The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-023-09706-6.


Introduction
Ciliates (protozoa of the phylum Ciliophora) are singlecelled organisms characterized by motile cilia, nuclear dimorphism and conjugation [1].Members of this group are very diverse and can inhabit a wide variety of environments.Species of Balantidium Claparède & Lachmann, 1858 have been widely found in the digestive tracts of animals, including amphibians, fishes, pigs, and humans [2][3][4].B. polyvacuolum is an endocommensal found in the hindgut of Xenocyprinae fishes [5,6], whose living environment is almost anaerobic.Only a few studies about this species are available, and they focus on its morphology and phylogeny [5][6][7].No genomic data are available for this ciliate species.
The hydrogenosomes of B. polyvacuolum and its endosymbionts remain poorly understood.In this study, we therefore investigated the energy metabolism of B. polyvacuolum based on single-cell transcriptome data.Cluster of Orthologous Group (COG) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and carbohydrate-active enzyme (CAZyme) identification were conducted.In addition, the ultrastructural morphology of hydrogenosomes and the endosymbiont bacteria was also revealed with the aid of transmission electron microscopy (TEM).

Specimen collection
The hosts (n = 9) of B. polyvacuolum, Xenocypris davidi, were collected from the Yangtze River in Xianning City, Hubei Province, China in July 2022 and transported alive to the laboratory for further examination.Fishes were anesthetized using 0.02% tricaine methane sulfonate (MS-222, Sigma) according to the manufacturer's protocol and dissected in accordance with the protocols approved by the Animal Ethics Committee of Institute of Hydrobiology, Chinese Academy of Sciences (IHB/ LL/2,023,036).The luminal contents of the fish intestines were transferred into Petri dishes and examined with the aid of a stereoscopic microscope Stemi SV6/AxioCam MRc5 (Zeiss, Oberkochen, Germany).Ciliates were collected with Pasteur micropipettes for morphological identification, ultrastructural analysis, and transcriptome sequencing.

Transmission electron microscopy
Balantidium polyvacuolum specimens were fixed with 2.5% glutaraldehyde.Post-fixation was performed in 1% (v/v) osmium tetroxide in phosphate buffer solution (PBS) for 2 h at 4℃, followed by dehydration in a gradient acetone series and embedded in Araldite.Ultrathin sections were then cut on a Leica Ultracut R ultramicrotome (Leica, Germany) and stained with uranyl acetate and lead citrate.The samples were viewed using a JEM-1230 Transmission Electron Microscope (JEOL, Japan).

Single-cell transcriptome amplification and sequencing
Single-cell samples, each comprising an individual B. polyvacuolum cell, were collected, lysed, and amplified to generate cDNA, according to the Smart-Seq2 protocol [18] and methods used in a previous study [19].Qualified cDNA libraries were then loaded on the Illumina Hiseq platform for PE150 sequencing (Illumina, CA, USA).
Open-reading frames (ORFs) were predicted with Transdecoder v5.5.0 (http:transdecoder.github.io).To further maximize sensitivity for capturing ORFs that may have functional significance, BLASTP searches against the Swiss-Prot, Pfam and NCBI NR protein databases were conducted.The unigenes were also annotated based on the COG database.A KEGG pathway enrichment analysis was conducted with the help of EggNOG-mapper (http://eggnog-mapper.embl.de)(accessed on 1st January 2023) [25] and TBtools v1.09 [26].Diamond v2.0.12 [27] was used for the BLAST search.Carbohydrate-active enzyme genes of B. polyvacuolum were identified using a hidden Markov model (HMM) search implemented in dbCAN HMMs 6.0 [28] with default parameters.The search results were then annotated against the CAZy database [29] using BLASTP.SignalP 5.0 was used to predict the protein peptide pre-sequences of these enzymes [30].

Identification of hydrogenosomal proteins
Hydrogenosomal proteins were predicted with methods relying on the homologs of mitochondrial proteomes and methods predicting N-terminal signal peptides.First, putative hydrogenosomal proteins were detected using BLAST.The decontaminated transcriptome was used to query BLASTP, searching for similarity against a database comprising the well-described homologs of mitochondrial proteins of the ciliate Tetrahymena thermophila [31] and anaerobic ciliates, the latter including C. porcatum, M. contortus, M. laminarius, P. frontata and Plagiopyla cf.narasimhamurtii [14,32] (see Additional file1-Table S5 for the sequences).Second, the identified sequences were used as queries in similarity searches against the NR database to confirm homology with other eukaryotes and to eliminate likely bacterial contaminants.All the BLAST searches used the default parameters, except for the e-value (which was 1e-5) and the query-gencode (which was 6).Third, TargetP [33] and MitoFates [34] and Deep-Loc-2.0 [35] were used to predict mitochondrial-targeting signals.Finally, hydrogenosomal metabolic pathways were depicted using Adobe Illustrator.

Transmission electron microscopy
Transmission electron micrographs of the body of B. polyvacuolum (Fig. 1A) showed the presence of many starch granules and a few lysosomes, randomly distributed within the cell.Endosymbiotic bacteria were also observed in the cytoplasm (Fig. 1B).The cell membrane and the nucleoid of the endosymbiotic bacteria could also be clearly seen (Fig. 1C).Numerous mitochondrionrelated organelles (MROs) were distributed in the vicinity of the starch granules (SGs), close to the cell membrane.Vacuoles of varying sizes were distributed around the MROs and kinetosomes could also be seen (Fig. 1D).The dividing MROs looked like a string of beads (Fig. 1E).Numerous rough endoplasmic reticula were located around the dumbbell-shaped and oval-shaped MROs (Fig. 1F).

Transcriptome assembly
We obtained 12,660,018 clean reads.The number of assembled contigs was 46,639, their total length was 32,465,220 bp, and their average GC content was 38%.The average contig length was 696 bp and the maximum length was 14,666 bp.The completeness of the assembly was 58.5% based on the BUSCO assessment.

COG annotation
In the COG annotation, 6,372 unigenes (Additional file 1-Table S1) were divided into 25 functional groups (Fig. S1).'Signal transduction mechanisms' was the largest group.A total of 998 unigenes were assigned to the 'metabolism' category (Fig. S1).Within this category, 'Carbohydrate transport and metabolism' , 'Energy production and conversion' , and 'Lipid transport and metabolism' were the most abundant subcategories.

KEGG annotation
The KEGG annotation results showed that 2,106 unigenes (Additional file 1-Table S2) were matched to the five functional categories of KEGG pathways (Fig S2 ): metabolism, genetic information processing, cellular processes, environmental information processing, and human diseases.Within the 'Metabolism' category, carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism, and nucleotide phosphorylation were the top five annotated subcategories (Fig. S2).

CAZymes identification
To understand the mechanism by which B. polyvacuolum contributes to the digestion of plant food in fish intestines, CAZymes were identified (Additional file 1-Table S3).Only 86 candidate CAZymes were identified in total (Fig. 2A), including 14 carbohydrate esterases (CEs), 22 with a carbohydrate-binding module (CBM), 22 glycoside hydrolases (GHs), and 28 glycosyltransferases (GTs).Among the identified CEs, CE1 accounted for the largest proportion (Fig. 2B).Among the identified CBMs, CBM20 accounted for 27% (Fig. 2C).The function of CBM20 is to bind to starch.Among the identified GHs, the proportions of GH13, GH22, and GH77 were the same as each other (Fig. 2D).The GH13 family is the major glycoside hydrolase family acting on substrates containing α-glucoside linkages.The GH5 and GH13 families act in cellulose degradation.The GH5 family can hydrolyze cellulose independently.Various GTs were also identified in this study (Fig. 2E).
In addition to the CAZymes, we also sought other evidence to show that B. polyvacuolum may help with plant biomass digestion.First, we used TEM for the detection of vacuoles with plant materials in B. polyvacuolum, which could indicate that the ciliate directly ingests plant biomass by phagocytosis.However, no such images were found, so we speculated that B. polyvacuolum may secrete enzymes outside in the gut environment to help with the digestion.We therefore investigated the presence of signal peptide pre-sequences on these CAZymes and found that some of them (CBM50, CBM57 and GH5) did indeed possess the signal peptide presequences.As these may act in the utilization and degradation of carbohydrates such as cellulose and chitin, this suggests that B. polyvacuolum helps its host digest plant biomass by secreting enzymes.

Hydrogenosome energy metabolism reconstruction
Multiple metabolic pathways of B. polyvacuolum were identified based on the homologous comparison in this study, including glycolysis, pyruvate metabolism, tricarboxylic acid (TCA) cycle, amino acid metabolism, and iron-sulfur cluster (ISC) biosynthesis (Fig. 3).All enzymes involved in glycolysis were identified.Pyruvate dehydrogenase (PDH), which is responsible for the oxidation of pyruvate, was also identified in MROs.The results showed that B. polyvacuolum had an incomplete TCA cycle.The electron transport chain (ETC) complexes I, III, and IV and V were completely absent.Only succinate dehydrogenase subunit A (SDHA) of complex II was identified.
Several enzymes and other proteins associated with the ISC biosynthesis system of B. polyvacuolum were identified.Among these, cysteine desulfurase (NFS1) could be the donor of sulfur and the Fe-S cluster could first be synthesized on the iron-sulfur cluster assembly protein 1 (ISU1).Iron-sulfur cluster biogenesis chaperone (Ssq1), GrpE protein (Mge1), and Jac1 could be responsible for the transfer of [2Fe-2S] clusters within the ISC biosynthesis pathway.Meanwhile, iron-sulfur cluster scaffold protein (Nfu1) could play a role in the assembly and transport of the [4Fe-4S] cluster.The Fe 2+ donor YAH1 was not identified.
Other enzymes involved in MRO pathways were also found, including the translocase of the inner mitochondrial membrane (Tim) complex, heat shock proteins (Hsp), ADP/ATP translocase (AAC), and the Mitochondrial Carrier Family Proteins (MCs).However, we did not detect the translocase of the outer mitochondrial membrane (Tom) complex.Superoxide dismutase (SOD) and peroxiredoxin (Prx), both of which are involved in the oxidative stress pathway, were also detected.

B. polyvacuolum may help host fish degrade plants and algae
Xenocyprinae fishes, the hosts of B. polyvacuolum, are omnivorous, feeding mainly on decaying sediment, as well as diatoms, attached algae and plant material [36].
Fig. 3 The predicted metabolic pathways of B. polyvacuolum.See Supplementary Table S4 for the full protein names.Proteins involved in different pathways are represented by different colors: yellow, glycolysis; pink, tricarboxylic acid (TCA) cycle; grey, electron transport chain (ETC); light-blue, amino acid metabolism; orange, pyruvate metabolism; dark-blue, fatty acid metabolism; green, iron-sulfur cluster (ISC) biosynthesis; purple, superoxide catalytic reaction; brown, other.The white shapes indicate that the protein is absent and the outlined circles indicate that some subunits of the protein have not been identified However, cellulose, as the main component of the plant cell walls, cannot be directly digested and utilized by fish [37].Thus, help from gut microorganisms including archaea, bacteria, and protozoa is particularly important.Previous studies have mostly focused on the beneficial effects of fish gut bacteria on nutrient metabolism, immune function, and health maintenance [37][38][39], but little is known about the role of protozoa in the fish gut.Ciliates have been extensively studied in domestic ruminants, and research has shown that these microorganisms may play a crucial role in the degradation of plant biomass, utilization of cellulose, and energy metabolism [40][41][42][43].On this basis, we hypothesized that ciliates may play similar roles in fish.
In indirect support of this hypothesis, we discovered many CAZymes in B. polyvacuolum through analysis of single-cell transcriptome data.Research has demonstrated that CAZymes act in the saccharification of polymeric carbohydrate resources, such as starch, cellulose or hemicellulose, by breaking them down into monosaccharides and oligosaccharides, which can be utilized by organisms [44].In this study, both COG annotation and KEGG pathway enrichment results showed that B. polyvacuolum produces many enzymes related to carbohydrate transport and metabolism and to energy metabolism.More specifically, we identified several enzyme families putatively associated with the metabolism of complex carbohydrates.The CE1 family is one of the largest and most diverse CE families, primarily responsible for the degradation of xylan [45,46].The CBM20 family comprises starch-binding domains and can disrupt the helical structure of amylose [47].The GH77 family contains starch-degrading enzymes, and it has also been identified in the rumen microbiome [48].
The presence of signal peptide pre-sequences suggests that B. polyvacuolum helps its host digest plant biomass by secreting enzymes.We also found a great number of SGs inside B. polyvacuolum and detected some glucose metabolism-related enzymes, which also indicates that B. polyvacuolum has the ability to degrade SGs.
Based on these findings, we hypothesize that a mutually beneficial symbiotic relationship may exist between the host fish and B. polyvacuolum.On the one hand, Xenocyprinae hosts provide B. polyvacuolum with underutilized food, a living environment, and protection from predators.On the other hand, B. polyvacuolum helps the host digest polysaccharides and improve the efficiency of nutrient use.In addition to B. polyvacuolum, however, there are also gut bacteria, fungi, and other protozoa living in the complex gut microhabitat, and the roles and interactions of these microorganisms remain unknown.More studies are therefore needed to investigate the relationship between these organisms and B. polyvacuolum.

B. polyvacuolum uses specialized MROs to adapt to the anaerobic environment of the digestive tract
Intestinal parasitic protists have evolved diverse MROs to survive in anaerobic or microaerophilic environments.For example, Nyctotherus ovalis living in the intestines of cockroaches has numerous hydrogen-producing mitochondria, which have both hydrogenosome-like characteristics and canonical mitochondrion-like characteristics [11,49,50].The MROs of the stramenopile Blastocystis hominis possess cristae, a mitochondrial genome, a TCA cycle and an incomplete ETC [8].Recent studies of rumen ciliates, such as the entodiniomorphids Entodinium furca and E. caudatum, and the vestibuliferid Isotricha intestinalis, have shown that their MROs have a partial ETC and can produce ATP via substrate phosphorylation [51].However, genomic data, both nuclear and MRO, for fish intestinal ciliates remain sparse.Thus, in this study, we tried to characterize the MROs of B. polyvacuolum from Xenocyprinae fish in order to contribute to a better understanding of the anaerobic adaptation of obligate endoparasites.
We found that the MROs of B. polyvacuolum lack almost all of the ETC complexes, except the SDHA of complex II.Similarly, only the SDHA of complex II has been identified in the anaerobic protozoa C. porcatum and P. cf. narasimhamurtii [14,32], and it is possible that the SDHA can act in reverse as a fumarate reductase [32].This suggests that a partial ETC could also possess functionality in the MROs of B. polyvacuolum.
Half of the TCA cycle-related enzymes from the canonical mitochondrial metabolisms were identified, which suggests that the MROs of B. polyvacuolum may possess canonical mitochondrial functions.Studies have shown that an incomplete TCA cycle can also run in reverse.When there are no complexes III and IV, fumarate can be converted into succinate by using electrons from rhodoquinol in anaerobic eukaryotes [52].The presence of a homologue of the putative rhodoquinol synthesis enzyme RquA may be a clue to the presence of rhodoquinol, but more experiments need to be done to validate this.
Our results have also provided some insights into the pyruvate metabolism of B. polyvacuolum.Although we did not identify Fe-hydrogenase, we did identify malic enzyme.This enzyme produces pyruvate for subsequent substrate-level phosphorylation, which suggests that the MROs of B. polyvacuolum may be able to generate ATP via substrate-level phosphorylation, in a similar way to other intestinal anaerobic ciliates [49].The canonical mitochondrial metabolism-related protein, the pyruvate dehydrogenase complex, is normally composed of three subunits (E1, E2, E3), but only one subunit (PDH-E1) was identified in B. polyvacuolum in this study.Evidence has shown that ciliates of the Spirotrichea, Armophorea, and Litostomatea (SAL) group likely use PDH, instead of the typical hydrogenosomal pyruvate:ferredoxin oxidoreductase (PFO) or pyruvate:NADP + oxidoreductase (PNO) [12,14].Acetyl CoA, the catalytic product of PDH, is then converted to succinyl-CoA (Su-CoA) by acetate:succinate CoA transferase (ASCT), and succinyl-CoA synthetase (SCS) then generates ATP by substrate level phosphorylation.Succinyl-CoA synthetase has been detected in this study, but not ASCT, and the alternative enzyme acetyl-CoA synthase was not detected either.This may be due to single-cell omics amplification bias, but more molecular experiments are needed in the future to validate this.
Based on the above findings, we assume that the MROs of B. polyvacuolum are hydrogenosomes with incomplete TCA cycles and a partial ETC complex.We plan to conduct further genomic analyses in the future, as well as studying the H 2 production, to verify this speculation.
In addition to identifying proteins related to the TCA cycle, the ETC, and pyruvate metabolism, we also identified proteins related to the ISC system in the genome of B. polyvacuolum.The biogenesis of cellular iron-sulfur proteins is an essential function of mitochondria and Fe-S cluster biogenesis pathways follow three main steps: de novo synthesis of a [2Fe-2S] cluster, trafficking of the cluster and insertion into [2Fe-2S] target apoproteins, and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster [53].The Fe-S cluster biogenesis pathways are highly conserved, with the ISC system operating in mitochondria, the CIA (Cytosolic Iron-sulfur protein Assembly) pathway operating in the cytosol and nucleus, and the SUF (SUlFur mobilization) system operating in plastid-containing organisms [54].In the parasitic protists, the hydrogenosomes of Trichomonas vaginalis can synthesize the Fe-S cluster via the ISC system [55].The mitosomes of Giardia contain components of the ISC system but lack YAH1 [56], and Blastocystis also contains a functional ISC system [57].In a study on N. ovalis, Hsp70 and type I [2Fe-2S] ferredoxin were the only proteins involved in the Fe-S cluster to be found [49].In this study, ISC biosynthesis system-related proteins, such as NFS1, ISU1, Ssq1, Mge1, Nfu1 and Hsp70, were detected.The only exception was YAH1, the donor of Fe 2+ .We therefore hypothesize that ISC biosynthesis may be functional in B. polyvacuolum.

Conclusion
In conclusion, the metabolic characteristics of B. polyvacuolum were studied.This ciliate may use specialized MROs (with an incomplete TCA cycle and a large part of the ETC missing) to adapt to the anaerobic digestive tract environment.Meanwhile, it may help the host fish with degradation of plant and algal material.

Data Availability
The raw Illumina sequences obtained during the current study are available in the Sequence Read Archive of GenBank under accession

Fig. 1
Fig. 1 Transmission electron micrographs of B. polyvacuolum.(A) The whole body of (B) polyvacuolum, with macronucleus (Ma) visible.(B) The distribution of starch granules (SG), a lysosome (Ly) and endosymbiotic bacteria (B), with the arrow showing dividing bacteria.(C) A magnification of bacteria, showing the cell wall and nucleoid.(D) The positional relationship between starch granules (SG), mitochondrion-related organelles (MRO), vacuoles (V) and kinetosomes (Ks).(E) Different shapes of dividing MROs.(F) Arrows show the rough endoplasmic reticulum (RER) around the MROs and arrowheads show the dumbbell-shaped MROs

Fig. 2
Fig. 2 Carbohydrate-active enzymes identified in B. polyvacuolum.(A)Four annotated carbohydrate hydrolase families (B) The type and proportion of annotated CEs.(C) The type and proportion of annotated CBMs.(D) The type and proportion of annotated GHs.(E) The type and proportion of annotated GTs This work was supported by the National Natural Science Foundation of China (No. 32170437), the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) (No. 2019QZKK0304), the earmarked fund for CARS (No. CARS-45), the Protist 10,000 Genomics Project (P10K) Consortium, and the National Aquatic Biological Resource Center (NABRC).