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Protistology 18 (2): 161-172 (2024) | doi:10.21685/1680-0826-2024-18-2-6 PPOtÎStOlOây
Original article
Ciliates from faeces of zebras Equus quagga from different zoos: species composition and intraspecies diversity
Olga Kornilova1, Olga Alekseeva2, Maria Skazina2, Mariia Belokon2 and Ludmila Chistyakova3*
1 Herzen State Pedagogical University of Russia, 191186 St. Petersburg, Russia
2 St. Petersburg State University, 199034 St. Petersburg, Russia
3 Zoological Institute RAS, 199034 St. Petersburg, Russia
| Submitted March 29, 2024 | Accepted May 6, 2024 |
Summary
Species composition of the endobiotic ciliates from the faeces of zebras Equus quagga from different zoos was investigated. The tendency towards a decrease in the species diversity of endobionts in captivity has been demonstrated. Most species of endobiotic ciliates typical for zebras have not been found in animals kept in zoos; the number of species of bacterivorous ciliates — representatives of the family Buetschliidae — is often small. At the same time, zebras in captivity may have species of ciliates not found in a given host in natural habitats. The level of differences in SSU and ITS region sequences in geographical isolates of ciliates Cycloposthium edentatum, Cochliatoxum periachtum, and Tripalmaria dogieli was analyzed.
Key words: endobiont ciliates, Equus quagga, captivity, geographical isolates
Introduction
Faunistic studies of endobiotic ciliates from the gastro-intestinal tract of herbivorous mammals kept in zoos are a promising research direction. Captivity conditions make it possible to analyse the influence of various environmental factors, such as diet, on the communities of endobiotic ciliates. Prolonged isolation of small groups of animal hosts provides a unique opportunity for a comparative analysis ofthe intraspecific variability of endobionts, which can be performed with the use of various genetic markers of populations of particular ciliate species. At the same time, co-habitation of several species of animal
https://doi.org/10.21685/1680-0826-2024-18-2-6
© 2024 The Author(s)
Protistology © 2024 Protozoological Society Affiliated with RAS
hosts increases the probability oftheir infection with non-specific ciliate species, allowing the study of its effect on the structure of endobiotic communities.
Comparative analyses of the species diversity of endobiotic ciliates in wild and captive hosts may also have practical importance. As a component of the microbiome ofthe gastro-intestinal tract, ciliates are actively involved in digestion. An assessment of changes in the structure of endobiotic communities during prolonged captivity may be useful for optimising animal care conditions. However, the communities of endobiotic ciliates in the gastrointestinal tract of captive mammals, including odd-toes ungulates, are almost unstudied.
Corresponding author: Ludmila Chistyakova. Zoological Institute RAS, Universitetskaya Emb. 1, 199034 St. Petersburg, Russia; pelomixa@mail.ru
Here, we present the results of our study of the species diversity of endobiotic ciliates of zebras Equus quagga from zoos in Vladivostok and Stary Oskol (Russia) and Vienna (Austria). We performed a comparative analysis of the species composition of endobiotic ciliates of wild and captive zebras and SSU sequence variability analysis for several geographical isolates of three species of endobiotic ciliates (Tripalmaria dogieli, Cycloposthium edenta-tum, Cochliatoxum periachtum) from zebras and from domestic horses.
Material and methods
Faecal samples were collected from zebras Equus quagga burchellii and E. quagga chapmani and domestic horses E. caballus (Table 1). A small portion of fresh faeces was placed in plastic tubes with a volume of up to 50 ml and fixed with 96° alcohol at a ratio of 1:1. In the laboratory, the fixative was changed, and the samples were then stored in the dark at room temperature.
Light-optical examinations and micropho-tography were performed using a Leica DM2500 microscope (Leica-Microsystems, Germany) equipped with a differential interference contrast and a Leica DFC495 (8.0MP) digital camera. During the study of cell morphology, the macronucleus was detected with the use of a 0.1% solution of methyl green in a 1% solution of acetic acid. The ciliates were identified following Gassovsky (1919), Hsiung (1930), Strelkow (1931, 1939), and Lynn (2008).
For DNA isolation, ciliate cells were picked one by one under a Nikon SMZ 1270 stereomicroscope (Nikon Corporation, Japan), washed in two changes of distilled water and placed in tubes with a lysing solution, 45—50 cells per tube. Genomic DNA was extracted using PureLink Genomic DNA Kit (Invitrogen) for DNA extraction according to the manufacturer's instructions. PicoPure™ DNA Extraction Kit (Thermo) was used for single-cell DNA isolation.
SSU rRNA gene fragment ~1400 bp long was amplified with the use of the forward primer 82F (5'-GAAACTGCGAATGGCTC-3'; Elwood et al., 1985) and the reverse primer EkyB (5'-TGATCCT TCTGCAGGTTCACCTAC-3'; Medlin et al., 1988) according to the protocol described by Ito et al., (2014). The PCR products were purified using Cleanup S-Cap kit (Evrogen). Sanger sequencing was performed using three primers: an amplification primer 82F, Jap2F (5'-TTTGCCA
AGGATGTTTTC-3'; Ito et al. 2014) and JaplR (5'-CTTGGCAAATGCTTTCGC-3'; Giribet et al., 1996). The sequences obtained in this way were used for the phylogenetic analysis. GenBank accession numbers for the SSU rRNA gene sequences newly obtained in this work are PP719287-PP719292. For the phylogenetic analysis, we combined the sequences obtained in our study with those available in GenBank (N=36, Supplement 1). The SSU rRNA gene alignment of the 42 sequences with a length of 1567 nucleotide sites was performed in AliView using MUSCLE under the default parameters (Larsson, 2014) (Supplement 2). The maximum likelihood tree reconstruction was performed in IQ-TREE v.1.6 (Nguyen et al., 2015), with the best evolutionary model (TIM2+F+I+G4) selected using Bayesian information criterion by the built-in ModelFinder (Kalyaanamoorthy et al., 2017). Branch support was estimated using the ultrafast bootstrap method (1000 replicates) (Hoang et al., 2018). Bayesian analyses were conducted under the GTR + I + G evolutionary model on the CIPRES portal ver. 3.1 (https://www.phylo.org/) with MrBayes software (Ronquist, Huelsenbeck, 2003) on XSEDE ver. 3.2.6 (Miller et al., 2010). Markov Chain Monte Carlo parameters were as follows: the two independent runs were done with four independent chains during 2,000,000 generations. Each 100th tree was sampled and 25% of the first trees were discarded as a burn-in.
Results and discussion
We found 19, 17, and 14 species and forms ofcili-ates in the faecal samples of the zebras from, respectively, Vladivostok Zoo, Stary Oskol Zoo, and Vienna Zoo (Table 1, Figs 1—3). All these ciliates have been previously reported from Equus spp. (Cedrola et al., 2019). However, we revealed certain differences between the species composition (lists of species and forms) of ciliate communities in wild and captive zebras. In the faunistic study of endo-biotic ciliates of the zebra E. quagga from Africa, Strelkow (1931, 1939) noted that five ciliate species (Trifascicularia cycloposthium, Triadinium elon-gatum, Ditoxum hamulus, Spirodinium nanum, and Spirodinium ferrumequinum) were found only in zebras and were apparently host-specific. However, the ciliates T. cycloposthium, T. elongatum, and S. ferrumequinum have never been detected in captive E. quagga. Another host-specific endobiotic ciliate, D. hamulus, has been recorded in E. quagga
Table 1. Occurrence of species of intestinal ciliates of zebras Equus quagga at various locations
in nature and in captivity.
№ Family/genus/species/morphotype A B C D E F G H I J K L
Buetschliidae Poche, 1913
Alloiozona Hsiung, 1930
1 trizona Hsiung, 1930 +
Hemiprorodon Strelkow, 1939
2 gymnoposthium Strelkow, 1939 +
Blepharosphaera Bundle, 1895
3 intestinalis Bundle, 1895 +
4 ceratotherii Van Hoven et al., 1998 +
Holophryoides Gassovsky, 1919
5 macrotricha Strelkow, 1939 +
Blepharoprosthium Bundle, 1895
6 pireum Bundle, 1895 + +
7 polytrichum Strelkow, 1939 +
Blepharoconus Gassovsky, 1919
8 sp. +
Polymorphellla Corliss, 1960
9 ampulla (Dogiel, 1929) + + +
Paraisotrichopsis Gassovsky, 1919
10 composita Gassovsky, 1919 + +
Bundleia Cunha & Muniz, 1928
subgen. Bundleia Strelkow, 1939
11 postciliata (Bundle, 1895) + + + + + +
12 piriformis Strelkow, 1939 +
13 nana Strelkow, 1939 + + +
subgen. Chlamydobundleia Strelkow, 1939
14 elongata Strelkow, 1939 + + + +
15 triangularis Strelkow, 1939 + +
subgen. Fibrillobundleia Strelkow, 1939
16 benbrooki Hsiung, 1930 + + +
17 inflata Strelkow, 1939 + + + + +
18 dolichosoma Strelkow, 1939 +
Paraisotrichidae da Cunha, 1917
Paraisotricha Fiorentini, 1890
19 minuta Hsiung, 1930 + +
20 colpoidea Fiorentini, 1890 +
Blepharocorythidae Hsiung, 1929
Blepharocorys Bundle, 1895
21 jubata Bundle, 1895 + + +
22 curvigula Gassovsky, 1919 + + + + + + + + + + +
Table 1. Continuation.
23 cardionucleata Hsiung, 1930 + + + +
24 angusta Gassovsky, 1919 + + + + + + + + + +
25 microcorys Gassovsky, 1919 + + + + + + +
26 valvata (Fiorentini, 1890) + +
Ochoterenaia Chavarria, 1933
27 appendiculata Chavarria, 1933 +
Circodinium Wolska, 1971
28 minimum (Gassovsky, 1919) + + +
Charonnautes Strelkow, 1939
29 equi (Hsiung, 1930) +
Cycloposthiidae Poche, 1913
Cycloposthium Bundle, 1895
30 bipalmatum (Fiorentini, 1890) + + + + +
31 edentatum Strelkow, 1928 + + + + + + +
32 affine Strelkow, 1929 +
33 piscicauda Strelkow, 1928 +
34 dentiferum Gassovsky, 1919 +
35 sp. +
Tripalmaria Gassovsky, 1919
36 dogieli Gassovsky, 1919 + +
37 dogieli zebrae Strelkow, 1931* + +
Trifascicularia Strelkow, 1931
38 cycloposthium Strelkow, 1931* + +
Spirodiniidae Strelkow, 1939
Ditoxum Gassovsky, 1919
39 hamulus Strelkow, 1931* + + +
40 brevinucleatum Strelkow, 1931 + + + +
Triadinium Fiorentini, 1890
41 caudatum Fiorentini, 1890 + + + + + + +
42 elongatum Strelkow, 1931* + +
Gassovskiella Grain, 1994
43 galea (Gassovsky, 1919) + + + + + + +
Cochliatoxum Gassovsky, 1919
44 periachtum Gassovsky, 1919 + + + + + +
Tetratoxum Gassovsky, 1919
45 excavatum Hsiung, 1930 +
46 parvum Hsiung, 1930 + + + + +
47 unifasciculatum Fiorentini, 1890 + + +
Spirodinium Fiorentini, 1890
48 nanum Strelkow, 1931 + + + + + + + +
49 ferrumequinum Strelkow, 1931* +
Notes:
A - Equus quagga burchellii, Naval Hill Franklin Nature Reserve, South Africa (Kornilova et al., 2021); B - E. q. burchellii, Limpopo, South Africa (Booyse and Dehority, 2017); C - E. q. burchellii, Vienna Zoo; D - E. q. burchellii, Vladivostok Zoo; E - E. q. burchellii, Gaziantep Zoo (Gurelli, 2023); F - E. q. burchellii, Sasali National Park, Izmir (Gurelli, 2023); G - Equus q. boehmi, Kenya (Strelkow, 1931); H - E. q. boehmi, Darica Faruk Yalçin Zoo (Gurelli, 2023); I - E. q. chapmani, Almaty Zoo, Kazakhstan (Kornilova, 2003); J - E. q. chapmani, Leningrad Zoo, Russia (Kornilova, 2003); K - E. q. chapmani, Askania-Nova biosphere reserve, Ukraine (Kornilova, 2003); L - E. q. chapmani, Stary Oskol Zoo. * - Morphotype found in zebras only.
+ Africa, + zoos, + natural parks and biosphere reserves.
Table 1. Continuation.
50 equi Fiorentini, 1890 + + +
51 confusum Hsiung, 1935 + + + +
Allantosomatidae Jankowski, 1967
Allantosoma Gassovsky, 1919
52 intestinalis Gassovsky, 1919 + + + +
53 cucumis Strelkow, 1939 +
Allantoxena Jankowski, 1978
54 biseriale (Strelkow, 1939) +
Arcosoma Jankowski, 1967
55 lineare (Strelkow, 1939) +
56 brevicorniger (Hsiung, 1928) +
57 dicorniger (Hsiung, 1928) + +
Total 19 7 14 19 10 18 16 20 4 3 23 17
Number of common species with wild zebras 5 13 4 6 9 2 2 8 7
burchellii only once, in Darica Faruk Yal?in Zoo (Gurelli, 2023). Interestingly, none of these four species of ciliates has ever been found in zebras from biosphere reserves (Askania Nova) and natural parks (Kornilova, 2003; Gurelli, 2023).
In our opinion, the most obvious explanation of these observations is that the ciliates T. cycloposthi-um, T. elongatum, D. hamulus, and S. ferrumequinum are highly sensitive to changes in the host diet. Spi-rodinium nanum, which is also presumably specific to E. quagga (Strelkow, 1939), was found in most of the captive zebras studied, as well as in the domestic horse and in the mountain zebra Equus zebra (Ike et al., 1985; Kornilova et al., 2020). Apparently, this species is less sensitive to changes in environmental factors, including host diet.
It should be noted that representatives of the family Buetschliidae that are usually numerous in ciliate communities of the horse intestine, are often almost absent in the ciliate communities of zebras kept in zoos. These ciliates, which feed on bacteria, are probably affected by the antibiotic treatment of the captive animals, which inhibits the development of bacteria in the intestine. A decrease in the total number of endobiotic ciliate species of various animals in captivity has often been noted and
primarily explained by antimicrobial treatments and changes in the diet (Modry et al., 2009).
At the same time, the faecal samples of zebras from zoos examined in our study contained some ciliate species that have not been recorded in zebras from natural habitats (Table 1). Ciliate communities in the equine gut are formed by coprophagy, mainly during the first weeks of life, with the vast majority of species passed from the mother to the foal (Ike et al., 1985). However, it has been suggested that horses may acquire certain endobionts throughout their lifetime by eating fresh faeces of other horses inhabiting the same area. For instance, the greater diversity of ciliates in the faecal samples of the zebra from Vladivostok Zoo may be due to the fact that before being placed in the zoo this animal spent a long time in a travelling circus, where the chances of different animals exchanging endobionts were fairly high. The zebra in question could have acquired some endobionts from, e.g., a domestic horse, enriching its endobiotic fauna. Unfortunately, the life history of the animals used in faunistic studies of endobiotic ciliates is usually unknown. However, it can be assumed that the chances of ciliate transmission between different equine species inhabiting the same territory are higher in natural
Fig. 1. Ciliates from the faeces ofzebras Equus quagga kept in captivity. Representatives ofthe family Buetschliidae and Paraisotrichidae; DIC. A - Bundleia inflata; B - B. nana; C - B. benbrooki; D - B. postciliata;E - B. elongata; F - Polymorphella ampulla; G - Hemiprorodongymnoposthium; H - Paraisotrichaminuta. A, B, C - staining by methyl green. Abbreviations: Ma - macronucleus, cv - concrement vacuole. Scale bars 10 ^m.
parks and biosphere reserves. Thus, although the species diversity of endobiotic ciliates in zebras from zoos, natural parks, and biosphere reserves may be quite high, the number of endobiotic species shared by the same host species from the natural and the artificial habitats is always small (Table 1).
In order to assess the differences between the isolates of the ciliate species, SSU sequences of Cochliatoxum periachtum (from the zebra in Vladivostok Zoo) and Tripalmaria dogieli (from the zebra in Stary Oskol Zoo) were obtained. For the sake of comparison, we also obtained SSU sequen-
ces of different geographical isolates of C. periachtum, T. dogieli, and Cycloposthium edentatum from the domestic horse (Table 2). We failed to obtain DNA suitable for analysis from the samples collected in Vienna Zoo, presumably due to the poor quality of the fixed material as the samples were collected several hours after defecation.
It should be noted that the SSU sequences of these species deposited in GenBank were isolated from endobiotic ciliates from the intestine of the Yakutian horse (Table S1), an endemic horse breed from Yakutia (Struder-Kypke et al., 2007). Com-
Fig. 2. Ciliates from the faeces ofzebras Equus quagga kept in captivity. Representatives ofthe family Spirodiniidae; DIC. A — Tetratoxumparvum; B — T. unifasciculatum; C — Ditoxum brevinucleatum; D — Triadinium caudatum; E — Gassovskiellagalea; F — Spirodinium equi; G — S. confusum; H — S. nanum. B—D, F, H — staining by methyl green. Abbreviations: Ma - macronucleus. Scale bars 20 ^m.
munities of endobiotic ciliates from Yakutian horses are known to harbour species absent in horses from other regions. Therefore, we had reasons to expect differences in the SSU sequences of isolates of C. periachtum, T. dogieli, and C. edentatum from the Yakut horse and other horses.
Different isolates of C. periachtum from the same host (domestic horse) were identical except for one unresolved site (File S2). The SSU sequences of C. periachtum from the zebra had three nucleotide substitutions compared with those of C. periachtum from the domestic horse, with the total fragment length being~1360 bp (File S2). The differences between T. dogieli from the zebra and the Yakut
horse made up seven substitutions per a fragment 1395 bp long (File S2). We failed to obtain a full-size SSU fragment of T. dogieli from the domestic horse, but two substitutions were detected in a fragment ~700 bp long compared to the other two SSU sequences included in the analysis (File S2). Among other members of the family Spirodiniidae, SSU sequences of different geographical isolates of the same species are available only for Spirodinium equi. The maximum number of substitutions as compared with other isolates (5) was detected in the ciliates from domestic horses in Japan (GenBank accession number AB 564642) (Ito et al., 2014) (File S2). On the phylogenetic trees, C. periachtum and
Fig. 3. Ciliates ffrom the faeces of zebras Equus quagga kept in captivity. Representatives of the families Blepharocorythidae, Cycloposthiidae, and Allantosomatidae; DIC. A - Blepharocorys cardionucleata; B - B. curvigula; C — B. angusta; D — Circodinium minimum; E — B. microcorys; F — Allantosoma intestinale; G -Cycloposthium edentatum; H — C. bipalmatum; I — Tripalmaria dogieli. A, C, F—I — staining by methyl green. Abbreviations: Ma — macronucleus. Scale bars 20 ^m.
T. dogieli from the zebras formed sister branches to the clades containing C. periachtum and T. dogieli from horses, respectively (Figs 4, 5).
Isolates of C. edentatum from horses in Yakutia and the Leningrad Region differed by one nucleotide substitution (File S2). Cycloposthium is the only group of endobiotic ciliates from odd-toed ungulates for which sufficient data are available for an analysis of the level of interspecific variation in the SSU sequence (Fregulia et al., 2021). It is noteworthy that the differences between C. minutum and C. lenticularis, which are quite distinct morphologically, also make up only one substitution per fragment with a length of ~1420 bp (Fregulia et al., 2020, 2021).
It is clear that a full analysis of the level of differences between the geographical isolates of spe-
cies of endobiotic ciliates is currently impossible. Nevertheless, we believe that the addition of new data to the molecular database is important both for verifying the topology of the phylogenetic tree of trichostomatid ciliates and, in the long term, for suggesting a meaningful hypothesis about the nature of intraspecific differences of endobiotic ciliates from different hosts and habitats.
Acknowledgments
The work was supported by the Russian Science Foundation (grant No 23-24-00240, https://rscf.ru/ project/23-24-00240/). The research was carried out using the equipment of the Core Facilities Centre "Taxon" at the Zoological Institute, Russian Academy of Sciences (St. Petersburg, Russia).
Table 2. List of the samples for DNA isolating and PCR
Host Collection site Year Genomic DNA isolation
Cochliatoxum periachtum Tripalmaria dogieli Cycloposthium edentatum
E. quagga burchellii Vladivostok Zoo, Russia 2021 + + +
E. quagga burchellii Vienna Zoo, Austria 2020 + +
E. quagga chapmani Stary Oskol Zoo, Russia 2022 + +
E. caballus South Africa 2019 + +
E. caballus Leningrad Region, Russia 2022-2023 + + + + + +
Notes: + DNA was isolated; ++ PCR fragment suitable for analysis was obtained.
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Supplementary materials
Table S1. List of SSU sequences from GenBank used for phylogenetic analysis.
File S2. Nucleotide alignment of18S rRNA gene sequences made in AliView using the MUSCLE algorithm (zip archive). Sequences taken from Gen Bank are designated by corresponding numbers.
