Balantidium grimi n. sp. (Ciliophora, Litostomatea), a new species inhabiting the rectum of the frog Quasipaa spinosa from Lishui, China

Balantidium grimi n. sp. is described from the rectum of the frog Quasipaa spinosa (Amphibia, Dicroglossidae) from Lishui, Zhejiang Province, China. The new species is described by both light microscopy (LM) and scanning electron microscopy (SEM), and a molecular phylogenetic analysis is also presented. This species has unique morphological features in that the body shape is somewhat flattened and the vestibulum is “V”-shaped, occupying nearly 3/8 to 4/7 of the body length. Only one contractile vacuole, situated at the posterior body, was observed. The phylogenetic analysis based on SSU-rDNA indicates that B. grimi groups together with B. duodeni and B. entozoon. In addition, the genus Balantidium is clearly polyphyletic.


Introduction
The genus Balantidium Claparède & Lachmann, 1858 consists of many species inhabiting the digestive tract in a wide number of hosts from both invertebrate and vertebrate animals as endocommensals. They are generally considered harmless, but factors depressing the resistance of the host enable them to invade the mucosa and cause ulceration. The representatives of Balantidium have some common morphological features: cell body sacciform or slightly elongated in shape, and completely covered with cilia forming dense longitudinal rows [21]. To our knowledge, 31 amphibian balantidial species have been reported so far (lists in Li et al. [20]).
Although many amphibian Balantidium species have been reported, few molecular data are available at present (only two species B. entozoon and B. duodeni have corresponding SSU-rDNA sequences in NCBI). Even less is known about phylogenetic relationships between different balantidial groups inhabiting different hosts (such as fishes, amphibians, mammals, etc.).
In the present study, a new Balantidium species inhabiting Quasipaa spinosa is described based on detailed light and scanning electron microscopy observation. This is also the first record of Balantidium species in the digestive tract of Quasipaa spinosa. Phylogenetic analysis based on SSU-rDNA was also carried out to reveal the relationships among Balantidium species as well as different clades of Trichostomatia.

Specimen collection and identification
The frogs used for this study were captured from Lishui City (27°25 0 -28°57 0 N, 118°41 0 -120°26 0 E), Zhejiang Province, southeast China in August, 2017. We obtained permits allowing us to capture and sacrifice these specimens. The frogs were transported alive to the laboratory, then all frog samples were anesthetized and dissected as soon as possible, the luminal contents of recta, intestines and duodena were collected respectively into different Petri dishes, and examined with the help of a stereomicroscope (Leica S8AP0, Germany). The ciliates were collected with Pasteur micropipettes and washed twice in 0.65% NaCl solution.

Light microscopy
Some specimens were fixed in 5% formalin for 10 min and soaked for about 30 min in 10% glycerin alcohol in a concave slide; the remaining specimens were fixed in Bouin's fluids and stained with a Protargol method [11].
Specimens were observed, measured and photographed using a microscope (Olympus BX53, Japan). All measurements are in micrometers.

Scanning electron microscopy
The fully washed specimens were fixed in 2.5% glutaraldehyde in 0.2 M PBS (pH 7.4) on a clean glass slide (1 cm Â 1 cm), which was previously treated with 0.1% poly-L-Lysine and dried completely in the air at room temperature. After being washed with PBS 3 times, they were post-fixed in 1% osmium tetroxide at 4°C for 1 h, followed by serial dehydration in acetone and critical point drying using the HCP-2 critical point dryer (Hitachi Science Systems, Japan). Subsequently, the glass slide was mounted on an aluminum-stub using a double-sided adhesive tape and sputter-coated with a thin layer of gold in IB-3 ion coater (Eiko Engineering, Japan), before observation and photography using a Quanta 200 SEM (FEI, Netherlands).

Extraction of genomic DNA and PCR amplification
About 50 individuals were harvested, suspended in lysis buffer (10 mM Tris-HCl, pH 8.0; 1 M EDTA, pH 8.0; 0.5 % sodium dodecyl sulfate; 60 mg/mL proteinase K), and incubated at 55°C for 12-20 h. DNA was extracted using a standard phenol/chloroform method, precipitated with ethanol, and resuspended in TE buffer. Polymerase chain reaction (PCR) amplifications were carried out using forward primer (5'-AACCTGGTTGATCCTGC-CAGT-3') and reverse primer (5'-TGATCCTTCTG-CAGGTTCACCTAC-3') [23]. The following cycling conditions included 5 min initial denaturation at 94°C; 35 cycles of 30s at 95°C, 1 min at 56-60°C, and 1-2 min at 72°C; with a final extension of 10 min at 72°C. The PCR products were isolated using 1% agarose gel electrophoresis and purified using the Agarose Gel DNA Purification Kit (TaKaRa Biotechnology, Dalian, Japan). The amplified fragment was cloned into a pMD ® 18-T vector (TaKaRa Biotechnology, Dalian) and sequenced in both directions using M13 forward and reverse primers on an ABI PRISM ® 3730 DNA Sequencer (Applied Biosystems, USA). The SSU rRNA gene sequence of B. grimi was deposited in GenBank with accession number MG837094.

Phylogenetic analysis
Besides the SSU-rDNA sequence of B. grimi that we obtained in this study, other litostomatean sequences were retrieved from the GenBank/EMBL databases ( Table 1). The sequence of Nyctotheroides deslierresae was used as the outgroup. The secondary structure-based SSU-rRNA sequence alignment of Litostomatea downloaded from the SILVA ribosomal RNA gene database project (https:// www.arb-silva.de/) [29] was used as the "seed" alignment to build a profile Hidden Markov Model (HMM) using HMMER Package, version 3.1. Then the HMM profile obtained was used to create an alignment of the 40 sequences using Hmmalign within the package. The masked regions that could not be aligned unambiguously were removed from the initial alignment using MEGA 6.0 [39]. A GTR+I+G model was selected as the best model by the program jModelTest 2.1.10 [8] based on the AIC criterion, which was used for both Maximum Likelihood (ML) and Bayesian (BI) inference analysis. An ML tree was constructed with the RaxML program [35]. The reliability of internal branches was assessed using the nonparametric bootstrap method with 1,000 pseudoreplicates. A Bayesian analysis performed with MrBayes v3.2.6 [32] was run for 1,000,000 generations sampling every 1,000 generations. All trees below the observed stationary level were discarded as a burn-in of 25% of the generations.

Results
Ninety-eight individuals of Q. spinosa were examined in the present study and 34 were found to be infected with Balantidium grimi (prevalence, 34.7%). These specimens were found mainly in the recta of frogs. Etymology: The new species was designated Balantidium grimi n. sp. in honor of the great contributions of Prof. J. Norman Grim to parasitic and symbiotic ciliates.  Figures 1A  and 2). A cytoproct present at the posterior end of the body (Figures 1A and 2). Detailed morphometric parameters are presented in Table 2.

Morphology under scanning electron microscope
B. grimi is thickly ciliated, but with uniform arrangement on the cell surface ( Figures 3A, B). Regular beat patterns of cilia that look like "waves" make the cell move smoothly ( Figure 3A). The "waves" and ridges Measurements are in mm.  formed an angle ranging from 0°(at the posterior) to 60°( at the anterior) ( Figures 3A, C, D). Numerous cortical grooves arranged alternately with cortical ridges, which are parallel to the longitudinal axis of the body ( Figure 3D). The cilia originate within grooves and are quite close together; those in Figure 3D are about 0.62 mm apart.

Phylogenetic analysis
The sequenced SSU-rRNA gene of B. grimi is 1,640 bases in length and the guanine-cytosine (GC) content is 42.26%. The topologies of our phylogenetic trees generated using MrBayes and PhyML algorithms are totally accordant (Figure 4). Species of the family Balantidiidae are separated into three clades. B. grimi grouped together with B. duodeni and the type species of the genus, B. entozoon, and form the first clade whose hosts are anuran amphibians (100% ML, 1.00 BI). B. polyvacuolum and B. ctenopharyngodoni form the second balantidial clade inhabiting fish hosts. The third group consisted of two isolates of B. coli, which were reported from many mammalian hosts, including pigs and humans.

Discussion
A new Balantidium species inhabiting Chinese anuran amphibians Quasipaa spinosa is recorded herein. To our knowledge, this is the first report of Balantidium species in Q. spinosa.
B. grimi is quite unique considering its remarkably flattened body and conspicuous slit-shaped vestibulum, which can distinguish it from other Balantidium species [7,12,21]. B. grimi resembles B.entozoon, B. duodeni, B. helenae and B. sinensis in some aspects. For example, B. grimi shares a similar Lv/Lb value with B. duodeni [7]. But in terms of body forms and dimensions, these two balantidial species could easily be discriminated from each other. As to the shape and dimension of the macronucleus, as well as the position of the contractile vacuole, B. grimi somewhat resembles B. helenae [33], but the latter species possesses a remarkable "knob" at the posterior end. Comparisons were also made between B. grimi and B. sinensis inhabiting the Chinese giant salamander Andrias davidianus [20] as well as B. entozoon, the type species of the genus Balantidium [12]. Detailed comparisons of morphometric parameters among corresponding Balantidium species are presented in Table 3.
According to the molecular phylogenetic analysis, the order Macropodiniida ciliates is closely related to fish balantidial species [14,19]. The affinity implies that macropodiniids may have been the result of separate invasions of terrestrial hosts by ciliates initially associated with aquatic hosts [19]. Macropodiniids, previously called "Australian clade", possess similar oral cavities to some vestibuliferids that are bordered by somatic kineties and analogous ultrastructure to the Isotrichidae [5,21,37,38]. Moreover, the strong molecular support of Macropodiniida assemblage as a sister clade to the Balantidiidae (fish balantidia) also gives us an indication that Macropodiniida ought to be incorporated into the order Vestibuliferida, which also coincides with the viewpoint of former studies [5,14,19].
Our results show that the genus Balantidium is clearly polyphyletic and all Balantidium species are separated into three distinct clades, according to host specificity: fish balantidia (B. ctenopharyngodoni and B. polyvacuolum), amphibian balantidia (B. grimi, B. entozoon and B. duodeni), and balantidia from warm-blooded vertebrates (Balantioides coli) [7]. Pomajbíková et al. [26] has proposed a new genus Neobalantidium for the third group. However, it was recently suggested to reinstate the genus Balantioides as this taxon has been named for a long time [7]. Here, we accepted the generic name Balantioides to describe this group. As to the amphibian balantidia, our new species clustered with the other two species, B. entozoon and B. duodeni with maximum molecular supports. On this point, our results are consistent with  , an interkinetal ridge (rd), the groove (gr) and the cilia (cl) extending from grooves and are close to one another. Scale bar = 5 mm. E. Selected enlargement of Figure 3D, showing a ridge (rd) between cilia. Scale bar = 2 mm. those of Chistyakova et al. [7], but differ from those of Li et al. [19]. We suspect that the key reason for this disagreement is the quantity of introduced species used for phylogenetic analysis. The greater the number of related species studied, the greater the accuracy of the resulting phylogeny. Thus, more molecular information on Balantidium species from fishes and amphibians as well as reptiles is needed to clarify their phylogenetic relationships.