Филогенетические отношения тетратиридиев Mesocestoides vaillant, 1863 от микромаммалий востока России и Аляски на основе 18S рРНК гена

Полный текст

The metacestode stage (tetrathyridium) of cestodes of the genus Mesocestoides, widespread parasites of predatory mammals, has no clear morphological characteristics that diagnostics usually relies on (in particular, there is no proboscis) (Kozlov, 1978; Dokuchaev, Gulyaev, 2004; Konyaev et al., 2011; Zaleśny, Hildebrand, 2012; Tokiwa et al., 2014).

It is not uncommon to find larval stages of Mesocestoides sp. in rodents, or M. lineatus in carnivorous mammals in the East of Russia (Gubanov, 1964; Gubanov, Fedorov, 1970; Domnich, 1985; Odnokurtsev, 2015). In addition, M. kirbyi Chandler, 1944 (Domnich, Obushenkov, 1983) and M. paucitesticulus (Konyaev et al., 2011) were recorded in this territory, and tetrathyridia that may belong to M. kirbyi, or M. perlatus (Goeze, 1782) were found in shrews from the Northern Okhotsk region and Chukotka (Dokuchaev, Gulyaev, 2004).

The first attempt to study the Mesocestoides tetrathyridia from Magadan region performed by molecular genetic methods (Pospekhova et al., 2018) had made it possible to outline some phylogenetic relationships within the genus. The subsequent work, based on the variability of the 12S rRNA gene fragment in Mesocestoides sp. (Pospekhova et al., 2023) suggested that none of the studied samples of tetrathyridia from insectivores and rodents belong to any of the confirmed species of the genus, namely M. lineatus (Goeze, 1782), M. litteratus (Batsch, 1786), M. canislagopodis (Rudolphi, 1810) (Krabbe 1865), M. corti (Hoeppli 1925) (= M. vogae) (Etges 1991)) и M. melesi Yanchev and Petrov 1985 (Yanchev, 1986; Gubány, Eszterbauer, 1998; Nickisch-Rosenegk et al., 1999; Padgett, Boyce, 2005; Literák et al., 2006; Hrčkova et al., 2011; Skírnisson et al., 2016; Bajer et al., 2020).

The purpose of this work was to determine the nucleotide sequences of the 18S rRNA nuclear gene fragment in tetrathyridia from 7 species of small mammals in the East of Russia and Alaska (USA) and to perform a preliminary analysis of the relationships between the haplotypes of the studied Mesocestoides with each other, and also with the data available in GenBank.

MATERIALS AND METHODS

Tetrathyridia samples were obtained as a result of theriological studies of rodents and insectivores conducted by N.E. Dokuchaev in 2002–2019; small mammal collection sites are shown in Fig. 1 and Table 1.

 

Figure 1. Collection sites for intermediate hosts of Mesocestoides. The numbers correspond to the sample numbers from Table 1.

 

Table 1. Mesocestoides tetrathyridia specimens used in this work

Sample number	Haplotypes	Host	Location	GenBank number
11	Mes18S_1	Clethrionomys rutilus	Kulu settlement, Magadan region	MK634547
12	Mes18S_1	Sorex isodon	Bolshoy Shantar Island, Khabarovsk Territory	MK634546
13	Mes18S_1	Craseomys rufocanus	Bolshoy Shantar Island, Khabarovsk Territory	MK634544
28	Mes18S_4	Craseomys rufocanus	Umara riwer floodplain, Magadan region	MN031874
47	Mes18S_3	Micromys minutus	Georgievka village Khabarovsk Territory	OP161928
48	Mes18S_1	Craseomys rufocanus	Umara riwer floodplain, Magadan region	OP161977
49	Mes18S_5	Sorex cinereus	Fairbanks, Alaska, USA	OP161923
50	Mes18S_1	Sorex caecutiens	«Contact» station, Magadan region	OP161924
51	Mes18S_2	Craseomys rufocanus	Nedorazumenia island, Magadan region	OP161926
52	Mes18S_1	Sorex caecutiens	Yakutsk, Republic of Sakha (Yakutia)	OP161922
53	Mes18S_1	Sorex tundrensis	Yakutsk, Republic of Sakha (Yakutia)	OP161927
54	Mes18S_2	Sorex caecutiens	Duckcha river floodplane, Magadan region	OP161921

Notes. Gathering locations, sample numbers and GenBank numbers are shown.

 

The work used samples of tetrathyridia from 4 species of shrews of the order Eulipotyphla (Insectivores), and three species of rodents (Rodentia). The localization of tetrathyridia is somewhat different in different hosts. According to our observations, in shrews they are more often located in the liver and the large lymphoid organ; in rodents, in the body cavity.

Some Mesocestoides host names that we previously contributed to GenBank (Myodes rutilus and M. rufocanus) do not correspond to the current classification system, thus, in the text we use the names Clethrionomys rutilus and Craseomys rufocanus, respectively (Kryštufek, Shenbrot, 2022).

Before DNA extraction from alcohol-fixed material, tetrathyridia with tissue localization were freed from cysts and washed in alcohol of the same concentration.

Isolation and purification of total DNA was carried out using the phenol-chloroform method (Sambrook et al., 1989). Amplification of 875 base pairs (bp) (131–1005 bp from the beginning of the gene) of the 18S rRNA gene fragment was carried out using newly designed primers Micr18SL61 gcc ttt ata cgg tga aac cgc gaa tgg (61–89 bp from the start of the gene) and Micr18SR14028 caa tct gtc aat cct cat agt gtc cgg cc (1428–1456 bp from the start of the gene). Polymerase chain reaction conditions followed the protocol of Literak et al., 2004: denaturing step 94^оC – 5 min; then 40 cycles: 94^оС – 1 min, 52^оС – 1 min, 72^оС – 2 min; final stage 72°C – 7 min. The amplified sections of nuclear DNA were purified and prepared for sequencing according to standard methods using the DiatomTM DNA Clean-Up reagent kit from Isogen Laboratory. The structure of the nucleotide sequence of the 18S rRNA gene was determined from 131 bp from the beginning of the gene using the Micr18SL61 primer according to the standard method using Big Dye Terminator DNA cyclic sequencing kits (Applied Biosystems, v. 3.1) and an ABI Prism 3500xL genetic analyzer (Applied Biosystems, USA). The 18S rRNA gene fragment was mapped relative to the complete nucleotide sequence of this gene in Mesocestoides corti GenBank No. AF286984 (Olson et al., 2001). Haplotypes of the 18S rRNA gene fragment of the studied samples of Mesocestoides sp. were assigned the abbreviation Mes18S.

For phylogenetic analysis of the obtained nucleotide sequences of the 18S rRNA gene, information about the corresponding fragment of this gene from samples belonging to the genus Mesocestoides was taken from GenBank (Table 2).

 

Table 2. Nucleotide sequences of 18S rRNA taken for comparison from GenBank

Cestode species	GenBank number, author	Country	Host
M. corti	GU442130 (Piseddu et al., unpublished)	Italy	Canis familiaris
M. corti	AF286984 (Olson et al., 2001)	Switzerland	laboratory mouse
M. litteratus	JN088190 (Zalesny, Hildebrand, 2012)	Poland	Myodes glareolus
M. litteratus	MN512711 (Bayer et al., 2020)	Poland	Vulpes vulpes
M. litteratus	MN512709 (Bayer et al., 2020)	Poland	Vulpes vulpes
M. melesi	MN401346 (Bayer et al., 2020)	Poland	Meles meles
M. melesi	MN401347 (Bayer et al., 2020)	Poland	Myodes glareolus
Mesocestoides sp.	EF095248 (Waeschenbach et al., 2007)	Bulgaria	Apodemus agrarius
Mesocestoides sp.	AF119678 (Crosbie et al., 2000)	USA	Canis latrans

 

The dendrogram of 18S rRNA gene haplotypes was constructed using the maximum likelihood (ML) method based on the Kimura biparametric distance model selected using the Bayesian information criterion. The stability of branch nodes was assessed using the bootstrap method (500 iterations).

Some of the data taken for comparison in GenBank had identical sequences; to simplify the structure of the ML–phylogenetic tree, we included in the analysis only one sequence from a number of identical ones.

The nucleotide sequence of the corresponding fragment of the 18S rRNA gene GQ260092 of Echinococcus granulosus (Jia, Yan, 2009, unpublished) was used as an outgroup.

Genetic data analysis was carried out using MEGA software packages 10.0.2.74 (Tamura et al., 2013) and ARLEQUIN ver. 3.5 (Excoffier et al., 2005).

RESULTS AND DISCUSSION

Characteristics of nucleotide sequences of Mes18S_1-Mes18S_5 haplotypes of the 18S rRNA gene fragment of Mesocestoides sp.

The 875 bp fragment of the 18S rRNA gene was sequenced for twelve samples of Mesocestoides sp. (Table 1). Five Mes18S haplotypes were identified. Among them, we deteced 27 nucleotide substitutions (ns) at 24 sites (Fig. 2).

 

Figure 2. Haplotypes Mes18S_1– Mes18S_5 of the 18S rRNA gene fragment of Mesocestoides sp. Substitution sites are shown relative to the Mes18S_1 nucleotide sequence from the beginning of the 18S rRNA gene.

 

The Mes18s_1 haplotype included 7 nucleotide sequences (MK634544, MK634546, MK634547, OP161922, OP161924, OP161927, OP161977) and Mes18s_2 included two sequences (OP161921 and OP161926), however, to simplify the analysis we used only one of identical sequences (for Mes18s_1 – MK634544, for Mes18s_2 – OP161926).

The number and type of ns indicate the presence of two haplogroups within the studied nucleotide sequences groups A (haplotypes Mes18S_1, Mes18S_3, Mes18S_4) and B (Mes18S_2, Mes18S_5). The presence of two haplogroups is a consequence of the high level of polymorphism in the nucleotide sequence of the 18S rRNA gene fragment in the general sample of Mesocestoides spp. In the haplogroup A, 8 polymorphic sites were identified (Fig. 2, Table 3). Mes18S_1 variant was predominant among the identified haplotypes, both within haplogroup A and in the general sample. It was found in samples from hosts belonging to different systematic groups (Clethrionomys rutilus, Craseomys rufocanus, Sorex caecutiens, S.isodon, S.tundrensis) with different levels of metabolism, and collected in geographically distant localities (Magadan region, Khabarovsk region and Yakutia) (Fig. 1, Table 1).

Haplogroup B is represented by two haplotypes Mes18S_2 and Mes18S_5, differing from each other by two transversions. Haplotype Mes18S_2 was the second in the proportion of identified nucleotide sequences; it was found in specimens parasitizing C. rufocanus and S. caecutiens from Magadan region. Haplotype Mes18S_5 was found in a specimen from alaskan S. cinereus (Table 1).

The total number of polymorphic sites in haplogroups A and B was 24. Taking into account all mutations in both haplogroups, haplogroup A differed from haplogroup B by 21 transitions and 6 transversions (Fig. 2).

Nucleotide diversity (π) and the average number of pairwise differences between haplotypes (Pi) are higher in haplogroup A than in haplogroup B. At the same time, haplotype diversity (h) is higher in group B (Table 3).

 

Table 3. Characteristics of Mes18s_1–Mes18s_5 haplotypes of the 18S rRNA gene fragment of Mesocestoides sp.

Haplogroup	Haplotipe	Sample share		N	n/m	k	Substitution share		Molecular diversity indices
		Haplotipe	Haplogroup				Transition	Transversion	π ± sd	h ± sd	Pi ± sd
А	Mes18s_1	0.5833	0.7500	9	10/8	3	0.500	0.500	0.0023 ± 0.0016	0.4167 ± 0.1907	1.9722 ± 1.2272
	Mes18s_3	0.0833
	Mes18s_4	0.0833
B	Mes18s_2	0.1667	0.2500	3	2/2	2	0	1.00	0.0015 ± 0.0016	0.6667 ± 0.3143	1.3333 ± 1.0983
	Mes18s_5	0.0833
Substitutions between haplogroups A and B		–		–	27/24	–	0.7779	0.2222	–	–	–
General sample		–		12	27/24	5	0.7779	0.2222	0.0095 ± 0.0053	0.6667 ± 0.1409	8.3333 ± 4.1537

Notes. N – sample size, n – number of substitutions, m – number of polymorphic sites, k – number of haplotypes in the sample, π – nucleotide diversity, h – haplotype diversity, Pi – average number of pairwise differences between haplotypes, sd – standard deviation, “–” – data is not calculated.

 

The genetic distance (pairwise Fst) between haplogroups A and B calculated by the pairwise differentiation method is 0.89583. The degree of genetic differences reliability (p _Fst = 0.00901 ± 0.0091) is less than 0.05, which indicates the genetic isolation of the nucleotide sequences of these haplogroups.

Phylogenetic relationships among Mesocestoides

Phylogenetic relationships within Mesocestoides spp. are presented in Fig. 3.

 

Figure 3. ML–phylogenetic tree constructed based on data on the variability of the nucleotide sequence of the studied haplotypes of the 18S rRNA gene fragment of Mesocestoides sp. and data from GenBank. The nodes indicate bootstrap indices (≥ 50%).

 

Five clades have been identified in the ML-phylogenetic tree of Mesocestoides spp. Clade I includes nucleotide sequences of the haplogroup A and EF095248 Mesocestoides sp. (Waeschenbach et al., 2007). Clade II contains the nucleotide sequences of M. corti: GU442130 (Piseddu et al., unpublished), AF286984 (Olson et al., 2001) and Mesocestoides sp. AF119678 (Crosbie et al., 2000). It can be assumed that the nucleotide sequences of the 18S rRNA gene fragment of the same Mesocestoides species representatives have a sufficiently similar structure to form subclades with statistically significant bootstrap indices on the branches of the ML phylogenetic tree. Therefore, it is possible, that AF119678 Mesocestoides sp. (Crosbie et al., 2000), which forms clade II together with AF286984 and GU442130, belongs to the species Mesocestoides corti. In addition, haplotype MK239661 (Heneberg et al., 2019) of Mesocestoides sp. has the same nucleotide sequence with GU442130, and AF119678 Mesocestoides sp. is identical to AF286984, AF119688-AF119690, which also allows us to classify these samples of Mesocestoides sp. as M. corti.

Clade III is formed by the nucleotide sequences of M. melesi. The MN401346 variant is identical to MN401345 and MN512707 (Bayer et al., 2020). Haplotype MN401347 differs from these nucleotide sequences by four transitions and five transversions.

Haplotypes of haplogroup B form clade IV with a bootstrap index of 100%. This indicates the genetic isolation of samples Mes18S_2 and Mes18S_5 from other nucleotide sequences. It should be emphasized that haplogroup B contains samples from remote habitats - from the vicinity of Magadan and Fairbanks (USA).

Information about 16 nucleotide sequences of the 18S rRNA gene of the species Mesocestoides litteratus was taken from the electronic database for phylogenetic analysis. These sequences form clade V, represented on the ML phylogenetic tree by three haplotypes. When comparing all analyzed nucleotide sequences in the MEGA software package, it was found that only representatives of M. litteratus have a deletion of 729–747 bp and an insertion of guanine at the 761 site. The results obtained suggest that the 729–747 bp segment of the rRNA nucleotide sequence does not significantly affect either the structure or functional activity of ribosomes. Perhaps the identified deletion and insertion can claim to be a genetic marker for the species M. litteratus.

The location of haplogroups A and B on the ML–phylogenetic tree and the significant difference between the nucleotide sequences of the haplotypes of these phylogroups allow us to assume that Mesocestoides sp., having haplotypes Mes18S_1, Mes18S_3 and Mes18S_4, belong to one species, and Mes18S_2 and Mes18S_5 to another.

As long as the Mesocestoides samples taken from GenBank correspond to the declared species, the data obtained as a result of genetic analysis (nucleotide substitutions, molecular diversity indices, topology of subclades on the ML phylogenetic tree) indicate that the tetrathyridia Mesocestoides sp. from our study do not belong to the species M. corti, M. melesi and M. litteratus. For two other confirmed species (M. canislagopodis and M. lineatus), there is no comparative material on 18S rRNA in GenBank, however, taking into account the results of our previous works (Pospekhova et al., 2018, 2023), there is a high probability that the studied samples of tetrathyridia from intermediate hosts of Eastern Russia and Alaska (USA) do not belong to any of the five confirmed Mesocestoides species.

ACKNOWLEDGEMENTS

The authors would like to thank the reviewers for their constructive and friendly comments, as well as Tatyana Sokolikova (Magadan, Russia) for proofreading the text.

FUNDING

The study was carried out in the course of fulfilling a state assignment on the topics: “Helminths in the biocenoses of North–East Asia: biodiversity, morphology and molecular phylogenetics”, registration No. 1021060307693-0 and “Mammals of the Arctic and Subarctic: structure and dynamics of communities, conservation problems”, State registration No. AAAA-A18-118010990006-3 (Institute of Biological Problems of the North, Far Eastern Branch of the Russian Academy of Sciences). No additional grants to carry out or direct this particular research were obtained.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

No humans or live animals were used in this work. The work used alcohol-fixed samples from the helminthological collection of our institute.

CONFLICT OF INTEREST

The authors of this work declare that they have no conflicts of interest.

Об авторах

Н. А. Поспехова

Institute of Biological Problems of the North, Far Eastern Branch of the Russian Academy of Sciences

Автор, ответственный за переписку.
Email: posna@ibpn.ru
Россия, Magadan, 685000

В. В. Переверзева

Institute of Biological Problems of the North, Far Eastern Branch of the Russian Academy of Sciences

Email: posna@ibpn.ru
Россия, Magadan, 685000

Н. Е. Докучаев

Institute of Biological Problems of the North, Far Eastern Branch of the Russian Academy of Sciences

Email: posna@ibpn.ru
Россия, Magadan, 685000

A. A. Примак

Institute of Biological Problems of the North, Far Eastern Branch of the Russian Academy of Sciences

Email: posna@ibpn.ru
Россия, Magadan, 685000

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