Паразитология, 2024, № 6

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Том 58  №  6  2024  Ноябрь–Декабрь
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СОДЕРЖАНИЕ
Том 58, № 6, 2024
Molecular and morphological description of Diplostomum spathaceum metacercariae 
from Abramis brama L. of Lake Syamozero (north-west Russia)
457
Lebedeva D. I., Sukhovskaya I. V., Kochneva A. A., Lysenko L. A., Kantserova N. P., 
Zaitsev D. O., Milyanchuk N. P., Ieshko E. P.
Фауна блох (Insecta, Siphonaptera) Республики Карелии
470
Медведев С. Г., Лютикова Н. А., Беспятова Л. А., Бугмырин С. В.
Видовой состав и особенности экологии мух-паучниц (Diptera, Nycteribiidae) – 
паразитов летучих мышей (Vespertilionidae, Chiroptera) в центре европейской части 
России (Владимирская область)
487
Павлов А. В., Быков Ю. А.
Блохи (Siphonaptera) насекомоядных на Кавказе
497
Котти Б. К., Иванов А. Л.
Иксодовые клещи (Parasitiformes, Ixodidae) красной полевки 
(Myodes rutilus Pallas, 1779) Западно-Сибирской равнины
508
Стариков В. П., Сарапульцева Е. С., Левых А. Ю.
История изучения кровососущих комаров (Diptera, Culicidae) 
Саратовской области (литературный обзор)
520
Кондратьев Е. Н., Корнеев М. Г., Поршаков А. М
Применение тигля Гуча при изготовлении тотальных препаратов из трематод
531
Ромашов Б. В., Ромашова Н. Б., Семенов С. Н., Бахтина А. В.
Профессор Кирилл Владимирович Галактионов. 
К 70-летию исследователя-паразитолога
535
Редакционная коллегия журнала «Паразитология»,  
Межрегиональная общественная организация Паразитологическое общество 
(Паразитологическое общество при РАН)

CONTENTS
Vol. 58, № 6, 2024
Molecular and morphological description of Diplostomum spathaceum metacercariae 
from Abramis brama L. of Lake Syamozero (north-west Russia)
457
Lebedeva D. I., Sukhovskaya I. V., Kochneva A. A., Lysenko L. A., Kantserova N. P., 
Zaitsev D. O., Milyanchuk N. P., Ieshko E. P.
The fauna of fleas (Insecta, Siphonaptera) of the Republic of Karelia
470
Medvedev S. G., Lyutikova N. A., Bespyatova L. A., Bugmyrin S. V.
Species composition and ecological features of bat flies (Nycteribiidae, Diptera),  
parasites of bats (Vespertilionidae, Chiroptera) in the center of the european part of Russia  
(Vladimir Province)
487
Pavlov A. V., Bykov Y. A.
Fleas (Siphonaptera) of Eulipotyphla in the Caucasus
497
Kotti B. K., Ivanov A. L.
Ixodid ticks (Parasitiformes, Ixodidae) of the nothern red-backed vole  
(Myodes rutilus Pallas, 1779) in the West Siberian Plain
508
Starikov V. P. , Sarapultseva E. S., Levykh A. Yu.
History of the study of blood-sucking mosquitoes (Diptera, Culicidae)  
in Saratov Province (literary review)
520
Kondratev E. N., Korneev M. G., Porshakov A. M.
Gooch crucible used for making total preparations of trematodes
531
Romashov B. V., Romashova N. B., Semenov S. N., Bakhtina A. V.
Professor Kirill Vladimirovich Galaktionov.  
To the 70th anniversary of the researcher-parasitologist
535
Editorial Board of «Parazitologia»,  
Society of Parasitologists belongs to the Russian Academy of Sciences 

ПАРАЗИТОЛОГИЯ, 2024, том 58, № 6, с. 457–469.
УДК 597.556.331.1:576.895.122 (282.247.211.6)
MOLECULAR AND MORPHOLOGICAL DESCRIPTION  
OF DIPLOSTOMUM SPATHACEUM METACERCARIAE 
FROM ABRAMIS BRAMA L. OF LAKE SYAMOZERO 
(NORTH-WEST RUSSIA)
© 2024 D. I. Lebedeva a, *, I. V. Sukhovskaya a, b, A. A. Kochneva a, 
L. A. Lysenko a, N. P. Kantserovaa, D. O. Zaitsev b, 
N. P. Milyanchuk a, E. P. Ieshko a
a Institute of Biology, Karelian Research Centre of the Russian Academy of Sciences, 
11 Pushkinskaya St., Petrozavodsk, 185910 Russia  
b Petrozavodsk State University, 
33 Lenin Ave., Petrozavodsk, 185910 Russia  
* e-mail: daryal78@gmail.com
Received October 16.2024  
Revised November 08.2024  
Accepted November 12.2024
Trematodes Diplostomum von Nordmann, 1832 are widely distributed parasites with complex 
life cycles involving freshwater snails as the first intermediate hosts, various fish species as the 
second intermediate hosts, and fish-eating birds as definitive hosts. Metacercariae of Diplostomum 
spp. are important fish pathogens with problematic morphological identification. The present research 
of Diplostomum larvae in the eyes of the freshwater bream Abramis brama was carried out within 
the framework of the long-term parasitological monitoring of fish in Lake Syamozero. The study has 
provided molecular and morphological characterization of Diplostomum spathaceum metacercariae 
from the lens of the freshwater bream. The partial cox1 mtDNA sequences used for molecular identification of the isolates were identical with those obtained previously, but morphological features 
of the metacercariae matched the literature data only in part. Comparison of different dimensions 
 
of parasites by discriminant analysis suggests that Diplostomum rutili is a junior synonym of Diplostomum spathaceum. New and archival data on diplostomids from bream’s eyes demonstrate a longterm increase in the infection prevalence.
Keywords: trematoda, cox1, freshwater bream, eye lens, Karelia
DOI: 10.31857/S0031184724060012, EDN: VjzGuE
Lake Syamozero (Baltic Sea drainage basin), located in South Karelia, has long been 
a unique water body for studying the dynamics of ecosystem eutrophication. Lake Syamozero is the third largest water body in South Karelia (Lake Onego drainage basin, 
Shuya River catchment). Its area including islands is 270.3 km2. The lake is mesotrophic. 
It is a relatively shallow reservoir with a slight decrease in oxygen concentration in the 
bottom layers. The lake has abundant zooplankton, and the benthic fauna is relatively 
457

rich, with a predominance of chironomids. The fauna of molluscs and insect larvae is also 
diverse. The ichthyofauna of the lake includes 24 species of fish, among which Cyprinids 
prevail (Novokhatskaya, 2008).
Various aspects of the lake ecosystem functioning, such as the fish fauna, hydrological 
parameters, fish parasites, have been studied by staff of the Karelian Research Centre RAS 
since the 1950s. Long-term multidisciplinary studies have shown that increased anthropogenic pressure in the 1970s–1980s has significantly altered the Syamozero ecosystem 
(Reshetnikov et al., 1982). In the 1990s, the lake has partly recovered ecologically as the 
anthropogenic pressure decreased (Sterligova et al., 2016).
Research on fish parasites in Syamozero has been underway for more than 50 years 
(Shulman, 1962; Shulman et al., 1974; Malakhova, Ieshko, 1977; Ieshko, Malakhova, 1982; 
Novokhatskaya, 2008; Novokhatskaya et al., 2008; etc.). The variations observed in the 
diversity and prevalence of parasites indicate the extreme lability of parasitic systems, 
which are responsive to any changes in the hydrological and hydrobiological conditions 
(Rumyantsev, 1996).
However, for an accurate overview of the changes in the parasite fauna of fish it is necessary to accurately identify these parasites, which is not always easy. For example, larvae 
of the genus Diplostomum von Nordmann, 1832 are difficult to identify morphologically. 
This is a large genus of widely distributed trematodes. Their life cycles involve freshwater 
snails as the first intermediate hosts, various fish species as the second intermediate hosts, 
and fish-eating birds as definitive hosts (Shigin, 1986).
Since the advance of the molecular methods, the systematics of many parasite species, 
including Diplostomum spp., has been revisited, and molecular analysis is now essential 
for the identification of metacercariae (Astrin et al., 2013; Faltýnková et al., 2014, 2022; 
Lebedeva et al., 2022; Sokolov et al., 2023; etc.).
The objective of our study is to provide a morphological and molecular description 
of Diplostomum metacercariae from the eyes of the freshwater bream Abramis brama L., 
1758 in Lake Syamozero as an integral part of investigating the parasite composition in 
the bream and long-term monitoring of changes in its parasite fauna.
MATERIALS AND METHODS
Sample collection
The work is based on the parasitological studies of bream from the south-western part of Lake 
Syamozero (Syargilahta Bay). Syargilahta Bay (Fig. 1) is a sheltered bay with small sparse aquatic 
vegetation and shares all of the abovementioned features with the lake.
Breams Abramis brama (20 specimens) were examined by complete parasitological dissection 
according to the method of Bykhovskaya-Pavlovskaya (1985). Fish aged from 4+ to 9+ years were 
caught with 30-60 mm mesh nets in June 2024. Diplostomum metacercariae were found in the 
eyes, removed under a preparation stereomicroscope, washed in distilled water and counted. Some 
larvae were immediately fixed in 96% ethanol and some stained with acetic acid carmine. We used 
definitions for vouchers in a molecular context proposed by Astrin et al. (2013). Metacercaria paragenophores (morphological voucher) stained with acetic acid carmine were mounted in Canadian 
balsam for the morphological investigation (Sudarikov, Shigin, 1965). Ten hologenophores (molecular 
voucher), each from an individual host, fixed in ethanol were used for the molecular studies. They 
are metacercariae with different body shapes corresponding to those of paragenophores.
The ecological metrics of fish infection, prevalence (Е) and mean abundance (M), were calculated 
according to Bush et al. (1997).
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Figure 1. Sampling location: Lake Syamozero. Syargilahta Bay is marked with a black circle.
Morphological examination
Photomicrographs of paragenophores of metacercariae were made with a Levenhuk C1400 NG 
digital camera attached to Olympus BX-53 microscope using LevenhukToupView image analysis 
software (V 3.5) at the Core Facility of the Karelian Research Centre of the Russian Academy of 
Sciences, Petrozavodsk, Russia. The morphology of 40 metacercariae from the eye lens of A. brama 
was investigated. Thirteen morphological characteristics according to Shigin (1986) were scored 
(in μm): body length (BL), body width (BW), oral sucker length (OSL), oral sucker width (OSW), 
ventral sucker length (VSL), ventral sucker width (VSW), holdfast organ length (HL), holdfast organ 
width (HW), pseudosucker length (PSL), pharynx length (PHL), pharynx width (PHW), distance from 
center of ventral sucker to anterior end of body (O), and number of excretory bodies. Six indices 
of the relative values of these parameters were used: BW × BL/HW × HL, BW × BL/VSW × VSL, 
OSW × OSL/VSW × VSL, HW × HL/ VSW × VSL, BW/BL (%), O/BL (%).
Morphological characteristics of the parasites were assessed by means of discriminant analysis 
in PAST v. 4.05 (Hammer et al., 2001). Values of ten morphometric parameters of metacercariae 
of different Diplostomum species were chosen for the canonical discriminant analysis (BL, BW, 
OSL, OSW, VSL, VSW, HOL, HOW). The choice was based on the availability of published data 
and their relevance for our study. We used average measurements of D. spathaceum metacercariae 
from Abramis brama (Shigin, 1986), Gasterosteus aculeatus Linnaeus, 1758, Salvelinus alpinus 
Linnaeus, 1758 (Faltýnková et al., 2014), Cyprinus carpio Linnaeus, 1758 (Niewiadomska, 2010; 
Pérez-del-Olmo et al., 2014), multiple hosts: Acipenser ruthenus Linnaeus, 1758; Abramis brama 
Linnaeus, 1758; Blicca bjoerkna Linnaeus, 1758; Chondrostoma nasus Linnaeus, 1758; Leuciscus 
aspius Linnaeus, 1758; Rutilus pigus (Lacépède, 1804); Rutilus rutilus Linnaeus, 1758; Vimba vimba 
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Linnaeus, 1758; Silurus glanis Linnaeus, 1758 (Kudlai et al., 2017); and average measurements of 
D. rutili Razmashkin, 1969 (Shigin, 1986) metacercaria from the lens of Hypophthalmichthys molitrix 
(Valenciennes, 1844) to determine their ordination in the canonical axes.
The taxonomy and nomenclature of Diplostomum spp. followed the latest studies by Schwelm 
et al. (2021), Achatz et al. (2022), Faltýnková et al. (2022) and Sokolov et al. (2023).
Slides of paragenophore specimens (DspAb1-DspAb6) of the parasites are deposited in the Helminthological Collections of the Karelian Research Centre, Russian Academy of Sciences (Petrozavodsk, Karelia, Russia).
Molecular and phylogenetic analysis 
Genomic DNA was isolated from 10 ethanol-fixed specimens using the DNA-Extran kits (Synthol, Moscow, Russia).
For each of these larvae, we amplified a fragment of the mtDNA cox1 gene using the primers 
Cox1_schist_5’ (5’-TCTTTR GAT CAT AAG CG-3’) and Cox1_schist_3’ (5’-TAA TGC ATM GGA 
AAA AAA CA-3’) of Lockyer et al. (2003). The PCR assay was carried in 20 μl of reaction mixture 
containing 5XScreenMix (Evrogen, Moscow Russia), 1.5 pmol of each primer, an 2 µl of DNA; 
 
an annealing temperature of 500C was used for the amplification. PCR products were purified using 
the Cleanup Standard Extraction Kit (Evrogen, Moscow, Russia) following the manufacturer’s instructions and then sequenced directly with the automatic sequencing system ABI PRISM 3100-Avant 
(Applied Biosystems Inc., Foster City, CA, USA). Consensus sequences were assembled within 
MEGA v. 10 (Kumar et al., 2018) to the 1170 bp length and deposited in GenBank under the accession numbers PQ461127 – PQ461136.
The identity of the newly generated sequences was checked with the Basic Local Alignment 
Search Tool (BLASTn) (www.ncbi.nih.gov/BLAST/, accessed on 10 October 2024). In total, the novel 
sequences were aligned with those of 32 representatives of the genus Diplostomum in MEGA v. 10 
and trimmed to the shortest length, 384 nt. Blasting of the sequences showed a more than 99% match 
with a large number of sequences of Diplostomum spathaceum. However, we used only 15 of them 
for the phylogenetic analysis. In this case, we used specimens of the species D. spathaceum, for which 
both molecular and morphological data are available, especially from bream, as well as specimens of 
this species in different development phases from different geographical locations (Georgieva et al., 
2013; Blasco-Costa et al., 2014; Faltýnková et al., 2014; Pérez-del-Olmo et al., 2014; Locke et al., 
2015; Kudlai et al., 2017; Achatz et al., 2022; Lebedeva et al., 2023; Diaz-Suarez et al., 2024). We 
also applied some sequences of Diplostomum spp. of “non-lens” localization (Achatz et al., 2022).
The cox1 sequence JX986909 of Tylodelphys clavata (Nordmann, 1832) was used as an outgroup.
To assess the phylogenetic relationships of the newly found Diplostomum metacercariae, we applied Bayesian inference analysis (BI) and Maximum Likelihood (ML). ML analyses of HKY+G+I 
parameter distances were carried out using MEGA v10; nodal support was estimated using 1000 
bootstrap resamplings. The best-fitting model for BI was identified as TVM + G + I with jModelTest 
v2.1.2 (Darriba et al., 2012). Bayesian inference analyses were conducted using MrBayes (v3.2.3) 
(Ronquist et al., 2012). Markov chain Monte Carlo (MCMC) simulations were run for 3,000,000 
generations, log-likelihood scores were plotted, and only the final 75% of trees were used to produce the consensus tree. Posterior probability was calculated to estimate nodal support. FigTree v1.4 
(Rambaut, 2012) was used to visualize the tree.
Distance matrices (p-distance) were calculated with MEGA v. 10 (Kumar et al., 2018). The 
cox1 haplotypes of Diplostomum spp. from the lens of the freshwater bream, Abramis brama, collected in the present study and previous study in Slovakia (Kudlai et al., 2017) countries were 
identified with DnaSP v. 6 (Rozas et al., 2017). The haplotype network was reconstructed using 
the Median-Joining method in PopART software v 1.7 (Population Analysis with Reticulate Trees, 
http://popart.otago.ac.nz).
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RESULTS
We investigated different eye tissues of bream, and Diplostomum metacercariae were 
found only in the lens. Prevalence was 100%, mean abundance 25.3 specimens per fish, 
intensity from 1 to 51 specimens.
Phylogenetic analysis included ten metacercariae taken from the eyes of different bream 
specimens fish and indicated that their partial sequences of the cox 1 gene were identical to 
the published ones of the species D. spathaceum (Rudolphi, 1819) Olsson, 1876 (Fig. 2).
Figure 2. Bayesian inference (BI) and maximum likelihood (ML) analyses tree for Diplostomum 
spp. based on the partial cox1 mtDNA sequences (384 bp). Nodal supports from both analyses are 
indicated as BI/ML. The scale bar indicates the expected number of substitutions per site. Only 
significant values of the posterior probabilities (≥ 0.5) are indicated. Newly obtained sequences 
are in bold. Abbreviations: ad – adult, mtc – metacercariae, Ab – Abramis brama, Bb – Blicca 
bjoerkna, Cm – Cyprinion macrostomum, La – Larus argentatus, Pp – Pungitius pungitius, 
Rr – Rutilus rutilus, Sa – Salvelinus alpinus, Ch – China, E – Estonia, G – Germany, 
H – Hungary, I – Iraq, Ic – Iceland, R – Russia, S – Slovakia, Sp – Spain.
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The metacercariae we studied joined in a clade with representatives of D. spathaceum 
at different developmental stages and from different countries, confirming a wide distribution and occurrence in different host species worldwide (Fig. 2). The p-distance between 
ten investigated parasites from Syamozero was 0.08 – 1%. And p-distances among samples 
of D. spathaceum were 0.03 – 1.5%.
There were seven haplotypes (Fig. 3) among the larvae from bream, with haplotype 
diversity at 0.93. Four of the sequences each had its own haplotype (PQ461127, PQ461129, 
PQ461130, PQ461134), and three more haplotypes were represented by two specimens 
each (PQ461132 and PQ461133; PQ461135 and PQ461136; PQ461128 and PQ461131, 
respectively).
Figure 3. Haplotype network for Diplostomum spathaceum based on novel and published partial 
cox1 mtDNA sequences of Kudlai et al. (2017). Unsampled intermediate haplotype is represented 
by a short intersecting line; each branch corresponds to a single mutational difference and 
connective lines represent one mutational step. Circle size is proportional to the number of isolates 
sharing a haplotype; black circles indicate transitive haplotypes that have not been found.
Comparison against the only available cox1 mtDNA sequences of D. spathaceum metacercariae from breams from Slovakia (Kudlai et al., 2017) showed that only one haplotype 
was shared between two Karelian metacercariae (PQ461128, PQ461131) and one Slovakian 
metacercaria (KY653964).
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