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СОДЕРЖАНИЕ
Том 58, № 2, 2024
Phylogenetic relationships of Mesocestoides Vaillant, 1863 
tetrathyridia from small mammals of eastern Russia and Alaska based on 18s rRNA gene
91
Pospekhova N. A., Pereverzeva V. V.,  
Dokuchaev N. E., Primak A. A.
Сингоспитальность и гостальные связи эриофиоидных клещей  
(Acariformes, Eriophyoidea): таксономический анализ комплексов видов  
клещей-галлообразователей на бореальных древесных двудольных 
101
Сухарева С. И., Аристов Д. А., Ганкевич В. Д., Десницкий А. Г.,  
Озман-Салливан С. К., Четвериков Ф. Е.
Оценка вирулентности изолятов некоторых видов энтомопатогенных 
анаморфных аскомицетов (Fungi, Ascomycota) в отношении взрослых особей  
клеща Ixodes persulcatus
124
Борисов Б. А., Беспятова Л. А., Леднёв Г. Р.,  
Левченко М. В., Бугмырин С. В.
Фауна и обилие иксодид (Parasitiformes, Ixodidae) на острове Аскольд  
(Приморский край): уникальность, инфицированность патогенами
136
Никитин А. Я., Зверева Т. В., Вержуцкая Ю. А., Кайсарова Н. А.,  
Солодкая Н. С., Сафонова Н. В., Гордейко Н. С., Андаев Е. И.,  
Колесникова В. Ю., Балахонов С. В.
Морфологические основы трех классификаций  
отряда блох (Insecta, Siphonaptera)
147
Медведев С. Г.
VII Съезд Паразитологического общества:  
Итоги и актуальные задачи (Петрозаводск, 16–20 октября 2023 г.)
169
Иешко Е. П., Бугмырин С. В.,  
Матвеева Е. М., Яковлева Г. А.

CONTENTS
Vol. 58, № 2, 2024
Phylogenetic relationships of Mesocestoides Vaillant, 1863 tetrathyridia 
from small mammalls of eastern Russia and Alaska based on 18s rRNA gene
91
Pospekhova N. A., Pereverzeva V. V.,  
Dokuchaev N. E., Primak A. A.
Synhospitality of eriophyoid mites (Acariformes, Eriophyoidea):  
taxonomic analysis of gall-forming mite species complexes  
on boreal woody dicotyledons 
101
Sukhareva S. I., Aristov D. A., Gankevich V. D., Desnitskiy A. G.,  
Ozman-Sullivan S. K., Chetverikov P. E.
Evaluation of virulence of isolates of certain species  
of Entomopathogenic anamorphic ascomycetes (Fungi: Ascomycota)  
in relation to adult individuals of the tick Ixodes persulcatus
124
Borisov B. A., Bespyatova L. A., Lednev G. R.,  
Levchenko M. V., Bugmyrin S. V.
Fauna and abundance of ixodids (Parasitiformes, Ixodidae)  
on Askold island (Primorsky krai): uniqueness, infection with pathogens
136
Nikitin A. Ja., Zvereva T. V., Verzhuckaja Ju. A., Kajsarova N. A.,  
Solodkaja N. S., Safonova N. V., Gordejko N. S., Andaev E. I.,  
Kolesnikova V. Yu., Balahonov S. V.
Morphological basis of the three classifications  
of the order of fleas (Insecta, Siphonaptera)
147
Medvedev S. G.
VII Congress of the Society of parasitologists: 
current results and challenges  (16–20 October, 2023. Petrozavodsk)
169
Ieshko E. P., Bugmyrin S. V.,  
Matveeva E. M., Yakovleva G. A.

ПАРАЗИТОЛОГИЯ, 2024, том 58, № 2, с. 91–100.
УДК 576.895.121:599.323+599.363
PHYLOGENETIC RELATIONSHIPS OF MESOCESTOIDES 
VAILLANT, 1863 TETRATHYRIDIA FROM SMALL MAMMALS  
OF EASTERN RUSSIA AND ALASKA BASED ON 18S rRNA GENE
© 2024 N. A. Pospekhova a, *, V. V. Pereverzeva a, 
N. E. Dokuchaev a, A. A. Primak a 
a Institute of Biological Problems of the North, Far Eastern Branch 
of the Russian Academy of Sciences,  
Magadan, 685000 Russia
*e-mail: posna@ibpn.ru
Received October 05, 2023  
Revised January 01, 2024  
Accepted January 12, 2024
A fragment of 875 bp of the 18S rRNA gene was studied in 12 samples of  Mesocestoides 
tetrathyridia from small mammals of 7 species collected in geographically distant localities. Five 
haplotypes were identified, differing from each other by 27 nucleotide substitutions in 24 sites. It has 
been found that the 18S rRNA haplotypes belong to two genetically distinct haplogroups. Molecular 
diversity indices were calculated for each of them. The conducted analysis allowed to suggest the 
following: 1) Mesocestoides sp. haplogroups A and B belong to two different species, but do not 
belong to any of the confirmed species of the genus; 2) the deletion of bp 729–747 and the insertion 
at site 761 of guanine can be regarded as a genetic marker for the species Mesocestoides litteratus.
Keywords: Mesocestoides, tetrathyridium, insectivores, rodents, 18S rRNA, phylogenetic analysis, 
genetic marker
DOI: 10.31857/S0031184724020017, EDN: ypsabz
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).
91

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.
92

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 94оC – 5 min; then 
40 cycles: 94оС – 1 min, 52оС – 1 min, 72оС – 2 min; final stage 72°C – 7 min. The amplified 
sections of nuclear DNA were purified and prepared for sequencing according to standard methods 
93

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).
94

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). 
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.
95

Substitution share
Molecular diversity indices
N
n/m 
k
Sample share
Haplotipe
Haplogroup
Transition
Transversion
π ± sd
h ± sd
Pi ± sd
Mes18s_1
0.5833
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
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
А
Haplogroup
Haplotipe
General sample
–
12
27/24
5
0.7779
0.2222
0.0095 ± 
0.0053
0.6667 ± 
0.1409
8.3333 ± 
4.1537
Substitutions between 
haplogroups A and B
–
–
27/24
–
0.7779
0.2222
–
–
–
Table 3. Characteristics of Mes18s_1–Mes18s_5 haplotypes of the 18S rRNA gene fragment of Mesocestoides sp.
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.
96

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).
97