Morphological and molecular characterization of an undescribed vestibuliferid ciliate from the captive-bred naked mole-rat, Heterocephalus glaber (Rodentia: Bathyergidae)

Abstract

The intestinal ciliates of the naked mole-rat (Heterocephalus glaber) remain poorly characterized. Previous reports identified a Balantidium or suggested an association with Meiostoma; however, their taxonomic identities have not been verified using morphological and molecular approaches. In March 2024, numerous ciliates were detected in fecal samples from a captive colony of naked mole-rats at a zoo in Japan. The ciliate was characterized using light microscopy, scanning electron microscopy, and molecular analyses based on the nuclear ribosomal RNA gene sequences.

The ciliate exhibited morphological characteristics typical of vestibuliferid ciliates. Molecular analyses based on ribosomal RNA regions revealed a distinct lineage related to Balantioides coli (syn. Balantidium coli) and other vestibuliferid ciliates. These morphological and molecular data indicate that the ciliate from the naked mole-rat represents a previously unrecognized vestibuliferid lineage. This study provides new insights into the diversity and taxonomy of intestinal ciliates associated with naked mole-rats.

Graphical abstract

Highlights

• •An intestinal ciliate was found in a captive naked mole-rat colony.
• •Morphological and molecular data indicate a previously unrecognized lineage.
• •18S and ITS1–5.8S–ITS2 sequences support a distinct phylogenetic position.
• •The ciliate was not identified as the primary cause of mortality.

1. Introduction

The naked mole-rat, Heterocephalus glaber (Rodentia: Hystricomorpha: Bathyergidae), is a eusocial subterranean rodent native to eastern Africa. This species has attracted considerable scientific interest because of its unusual biological characteristics, including poor thermoregulation, exceptional longevity, and remarkable resistance to cancer and age-related diseases (Hill et al., 1957; Buffenstein, 2005). Consequently, naked mole-rats are maintained in zoological collections and research facilities worldwide.

In March 2024, an episode of consecutive deaths occurred in a captive colony of naked mole-rats maintained at Ueno Zoo, Tokyo, Japan. Microscopic examination of intestinal contents and fecal samples from affected animals revealed the presence of a Balantidium-like ciliate and trichomonad-like flagellates. The first detailed description of a Balantidium-like ciliate from the naked mole-rat was provided by Porter (1957). Subsequent reports also described Balantidium-like ciliates from this host (Hill et al., 1957; de Graaff, 1964; Buffenstein and Yahav, 1991; Dyer, 1998; Buffenstein et al., 2012; Rakhimov et al., 2026). The taxonomy of mammalian Balantidium has since been revised: Balantidium coli was transferred first to Neobalantidium (Pomajbíková et al., 2013) and subsequently to Balantioides (Chistyakova et al., 2014). Dyer (1998), however, suggested that this Balantidium-like ciliate from the naked-mole rat might have been misidentified and could instead belong to the genus Meiostoma, a monotypic genus represented by Meiostoma georhychi, originally described from the caecum of the Cape mole-rat (Georychus capensis) in South Africa (Sandon, 1941). Unlike Balantioides (family Balantidiidae), M. georhychi was originally described as possessing a concrement vacuole, a diagnostic feature more typically associated with the family Buetschliidae (Lynn, 2008). In the original description, the genus name was spelled Meistoma in the title but Meiostoma in the text, and the specific epithet was based on Georhychus, an incorrect subsequent spelling of the host genus Georychus Illiger, 1811 (Bennett et al. (2006). This proposed reassignment to Meiostoma, however, has not been verified by detailed morphological or molecular analyses. To date, therefore, only two ciliate taxa have been proposed for the naked mole-rat: Balantidium/Balantioides sp., based on repeated morphological observations since Porter (1957), and Meiostoma, based on Dyer's (1998) unverified hypothesis. The taxonomic identity of intestinal ciliates infecting naked mole-rats therefore remains unresolved. In the present study, we investigated the protozoan organisms detected during the diagnostic investigation of a mortality event in a captive naked mole-rat colony and characterized the intestinal ciliate using morphological and molecular approaches.

2. Materials and methods

2.1. Parasitological examination

In March 2024, more than 10 naked mole-rats from a captive colony of approximately 50 individuals maintained at Ueno Zoo, Tokyo, Japan, died within a 10-day period. The colony was established from naked mole-rats imported from Singapore in 2001 for zoological exhibition and captive breeding purposes and has been maintained through successive captive breeding at the facility.

Fecal examinations of dead or moribund animals revealed numerous large ciliates (Video). To investigate the ciliates detected during the diagnostic examination, fecal samples were collected and fixed in either 10% neutral buffered formalin or 70% ethanol and transported to the Laboratory of Veterinary Parasitology, Nippon Veterinary and Life Science University, for morphological and molecular analyses. Because fecal samples were collected from the colony without individual identification, the infection status of each animal could not be determined. Histopathological examinations were conducted by commercial diagnostic laboratories. Lesions suggestive of herpesvirus infection were observed in several individuals; however, the definitive cause of death could not be determined. Following treatment of the colony with metronidazole, protozoa were no longer detected in fecal examinations, and further sampling was not possible.

2.2. Morphological analyses

2.2.1. Light microscopy

Formalin-fixed specimens were subjected to MFS staining according to Imai (1998) and silver impregnation using a modified Fernandez-Galiano method (Augustin et al., 1984). Morphological observations were performed using an Olympus BX41 light microscope (Olympus, Tokyo, Japan) and photomicrographs were captured with an Olympus DP74 digital camera. The orientation of the cell body was determined based on the position of the oral apparatus when observable. For specimens lacking a discernible oral apparatus, the orientation was assigned based on general body morphology. Morphometric measurements were obtained from 30 individuals using cellSens software (Olympus) and presented in micrometers as the mean ± standard deviation, followed by the range (minimum-maximum). For the curved macronucleus, the actual curved length was measured rather than the maximum straight-line distance between the two farthest points.

2.2.2. Scanning electron microscopy

Scanning electron microscopy (SEM) was performed essentially as described by Tokiwa et al. (2010). Briefly, formalin-fixed specimens were post-fixed in 2% osmium tetroxide, dehydrated through a graded ethanol series, freeze-dried, sputter-coated with platinum–palladium, and examined using a JSM-6380LV scanning electron microscope (JEOL, Tokyo, Japan).

2.3. Molecular analyses

2.3.1. DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from a single fecal specimen preserved in 70% ethanol using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Partial sequences of the nuclear 18S rRNA gene (18S) and the complete ITS1–5.8S rRNA–ITS2 region (ITS1–5.8S–ITS2) were amplified by polymerase chain reaction (PCR). PCR was performed in a final reaction volume of 20 μL containing 0.2 μL TaKaRa Ex Taq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), 2.0 μL of 10× Ex Taq buffer, 1.6 μL dNTP mixture (2.5 mM each), 0.2 μL each of forward and reverse primer (50 μM, final concentration 0.5 μM each), 1.0 μL template DNA, and 14.8 μL nuclease-free water. The 18S was amplified using the universal eukaryotic primers Medlin A (5′-AACCTGGTTGATCCTGCCAGT-3′) and Medlin B (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) (Medlin et al., 1988). PCR amplification consisted of an initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 62°C for 2 min, and 72°C for 2 min, with a final extension at 72°C for 7 min. The ITS1–5.8S–ITS2 was amplified using primers B5D (5′-GCTCCTACCGATACCGGGT-3′) and B5R (5′-ATATGCTTAAGTTCAGCGGG-3′) (Ponce-Gordo et al., 2008). PCR conditions consisted of an initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products were separated by electrophoresis on 1.5% agarose gels and stained with GRGreen (BioCraft, Tokyo, Japan). DNA bands were visualized using an LED transilluminator, and fragment sizes were estimated with a 100-bp DNA ladder (Maestrogen, Taiwan). PCR products were treated with ExoSAP-IT (Thermo Fisher Scientific, USA) prior to direct sequencing in both directions using the amplification primers on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

2.3.2. Sequence and phylogenetic analyses

The boundaries of the rRNA gene regions were determined by comparison with the corresponding annotated sequence of Balantioides coli (GenBank accession no. KM098124). The obtained sequences were subjected to similarity searches using the BLASTn program against sequences available in the International Nucleotide Sequence Database Collaboration (INSDC) database.

For phylogenetic analysis, a total of 261 representative 18S sequences of trichostomatid ciliates were selected, together with one haptorian sequence, Didinium nasutum (Didiniidae; U57771), which was used as the outgroup following Ito et al. (2014). For the phylogenetic analysis based on the ITS1–5.8S–ITS2 region, 123 representative sequences were selected, and the tree was rooted with Isotricha prostoma (Isotrichidae; PP067626), following Belokon et al. (2026). These sequences were retrieved from the INSDC database using the BLAST search function in Geneious Prime 2025.2 (Biomatters Ltd., Auckland, New Zealand), and sequences considered inappropriate or of questionable quality based on the BLAST search results were manually excluded. The resulting datasets, including the sequence alignments used for phylogenetic reconstruction, have been deposited in Mendeley Data.

Multiple sequence alignment was performed using the L-INS-i algorithm implemented in MAFFT v7 (Katoh and Standley, 2013). A maximum-likelihood (ML) phylogenetic tree was reconstructed using IQ-TREE (Trifinopoulos et al., 2016) via the W-IQ-TREE web server implemented at the Los Alamos HIV Sequence Database. The best-fit nucleotide substitution models (GTR + F + R8 for 18S, TIM2+F + G4 for ITS1-5.8S-ITS2) were selected automatically. Nodal support was assessed using 1000 ultrafast bootstrap (UFBoot) (Minh et al., 2013) replicates and 2000 SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al., 2010) replicates. The resulting tree was visualized and edited using the Interactive Tree Of Life (iTOL) (Letunic and Bork, 2024). Taxonomic classification above the family level follows Lynn (2008).

3. Results

3.1. Morphological characteristics

Trophozoites showed considerable variation in body shape and size. Detailed morphometric measurements are summarized in Table 1. In typical specimens, the body was elongated oval to spindle-shaped, with the anterior end often slightly curved. The entire body surface was covered with numerous cilia (Fig. 1A, 3A and 3B) arranged in longitudinal rows (Fig. 2A–C, 3D). Silver impregnation revealed numerous somatic kineties extending longitudinally along the body surface (Fig. 2A–C), with 56–66 kineties observed. The macronucleus, examined in MFS-stained (Fig. 1B) and silver-impregnated specimens (Fig. 2A, B, 2D–F), was elongated and curved, and was located in the central region or occasionally shifted toward the anterior or posterior region of the body. An ellipsoidal to spherical micronucleus (Fig. 2E and F), measuring 3–7 μm in length, was observed adjacent to the macronucleus. The cytostome and vestibule were inconspicuous and could not be clearly observed in most specimens. In a few specimens where the oral region was discernible, the cytostome appeared as a small opening at the anterior region of the body (Figs. 2C and 3C). In unstained preparations, a non-ciliated sac-like structure (Fig. 1C) was occasionally observed protruding from this region. Numerous rod-shaped bacteria were observed within the body (Fig. 2A, D, 2E).

Table 1.

Morphometric comparison of the present ciliate with previously reported ciliates from naked mole-rats (Heterocephalus glaber) and Meiostoma georhychi.

Species	Present study	Balantidium sp.	Meiostoma georhychi
Host	Heterocephalus glaber	Heterocephalus glaber	Georychus capensis
Locality	Japan (captive)	Somaliland and Kenya	South Africa
Reference	Present study	Porter (1957)	Sandon (1941)
Body length (L)	211.7 ± 46.3 (102.3–306.1)	(30–320)	200–800
Body width (W)	89.4 ± 23.4 (48.1–131.0)	11.5–60	NR
L/W ratio	2.5 ± 0.6 (1.6–3.6)	NR	2.6–8.2
Macronucleus	40.3 ± 10.0 (23.6–68.3)	11.85–60	kidney-shaped (25–45) to sausage-shaped (88 × 27.5)
Concrement vacuole	ND	NR	Present
Somatic kineties	56–66	NR	NRa
Cyst	ND	17.7 × 16.3 – 76.6 × 66.6	NR

ND, not detected; NR, not reported.

^aThe original description reported only the spacing between adjacent somatic kineties (≤1 μm), whereas the total number of somatic kineties was not given.

Fig. 1.

Light micrographs of the intestinal ciliate from the naked mole-rat (Heterocephalus glaber). (A) Formalin-fixed trophozoite showing an elongated oval to spindle-shaped, bilaterally asymmetrical body covered with cilia. (B) MFS-stained trophozoite showing the curved macronucleus (arrowhead). (C) Small trophozoite showing a protruding structure at the anterior end (arrow). Scale bars = 50 μm.

Fig. 3.

Scanning electron micrographs of trophozoites of the intestinal ciliate from the naked mole-rat (Heterocephalus glaber). (A) Left lateral view. (B) Ventrolateral view. (C) Anterior region showing the oral opening. (D) Somatic kineties. Scale bars = 20 μm (A, B) and 5 μm (C, D).

Fig. 2.

Silver-impregnated trophozoites of the intestinal ciliate from the naked mole-rat (Heterocephalus glaber). (A) Dorsal view. (B) Ventral view. (C) Ventral view of the anterior region showing the small anterior cytostome opening. (D–F) Macronucleus and micronucleus. Dorsoventral (D, E) and lateral (F) views of the macronucleus (arrowheads) and micronucleus (arrows). Scale bars = 50 μm (A, B) and 10 μm (C–F).

3.2. Molecular characterization and phylogenetic analysis

Genomic DNA was extracted from a fecal sample containing multiple ciliate individuals. For both the 18S and ITS1–5.8S–ITS2 sequences, no ambiguity (e.g., overlapping peaks) was observed in the sequencing chromatograms, indicating that a single ciliate lineage was present in the sample.

A total of 1063 bp of the 18S gene sequence was determined. The guanine–cytosine (GC) content was 41.5%. BLASTn analysis showed that the obtained sequence shared the highest sequence identity with vestibuliferid ciliates. The highest sequence identities were observed with Balantioides coli (96.1–96.6%), followed by members of the family Pycnotrichidae, including Buxtonella sulcata (≤96.0%) and Infundibulorium cameli (≤95.9%). The maximum-likelihood tree placed the sequence obtained in this study as an independent lineage that did not cluster within any previously described genus. This lineage was recovered near the base of a clade comprising the vestibuliferid families Balantidiidae, Paraisotrichidae, and Pycnotrichidae together with members of the entodiniomorph families Ditoxidae and Buetschliidae, but nodal support for this placement was low (UFBoot 73%, SH-aLRT 51%), consistent with an unstable phylogenetic position (Fig. 4).

Fig. 4.

Maximum-likelihood phylogenetic tree inferred from 18S rRNA gene sequences of trichostomatid ciliates. Bootstrap support values (UFBoot/SH-aLRT) ≥ 50% are shown at the nodes. Clades containing multiple sequences were collapsed for clarity. The scale bar indicates the number of substitutions per site.

A separate fragment spanning the ITS1–5.8S–ITS2 region was amplified using the primers B5D and B5R. This amplified fragment was 493 bp in length (GC content of 32.3%) and, owing to the primer binding sites, also included short partial 18S (109 bp) and 28S (23 bp) sequences flanking the ITS1–5.8S–ITS2 region proper. This latter comprised a complete ITS1 sequence (111 bp; GC content, 14.5%), a complete 5.8S gene sequence (144 bp; GC content, 41.0%), and a complete ITS2 sequence (106 bp; GC content, 22.6%). The ITS1 sequence showed the highest sequence identity to B. coli sequences, with a maximum identity of 86.1%. The 5.8S gene sequence showed 100% sequence identity to Balantidium ctenopharyngodoni (KU170972), which was reported from grass carp (Ctenopharyngodon idella). The maximum sequence identities to Buxtonella sequences from different monkey species and B. coli sequences were 97.9% and 97.2%, respectively. The ITS2 sequence shared a maximum sequence identity of 87.7% with B. coli sequences. In the phylogenetic tree based on the ITS1–5.8S–ITS2 region (Fig. 5), Balantidiidae and Pycnotrichidae formed a monophyletic clade (UFBoot 95.5%, SH-aLRT 97%). Within this clade, B. coli branched first (UFBoot 79.8%, SH-aLRT 89%), followed by the ciliate detected in the present study (UFBoot 60.8%, SH-aLRT 82%). The remaining taxa (UFBoot 88.2%, SH-aLRT 91%) branched sequentially. Both Balantidium and Buxtonella were each recovered as polyphyletic.

Fig. 5.

Maximum-likelihood phylogenetic tree inferred from ITS1–5.8S–ITS2 rRNA gene sequences of trichostomatid ciliates. Bootstrap support values (UFBoot/SH-aLRT) ≥ 50% are shown at the nodes. Clades containing multiple sequences were collapsed for clarity. The scale bar indicates the number of substitutions per site.

4. Discussion

A ciliate morphologically similar to the present organism was reported by Porter (1957) as a Balantidium sp. from the naked mole-rat. Porter noted that the morphological variation in this ciliate was greater than that observed in other species of Balantidium and suggested that the criteria proposed by Hegner (1934) for the genus Balantidium were not applicable to this ciliate. The variation in body shape and nuclear morphology was comparable to that observed in the present specimens (Table 1). According to Porter (1957), the oral apparatus was clearly visible in living specimens and in specimens examined in water or alcohol preparations. A transparent droplet-like structure was also occasionally observed protruding from the cytostome, resembling that observed in the present study. However, Porter described a ciliated vestibular region and noted that the posterior end of the body lacked cilia. Although the vestibular apparatus could not be clearly observed in the present specimens, the present specimens differed from Porter's specimens in that the entire body surface, including the posterior region, was covered with cilia. This discrepancy may be attributable to differences in fixation and preservation conditions, although the possibility that the two ciliates represent distinct taxa cannot be excluded. Despite this morphological variation, molecular analysis of the fecal sample yielded unambiguous 18S and ITS1–5.8S–ITS2 chromatograms, consistent with the presence of a single ciliate lineage in the sample. The observed variation in body shape and size is therefore likely to reflect intraspecific plasticity within this lineage, as similarly noted by Porter (1957), rather than the co-occurrence of multiple ciliate taxa.

Dyer (1998) later suggested that the Balantidium-like ciliate reported by Porter might instead represent M. georhychi, a ciliate originally described from another subterranean hystricomorph rodent, the Cape mole-rat (Georychus capensis; Sandon, 1941). Meiostoma georhychi shares some similarities with the present ciliate, such as the presence of a small anterior cytostome and an occasional transparent droplet-like structure protruding from the oral region. The cytostome of M. georhychi was described as a very small circular opening in the pellicle at the anterior end of the body, with an occasional faint transparent process or oral papilla. In addition, M. georhychi has a considerably larger body size (200–800 μm) and occasionally possesses a concrement vacuole, a structure characteristic of the family Buetschliidae. These characteristics suggest that M. georhychi may not belong to the same lineage as the present ciliate and may instead be related to members of Buetschliidae. Based on these morphological characteristics, the ciliate identified in the present study was assigned to an undescribed vestibuliferid lineage.

Molecular data further support the distinctiveness of the present ciliate from currently recognized vestibuliferid taxa. Although the 18S sequence showed the highest similarity to B. coli, the relatively low sequence identity, together with the marked divergence observed in the ITS1 and ITS2 regions, suggests that the present organism represents a genetically distinct lineage. The complete identity of the 5.8S gene sequence with Balantidium ctenopharyngodoni should be interpreted cautiously because this region is highly conserved and provides limited resolution for species-level identification. The combination of divergence across multiple ribosomal markers and the organism's distinct phylogenetic position suggests that this organism is genetically distinct from all currently characterized vestibuliferid taxa. The unstable placement of the present lineage in the 18S tree is consistent with previous analyses of trichostome ciliates, in which the orders Vestibuliferida and Entodiniomorphida, as well as several families such as the Cycloposthiidae and Paraisotrichidae, were recovered as non-monophyletic (Ito et al., 2014; Kornilova et al., 2023). In the ITS1–5.8S–ITS2-based tree, the present lineage was similarly recovered as a distinct branch within the clade comprising Balantidiidae and Pycnotrichidae, diverging after B. coli, although nodal support for this placement was likewise moderate to low. This concordance between the two independent markers, despite their differing rates of evolution, lends some support to the present lineage occupying a genuinely distinct position near the base of this clade, although, as discussed above, the reliability of the ITS-based topology at this taxonomic depth should be interpreted with caution. This limited resolution likely reflects both the rapid divergence of these lineages and the sparse taxon sampling currently available for many vestibuliferid groups, and denser sampling combined with additional molecular markers will be required to resolve the phylogenetic position of the present ciliate.

Taken together, the morphological and molecular evidence suggests that the ciliate detected from the naked mole-rat represents a previously unrecognized lineage within the Vestibuliferida. However, detailed observations of the vestibular apparatus, which is an important diagnostic character in vestibuliferid ciliates, are required before formally establishing a new taxon. For the same reason, a detailed morphological comparison with the broader range of vestibuliferid families recovered in the present phylogenetic analyses is not currently feasible, as such a comparison would similarly rely on characters of the oral infraciliature and vestibular apparatus that could not be reliably assessed in the available specimens. Additional specimens from wild or captive populations of naked mole-rats will be necessary to confirm the morphological characteristics and taxonomic position of this ciliate. Furthermore, a detailed redescription of Meiostoma, including comprehensive morphological and molecular characterization, is required to clarify its phylogenetic position. Because these morphological data are currently insufficient for a formal description, we refrain from erecting a new taxon here. To facilitate reference in future studies, however, we provisionally designate this ciliate as the naked mole-rat vestibuliferid HgV. This is an informal designation and does not constitute an available name under the International Code of Zoological Nomenclature; the ciliate can be unambiguously identified by the nucleotide sequences deposited in the present study.

Adult naked mole-rats are known to defecate in specialized latrine chambers within their burrow systems. Rather than consuming feces deposited in the latrines, they typically ingest fecal pellets directly as these are voided, both from themselves (autocoprophagy) and from other colony members (allocoprophagy) (Dyer, 1998; Tanes et al., 2026). This coprophagic behavior is thought to help establish and stabilize the gut microbial community, and the ingested feces contain abundant microorganisms derived from hindgut fermentation (Tanes et al., 2026). Considering the biology of related intestinal ciliates, these behaviors may facilitate fecal–oral transmission and maintenance of vestibuliferid ciliates within naked mole-rat colonies. Although cysts were not observed in the present study, Porter (1957) reported cysts in fecal samples of naked mole-rats. If such structures are also produced by the present ciliate, encystment could further contribute to transmission within colonies.

In the present study, numerous ciliates were observed in fecal samples from affected animals; however, no evidence of tissue invasion or specific pathological changes associated with the ciliate was obtained (data not shown). Therefore, the vestibuliferid ciliate detected in this study is considered unlikely to have been the primary cause of mortality. A similar increase in ciliate abundance has also been reported in naked mole-rats by Delaney et al. (2013), suggesting that such proliferation may reflect dysbiosis of the intestinal microbial community. Further investigations are required to determine its potential pathogenicity and ecological role in naked mole-rats.

CRediT authorship contribution statement

Mami Takase: Writing – review & editing, Visualization, Investigation, Formal analysis. Toshihiro Tokiwa: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Data availability

The nucleotide sequences generated in this study have been deposited in the DNA Data Bank of Japan under accession numbers LC948552 (18S) and LC948553 (18S–ITS1–5.8S–ITS2–28S). The sequence alignments used for the phylogenetic analyses have been deposited in Mendeley Data (10.17632/89p2rmjmj8.1).

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Claude (Anthropic) solely for English language editing and proofreading. After using the AI tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Declaration of competing interests

The authors declare no competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Video

Motile trophozoites of the intestinal ciliate observed in a direct fecal smear of a naked mole-rat.