Molecular Characterization of Ctenotaenia marmotae (Frölich, 1802) Railliet, 1893 (Cyclophyllidea: Anoplocephalidae) Parasitizing Rodents of the Genus Marmota and Spermophilus from Eurasia

Abstract

Cestodes Ctenotaenia marmotae are a widespread parasite of Eurasian ground squirrels. Being the only valid species of its genus, it has been recorded in ten host species from the genera Marmota and Spermophilus. Its definitive hosts live in similar ecological conditions and their area covers a wide geographical territory. Therefore, it remained unclear whether C. marmotae is a single species and how high the level of its genetic variability is. The present study analyzed the variability of two mitochondrial cestode genes from four host species from geographically distant localities. Phylogenetic analysis revealed that the newly obtained sequences form a species-level clade with already known sequences from the GenBank. Small genetic distances between the studied sequences indicate low intraspecific genetic variability within the C. marmotae, which may be a consequence of rapid expansion of the cestodes in ground squirrel species inhabiting the flat and mountain steppe landscapes in the past.

1. Introduction

Cestodes of the genus Ctenotaenia Railliet, 1893 (Anoplocephalidae family) are dominant and widespread endoparasites of the small intestine of ground squirrels of Eurasia (Marmotini tribe) [1]. They have been previously shown to parasitize 5 of 8 species of marmots Marmota (Blumenbach, 1779) and 5 of 16 species of susliks Spermophilus (F. Cuvier, 1825) [2] inhabiting Eurasia [3,4]. A study of preparations from the collection of the University of Neuchátel, Switzerland showed that there is one valid species in the genus Ctenotaenia, C. marmotae (Frölich, 1802) Railliet, 1893 [5]. Anoplocephalid cestodes that parasitize rodents are known to exhibit latent molecular diversity [6], geographic isolation, and/or host specificity [7,8]. Thus, P. omphalodes and P. jarrelli were previously considered as one species because of their high morphological similarity [8]. However, P. omphalodes parasitize five species of rodents of different genera [9], in contrast to P. jarrelli, which are only found in voles of the Alexandromys subgenus.

The aim of this study is to describe the variability of two mitochondrial genes of Ctenotaenia marmotae parasitizing marmots and susliks of geographically remote regions of Eurasia.

2. Materials and Methods

Data collection. The small number of investigated specimens of definitive hosts is due to the fact that some species of susliks and marmots currently have low numbers, and some are protected. For example, the mottled suslik is included in the Red Data Book of the Republic of Belarus [10], and the number of the red-cheeked suslik in the south of Western Siberia, according to our observations, has decreased significantly over the past three decades. The carcasses of the two suslik species from natural populations were examined for the presence of endoparasites. The carcass of the red-cheeked suslik Spermophilus erythrogenys Brandt, 1841 was taken from a dog that captured it near a burrow on 13 May 2020, near Doronino village (Toguchinsky District, Novosibirsk Oblast, Russia) (Figure 1). Twelve adult cestodes were found in the intestines of this host. The carcass of a mottled suslik S. suslicus (Guldenstaedt, 1770) was taken from a gray crow on 20 May 2020 near Nesvizh town (Minsk Region, Republic of Belarus) (Figure 1). The gastrointestinal tract of the suslik was extracted and frozen. During its autopsy, seven specimens of cestodes were found.

In August 2013, cestode proglottids were found in feces of grey marmot Marmota baibacina near its burrow in Ulandryk (Kosh-Agachsky district, Altai Republic) (Figure 1). The systematic position and Latin names of the definitive hosts are given according to [2]. The discovered specimens of cestodes were relaxed in the water and fixed in 70% ethanol. The tapeworms were stained in Ehrlich’s acid haematoxylin, differentiated in a 3% aqueous solution of ferric ammonium sulphate, dehydrated in a graded ethanol series, cleared in eugenol, and mounted in Damar gum. All found specimens were morphologically identified as Ctenotaenia marmotae and vouchers were deposited in the parasitological collection of the Zoological Museum of the Institute of Systematics and Ecology of Animals SB RAS (ISEA AK Ctm01, AK Ctm02).

Molecular identification and phylogeny analysis. Total DNA was isolated using a 5% Chelex-100 solution (Bio-Rad Laboratories, Hercules, CA, USA). The isolation protocol followed the manufacturer’s recommendations for InstaGene™ Matrix (Bio-Rad Laboratories, Hercules, CA, USA). We used 2–5 μL of the obtained supernatant to perform PCR. Fragments of two mitochondrial genes were amplified using primers COX-F, COX-R (cox1), and Cyclo_Nad1F, Cyclo_trnNR (nad1) [6]. The amplicons were purified and sequenced directly on an ABI 3130XL Genetic Analyser at the Genomics Core Facility ICBFM SB RAS (Novosibirsk, Russia). Original sequences were submitted to the GenBank (

Table 1. List of examined specimens of Ctenotaenia marmotae and GenBank accession numbers according to geographical origin. Specimens, except for AY568187 and HM134271, were collected and sequenced by the authors.

Locality (Number)	Coordinates	GenBank Acc. No. (Cox1/Nad1)	Final Host
France (1)	--	AY568187/HM134271	Marmota marmota (Linnaeus, 1758)
Nesvizh, Minsk Oblast, Belarus (2)	53.24530438 N 26.76108783 E	ON459626/ON505824	Spermophilus suslicus (Guldenstaedt, 1770)
Doronino, Novosibirsk Oblast, Russia (4)	54.98292101 N 84.58460474 E	ON459627/ON505825	Spermophilus erythrogenys Brandt, 1841
Ulandryk, Altai Republic, Russia (3)	49.60679776 N 89.07580720 E	ON459628/ON505826	Marmota baibacina Kastschenko, 1899

). In addition, sequences of anoplocephalid cestodes from the GenBank database were used (

Table 2. The sequences list the anoplocephalid cestodes involved in the phylogenetic analysis. The list is given according to [6,11,14].

Cestode Species	Cox1	Nad1	Final Host
Paranoplocephala jarrelli	AY181523, AY181540, AY181533, EF688302, EF688303, EF583964	KJ778952	Microtus oeconomus
Paranoplocephala kalelai	EF583961, ON533421, ON533414, ON533416, LC535248	ON548168, OM548169, ON548176, ON548178	Myodes rufocanus
Paranoplocephala macrocephala	KJ778922, AY1811515,	KJ778954	Microtus longicaudus
	AY181517		Microtus pennsylvanicus
Paranoplocephala omphalodes	AY18122, AY18124,		Microtus agrestis
	AY18139		Myodes glareolus
Rauschoides alternata	AY181502	KJ778959	Dicrostonyx groenlandicus
Rauschoides arctica	AY181505	KJ778958	Dicrostonyx groenlandicus
Gulyaevia buryatiensis	DQ445259	KJ778940	Myodes rufocanus
Gulyaevia longivaginata	DQ445261	KJ778941	Myodes rutilus
Douthittia nordenskioeldi	AY568204	KJ778933-KJ778937	Microtus miurus
Douthittia nearctica	KJ778910	KJ778931, KJ778932	Myodes rutilus
Douthitta sp.		KJ778938	Myodes rutilus
Anoplocephaloides lemmi	EU744307	KJ778962	Lemmus sibiricus
Anoplocephaloides kontrimavichusi	AY568195	HM134274	Synaptomys borealis
Parandrya oeconomi	AY568205	KJ778949	Microtus oeconomus
Parandrya feodorovi	EF688300	KJ778948	Microtus sp.
Andrya rhopalocephala	AY189958	HM134265	Lepus europaeus
Equinia mamilliana	---	HM134268	Equus caballus
Anoplocephala perfoliata	AY568189	---	Equus caballus

Cestode species from the sister group of Andrya rhopalocephala & Ctenotaenia marmotae were included in the phylogenetic analysis [11]. Sequences AY568189 Anoplocephala perfoliata and HM134268 Equinia mamillana (for cox1 and nad1, respectively) were used as an outgroup.

). Sequence alignment by Clustal Omega [12], phylogenetic analysis by Maximum Likelihood method was conducted in MEGA 11 [13]. The best substitution model for unpartitioned loci (cox1 and nad1: GTR + G + I) were identified in MEGA 11 according to the Akaike information criterion. Branch support was estimated with 1000 bootstrap replicates. All positions containing missing data were eliminated (complete deletion option).

3. Results and Discussion

We have obtained the sequences of two mitochondrial genes of the cestode Ctenotaenia marmotae (cox1 and nad1) from three rodent species from Russia and Belarus (Table 1). When comparing these sequences with those of mitochondrial genes of C. marmotae from the alpine marmot Marmota marmota (AY568187/HM134271), we have revealed a high percentage of identity for both markers (

Table 3. Sequence identity rates (%) of mitochondrial genes cox1 (above the diagonal) and nad1 (below the diagonal) of Ctenotaenia marmotae from different host species.

	Host M. baibacina (Altai, Russia)	Host M. marmota (Alps, France)	Host S. erythrogenus (West Siberia, Russia)	Host S. suslicus (Belarus)
Host M. baibacina (Altai, Russia)		96.6	97.2	96.6
Host M. marmota (Alps, France)	97.7		95.2	94.8
Host S. erythrogenus (West Siberia, Russia)	98.0	97.2		99.5
Host S. suslicus (Belarus)	97.7	96.9	99

). Thus, the percentage of identity was 94.8–99.5% for the cox1 gene, and 96.9 to 99% for the nad1 gene, with reading lengths of 591– 629 and 784–795 nucleotides, respectively.

Analyzing the sequences of the cox1 gene of C. marmotae, we found that the closest to each other are the sequences of cestodes from both suslik species with 99% identity. The sequence from C. marmotae from the Alpine marmot was the most distant sequence from susliks cestodes. However, it is close to the sequence of C. marmotae from M. baibacina inhabiting the Altai Mountains (Ulandryk). The level of divergence of cestode from Alpine marmota from other cestode specimens suggests that isolation of M. marmota in European mountain ranges affected cestodes but did not lead to divergence within C. marmotae species. We observed the same trend by comparing the sequences of the nad1 gene.

All eight sequences of the two C. marmotae mitochondrial genes are unique, separate haplotypes. For the cox1 gene, 35 polymorphic sites were identified, with an aligned sequence length of 591 nucleotide pairs. For the nad1 gene, 33 polymorphic sites were identified (sequence length of 776 nucleotide pairs).

On a phylogenetic tree (Figure 2A,B) constructed for each of the molecular markers used, all C. marmotae specimens from the four definitive host species form a single clade. Given the scale of the trees, the differences between the nad1 gene sequences in the C. marmotae clade are comparable to those of the cox1 gene. However, in both cases, the level of divergence within the C. marmotae clade, visually assessed by branch length, does not exceed the level of divergence within clades representing species of the same genus (e.g., in the genus Paranoplocephala). The sequences of C. marmotae from S. suslicus from Belarus and S. erythrogenys from Western Siberia turned out to be as close as possible to each other, while the distance between the localities is approximately 3680 km. We note that there is a weak tendency to form two clusters corresponding to different taxonomic groups of hosts—susliks and marmots. This can be traced for both genes. This is especially true for the C. marmotae subcluster from susliks. However, the revealed differences between different host groups do not allow a definitive conclusion. Additional samples of C. marmotae should be investigated to verify this assumption, preferably from the final host species not involved in this study.

Widespread distribution of close haplotypes over a large territory taking into account parasitism in four species of the final hosts may indicate rapid geographical expansion in the host areas. For example, it was shown that the cestode Paranoplocephala omphalodes, widely represented in Eurasia and parasitizing in 5 rodent species, has a low haplotype divergence of the mitochondrial gene cox1 [9].

According to available data, C. marmotae has been found in various species of ground squirrels: Marmota baibacina, M. bobac, M. marmota, M. sibirica, M. himalayana, Spermophilus pigmaeus, S. erythrogenys, S. suslicus, S. citellus, S. undulatus, S. xanthoprymnus [2]. Marmots and susliks are known to be widely distributed in Eurasia, predominantly inhabiting mountains and plain steppe habitats [15]. In Eurasia, an extensive steppe belt, formed after the last glaciation, extends from Eastern Europe almost to the Far East. Social way of life and the use of burrows promotes the spread of cestodes between animals (including different species) in host populations [16].

The analysis of the current distribution of marmots and ground squirrels [2], in which Ctenotaenia marmotae has been found, concludes that their ranges, located in plain or mountain-steppe landscapes of Eurasia, either overlap or are close to each other. This may have contributed to the spread of C. marmotae among various marmot and suslik species.

Thus, the study of the cestode C. marmotae, which parasitizes two species of marmots and two species of susliks in different parts of Eurasia, revealed a low divergence of the mitochondrial genes cox1 and nad1. The closer distances between the gene sequences of cestodes of suslik species compared to those of marmot species may reflect a greater level of divergence between cestodes parasitizing host animals belonging to different genera. However, the limited sampling of specimens and localities does not allow further phylogeographical inferences. Future research should prioritize expanded sampling to clarify the evolutionary, host, and biogeographical dynamics within C. marmotae.