S, Modeo L, Verni F, Sandeep B, Kalavati C, Petroni G. Critical revision of the family Plagiopylidae (Ciliophora: Plagiopylea), including the description of two novel species, Plagiopyla ramani and Plagiopyla narasimhamurtii, and redescription of Plagiopyla nasuta Stein, 1860 from India. Zool J Linn Soc-Lond. 2019;186:1–45. doi: 10.1093/zoolinnean/zly041. [DOI] [Google Scholar]
  66. Orsi W, Edgcomb V, Faria J, Foissner W, Fowle WH, Hohmann T, Suarez P, Taylor C, Taylor GT, Vd'ačný P, Epstein SS. Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela. Int J Syst Evol Microbiol. 2012;62:1425–1433. doi: 10.1099/ijs.0.034710-0. [DOI] [PubMed] [Google Scholar]
  67. Pan B, Chen X, Hou L, Zhang Q, Qu Z, Warren A, Miao M. Comparative genomics analysis of ciliates Provides insights on the evolutionary history within “Nassophorea-Synhymenia-Phyllopharyngea” assemblage. Front Microbiol. 2019;10:2819. doi: 10.3389/fmicb.2019.02819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Park T, Wijeratne S, Meulia T, Firkins JL, Yu Z. The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment. Genomics. 2021;113:1416–1427. doi: 10.1016/j.ygeno.2021.03.014. [DOI] [PubMed] [Google Scholar]
  69. Ricard G, McEwan NR, Dutilh BE, Jouany JP, Macheboeuf D, Mitsumori M, McIntosh FM, Michalowski T, Nagamine T, Nelson N, Newbold CJ, Nsabimana E, Takenaka A, Thomas NA, Ushida K, Hackstein JH, Huynen MA. Horizontal gene transfer from bacteria to rumen ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment. BMC Genomics. 2006;7:22. doi: 10.1186/1471-2164-7-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Rotterová J, Salomaki E, Pánek T, Bourland W, Žihala D, Táborský P, Edgcomb VP, Beinart RA, Kolísko M, Čepička I. Genomics of new ciliate lineages provides insight into the evolution of obligate anaerobiosis. Curr Biol. 2020;30:1–14. doi: 10.1016/j.cub.2020.03.064. [DOI] [PubMed] [Google Scholar]
  71. Rotterová J, Edgcomb VP, Čepička I, Beinart R. Anaerobic ciliates as a model group for studying symbioses in oxygen-depleted environments. J Eukaryot Microbiol. 2022;24:e12912. doi: 10.1111/jeu.12912. [DOI] [PubMed] [Google Scholar]
  72. Sela I, Ashkenazy H, Katoh K, Pupko T. GUIDANCE2: accurate detection of unreliable alignment regions accounting for the uncertainty of multiple parameters. Nucleic Acids Res. 2015;43:W7–14. doi: 10.1093/nar/gkv318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Sheng Y, He M, Zhao F, Shao C, Miao M. Phylogenetic relationship analyses of complicated class Spirotrichea based on transcriptomes from three diverse microbial eukaryotes: Uroleptopsis citrina, Euplotes vannus and Protocruzia tuzeti. Mol Phylogenet Evol. 2018;129:338–345. doi: 10.1016/j.ympev.2018.06.025. [DOI] [PubMed] [Google Scholar]
  74. Smith DG, Gawryluk RM, Spencer DF, Pearlman RE, Siu KW, Gray MW. Exploring the mitochondrial proteome of the ciliate protozoon Tetrahymena thermophila: direct analysis by tandem mass spectrometry. J Mol Biol. 2007;374:837–863. doi: 10.1016/j.jmb.2007.09.051. [DOI] [PubMed] [Google Scholar]
  75. Stairs CW, Eme L, Brown MW, Mutsaers C, Susko E, Dellaire G, Soanes DM, van der Giezen M, Roger AJ. A SUF Fe-S cluster biogenesis system in the mitochondrion-related organelles of the anaerobic protist Pygsuia. Curr Biol. 2014;24:1176–1186. doi: 10.1016/j.cub.2014.04.033. [DOI] [PubMed] [Google Scholar]
  76. Stairs CW, Leger MM, Roger AJ. Diversity and origins of anaerobic metabolism in mitochondria and related organelles. Philos Trans R Soc Lond B Biol Sci. 2015;370:20140326. doi: 10.1098/rstb.2014.0326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, Xie XS. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science. 2010;329:533–538. doi: 10.1126/science.1188308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016;44:W232–235. doi: 10.1093/nar/gkw256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Vďačný P, Orsi W, Bourland WA, Shimano S, Epstein SS, Foissner W. Morphological and molecular phylogeny of dileptid and tracheliid ciliates: resolution at the base of the class Litostomatea (Ciliophora, Rhynchostomatia) Eur J Protistol. 2011;47:295–313. doi: 10.1016/j.ejop.2011.04.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Vďačný P, Rajter Ľ, Stoeck T, Foissner W. A proposed timescale for the evolution of armophorean ciliates: clevelandellids diversify more rapidly than metopids. J Eukaryot Microbiol. 2019;66:167–181. doi: 10.1111/jeu.12641. [DOI] [PubMed] [Google Scholar]
  81. Wang G, Wang S, Chai X, Zhang J, Yang W, Jiang C, Chen K, Miao W, Xiong J. A strategy for complete telomere-to-telomere assembly of ciliate macronuclear genome using ultra-high coverage Nanopore data. Comput Struct Biotechnol J. 2021;19:1928–1932. doi: 10.1016/j.csbj.2021.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wang J, Zhang T, Li F, Warren A, Li Y, Shao C. A new hypotrich ciliate, Oxytricha xianica sp. nov., with notes on the morphology and phylogeny of a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988 (Ciliophora, Oxytrichidae) Mar Life Sci Technol. 2021;3:303–312. doi: 10.1007/s42995-020-00089-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Wu T, Li Y, Lu B, Shen Z, Song W, Warren A. Morphology, taxonomy and molecular phylogeny of three marine peritrich ciliates, including two new species: Zoothamnium apoarbuscula n. sp. and Z. apohentscheli n. sp. (Protozoa, Ciliophora, Peritrichia) Mar Life Sci Technol. 2020;2:334–348. doi: 10.1007/s42995-020-00046-y. [DOI] [Google Scholar]
  84. Xu Y, Shen Z, Gentekaki E, Xu J, Yi Z. Comparative transcriptome analyses during the vegetative cell cycle in the mono-cellular organism Pseudokeronopsis erythrina (Alveolata, Ciliophora) Microorganisms. 2020;8:108. doi: 10.3390/microorganisms8010108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Yan Y, Maurer-Alcala XX, Knight R, Kosakovsky Pond SL, Katz LA. Single-cell transcriptomics reveal a correlation between genome architecture and gene family evolution in ciliates. Mbio. 2019;10:e02524-19. doi: 10.1128/mBio.02524-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Zhang T, Li C, Zhang X, Wang C, Roger AJ, Gao F. Characterization and comparative analyses of mitochondrial genomes in single-celled eukaryotes to shed light on the diversity and evolution of linear molecular architecture. Int J Mol Sci. 2021;22:2546. doi: 10.3390/ijms22052546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Zhu C, Liu W, Li X, Xu Y, El-Serehy HA, Al-Farraj SA, Ma H, Stoeck T, Yi Z. High salinity gradients and intermediate spatial scales shaped similar biogeographical and co-occurrence patterns of microeukaryotes in a tropical freshwater-saltwater ecosystem. Environ Microbiol. 2021;23:4778–4796. doi: 10.1111/1462-2920.15668. [DOI] [PubMed] [Google Scholar]
  88. Zhuang W, Li S, Bai Y, Zhang T, Al-Rasheid KAS, Hu X. Morphology and molecular phylogeny of the anaerobic freshwater ciliate Urostomides spinosus nov. spec. (Ciliophora, Armophorea, Metopida) from China. Eur J Protistol. 2021;81:125823. doi: 10.1016/j.ejop.2021.125823. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 1089 KB) (1.1MB, docx)
Supplementary file2 (XLSX 128 KB) (127.9KB, xlsx)
Supplementary file3 (XLSX 129 KB) (128.8KB, xlsx)
Supplementary file4 (XLSX 80 KB) (79.6KB, xlsx)
Supplementary file5 (XLSX 64 KB) (64.1KB, xlsx)
Supplementary file6 (XLSX 14 KB) (13.3KB, xlsx)

Data Availability Statement

The 18S rDNA sequences were submitted into GenBank (accession numbers are SAMN29232525 for Metopus laminarius and SAMN29232526 for Plagiopyla cf. narasimhamurtii). And the single-cell transcriptomes and mass-culture transcriptomes of two species were submitted to GenBank under BioProject ID PRJNA851543.

Articles from Marine Life Science & Technology are provided here courtesy of Springer

RESOURCES