Introduction
Parasites represent an integral and functionally important component of natural ecosystems. They play key roles in multiple ecological processes, including trophic interactions, host behaviour regulation, and immune and metabolic modulation (e.g., Born-Torrijos et al. Reference Born-Torrijos, Van Beest, Merella, Garippa, Raga and Montero2023; Poulin Reference Poulin2010; Sikder et al. Reference Sikder, Pierce, Sarkar, McHugh, Quinlan, Giacomin and Loukas2024). Additionally, parasites serve as valuable ecological markers for tracking intermediate and definitive host migrations, as well as pathogen distribution patterns. Of particular interest in this context are parasites with complex life cycles, particularly the cosmopolitan genus Diplostomum Nordmann, 1832, which is transmitted within ecosystems through avian, fish, and snail hosts. In recent years, this genus has garnered increasing attention in studies of cryptic diversity, phylogenetics, and evolution (e.g., Blasco-Costa et al. Reference Blasco-Costa, Faltýnkova, Georgieva, Skírnisson, Scholz and Kostadinova2014; Faltýnková et al. Reference Faltýnková, Kudlai, Pantoja, Yakovleva and Lebedeva2021; Georgieva et al. Reference Georgieva, Soldanova, Pérez-del-Olmo, Dangel, Sitko, Sures and Kostadinova2013; Kudlai et al. Reference Kudlai, Oros, Kostadinova and Georgieva2017; Locke et al. Reference Locke, Al-Nasiri, Caffara, Drago, Kalbe, Lapierre, McLaughlin, Nie, Overstreet, Souza, Takemoto and Marcogliese2015; Selbach et al. Reference Selbach, Soldanova, Georgieva, Kostadinova and Sures2015; Sokolov et al. Reference Sokolov, Ieshko and Lebedeva2023). However, investigating the distribution of Diplostomum spp. across regions remains challenging due to unresolved cryptic diversity not accounted for by traditional taxonomic frameworks (Faltýnková et al. Reference Faltýnková, Kudlai, Pantoja, Yakovleva and Lebedeva2021; Georgieva et al. Reference Georgieva, Soldanova, Pérez-del-Olmo, Dangel, Sitko, Sures and Kostadinova2013; Schwelm et al. Reference Schwelm, Georgieva, Grabner, Kostadinova and Sures2021).
Recent research demonstrates that an integrative approach – combining ecological data (e.g., infection conditions, host specificity), morphology, and molecular analysis – enhances the accuracy of species identification within this group (Faltýnková et al. Reference Faltýnková, Kudlai, Pantoja, Yakovleva and Lebedeva2021). Molecular methods, particularly DNA barcoding of the mitochondrial cox1 gene and, less frequently, the complete ITS1-5.8S-ITS2 ribosomal cluster, have become standard tools. These techniques have facilitated the rapid accumulation of sequence data from diverse geographic regions (Faltýnková et al. Reference Faltýnková, Kudlai, Pantoja, Yakovleva and Lebedeva2021; Hoogendoorn et al. Reference Hoogendoorn, Smit and Kudlai2020; Kudlai et al. Reference Kudlai, Oros, Kostadinova and Georgieva2017; Lebedeva et al. Reference Lebedeva, Chrisanfova, Ieshko, Guliaev, Yakovleva, Mendsaikhan and Semyenova2021; Locke et al. Reference Locke, Al-Nasiri, Caffara, Drago, Kalbe, Lapierre, McLaughlin, Nie, Overstreet, Souza, Takemoto and Marcogliese2015).
The Russian Far North has garnered increasing scientific and economic interest due to expanding resource extraction and the heightened sensitivity of Arctic ecosystems to climate change (ACIA 2005). The Arctic and subarctic zones are hypothesized to serve as hotspots for intercontinental pathogen dispersal, including parasitic fauna, owing to dense populations of migratory birds (Gass et al. Reference Gass, Dusek, Hall, Hallgrimsson, Halldorsson, Vignisson, Ragnarsdóttir, Jónsson, Krauss, Wong, Wan, Akter, Sreevatsan, Trovão, Nutter, Runstadler and Hill2022). Notably, over half of the Arctic avian fauna comprises migratory Charadriiformes and Anseriformes (Chernov Reference Chernov1999) – key definitive hosts for Diplostomum spp. Despite abundant avian hosts, extreme environmental conditions limit the diversity and availability of intermediate hosts (Galaktionov Reference Galaktionov1996), constraining the completion of complex trematode life cycles, particularly for diplostomids. These factors profoundly shape parasite biodiversity in Arctic and subarctic aquatic ecosystems.
Despite the ecological significance of Diplostomum spp., their diversity in Arctic regions remains poorly understood. Molecular-based studies of northern Eurasian diplostomids have been conducted in Norway, Finland, Estonia, Iceland, and the Republic of Karelia (Russia) (e.g., Blasco-Costa et al. Reference Blasco-Costa, Faltýnkova, Georgieva, Skírnisson, Scholz and Kostadinova2014; Kudlai et al. Reference Kudlai, Oros, Kostadinova and Georgieva2017; Lebedeva et al. Reference Lebedeva, Popov, Yakovleva, Zaicev, Bugmyrin and Makhrov2022; Martinek et al. Reference Martinek and Hernández-Orts2024; Sokolov et al. Reference Sokolov, Ieshko and Lebedeva2023), yet comprehensive assessments are lacking for most Arctic localities. Although helminth distributions in Arctic avian hosts have been examined (Galaktionov Reference Galaktionov1996), no Diplostomum spp. have been formally described from these birds. A distinctive feature of Far Northern waters is their cold-adapted salmonid populations, including Atlantic salmon Salmo salar Linnaeus, 1758 and brown trout Salmo trutta Linnaeus, 1758, which serve as second intermediate hosts for diplostomids (Blasco-Costa et al. Reference Blasco-Costa, Faltýnkova, Georgieva, Skírnisson, Scholz and Kostadinova2014; Rochat et al. Reference Rochat, Paterson, Blasco-Costa, Power, Adams, Greer and Knudsen2022). Nevertheless, molecular data on Diplostomum spp. infecting these fish in the Russian Far North are absent.
To address these knowledge gaps, we investigated the species composition of Diplostomum in parrs of S. salar and S. trutta from rivers of the European Russian Far North draining into the Barents and White Seas. Our study simultaneously evaluates the Eurasian distribution patterns of these parasites.
Materials and methods
Parrs of Atlantic salmon and Brown trout were collected from eight rivers of Murmansk Region, the far North of the European part of Russia: Kola River (N68°52′34.96″; E33°01′54.07″), Pecha River (N68°31′37.76″; E31°35′20.41″), Kutujoki River (N66°57′24.79″; E30°24′25.42″), Mashjoki River (N68°56′40.49″; E28°39′24.56″), Nautsijoki River (N68°55′27.63″; E28°59′13.86″), Noleshjoki River (N68°39′50.13″; E29°48′19.72″), Sojgjoki River (N68°39′50.13″; E29°48′19.72″), and Srednii Verman River (N67°01′12.96″; E30°48′02.22″) (Table 1).
Infection indices on occurrence of Diplostomum spp. metacercariae in salmonid parrs from the far North of the European part of Russia. P: prevalence, I: infection intensity, M: mean intensity
Fish specimens were collected in October 2021 using a backpack electrofisher (FA3 model, serial no. 88/92; GEOMEGA a/s, Trondheim, Norway). The equipment delivered a pulsed DC current of 700/1400 V at a frequency of 35–70 Hz. The fish was sacrificed by a blow to the head and dissected according to Bykhovskaya-Pavlovskaya (Reference Bykhovskaya-Pavlovskaya1985); eyes were extracted and investigated under a stereomicroscope. Metacercariae from lens, vitreous humor, and retina were collected separately and fixed in 96% ethanol. The abundance of diplostomids in each fish was quantified, and ecological metrics of infestation – prevalence (P), infection intensity (I), and mean intensity (M) – were calculated following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997). Infestation data were analyzed separately for ‘lens’ and ‘non-lens’ metacercariae across surveyed rivers. Quantitative metrics were calculated only for fish species collected from rivers with sample sizes >13 individuals; rivers with sporadic collections were excluded from analysis.
In addition, the analysis included samples of Diplostomum spp. from lens and vitreous humor of Coregonus lavaretus pidschian (Gmelin), Leuciscus leuciscus (Linnaeus, 1758), and Thymallus arcticus (Pallas, 1776) caught in Teletskoye Lake (51°79′N, 87°30′E), a continental waterbody in Asian Russia geographically isolated from marine influences.
Genomic DNA was extracted from 27 single Diplostomum spp. specimens (16 from rivers of Murmansk Region and 11 from Teletskoye Lake) using a 5% aqueous suspension of Chelex 100 resin (Sigma-Aldrich, Massachusetts, USA) supplemented with proteinase K (0.1 mg/mL). Double-stranded DNA was amplified using the BioMaster HS-Taq PCR-Color (2×) kit (Novosibirsk, Russia) according to the manufacturer’s instructions. The partial cox1 region was amplified using Dice1R (5′-ATTAACCCTCACTAAATTWCNTTRGATCATAAG-3′) and Dice11R (5′-TAATACGACTCACTATAGCWGWACHAAATTTHCGATC-3′) primers (Steenkiste et al. Reference Steenkiste, Locke, Castelin, Marcogliese and Abbott2014). The cycling conditions were as follows: 94°C for 2 min; 3 cycles of 94°C for 40 s, 51°C for 40 s, 72°C for 1 min; 5 touchdown cycles of 94°C for 40 s, 50°C to 46°C for 40 s (dropping 1°C per cycle), 72°C for 1 min; 35 cycles of 94°C for 40 s, 45°C for 40 s, 72°C for 1 min; and a final extension at 72°C for 10 min. PCR amplification of the complete ITS1-5.8S-ITS2 cluster was conducted using the D1 (5′-AGG AAT CC TGG TAA GTG CAA G-3′) and D2 (5′-CGT TAC TGA GGG AAT CCT GGT-3′) primers (Galazzo et al. Reference Galazzo, Dayanandan, Marcogliese and McLaughlin2002). The cycling conditions were as follows: 94°C for 2 min, 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min; and a final extension at 72°C for 10 min. The PCR products were purified and sequenced in Eurogen (Moscow, Russia). Newly obtained sequences were deposited into GenBank (NCBI).
Sequence chromatogram quality was assessed using Chromas v. 2.5 (Technelysium Pty, South Brisbane, Australia) based on peak sharpness and nucleotide resolution at each position. For alignment MUSCLE algorithm implemented in MEGA v. 11 (Tamura et al. Reference Tamura, Stecher and Kumar2021) was used. Ambiguous and uninformative sites were removed using Gblocks service (Castresana Reference Castresana2000). Species affiliation of the newly collected Diplostomum specimens was tested using NCBI BLAST.
Genetic distances were calculated using the p-distance model in MEGA 11 (Tamura et al. Reference Tamura, Stecher and Kumar2021). Haplotype analyses were performed with DnaSP 6 (Rozas et al. Reference Rozas, Ferrer-Mata, Sánchez-DelBarrio, Guirao-Rico, Librado, Ramos-Onsins and Sánchez-Gracia2017). Bayesian Inference and Maximum Likelihood were used to infer phylogenetic trees, resulting in a consensus tree. Maximum Likelihood inference was performed using IQ-TREE v.2.2.2.6 (Minh et al. Reference Minh, Schmidt, Chernomor, Schrempf, Woodhams, von Haeseler and Lanfear2020). The best-fit substitution model was selected with the program’s integrated ModelFinder algorithm (Kalyaanamoorthy et al. Reference Kalyaanamoorthy, Minh, Wong, von Haeseler and Jermiin2017). Nodal support was assessed with 10,000 ultrafast bootstrap replicates and the SH-like approximate likelihood ratio test. For Bayesian Inference we used MrBayes v.3.2.7 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), identifying appropriate model with jModelTest v.2.1.10 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). cox1 tree was constructed with HKY+I+G model, ITS cluster tree – using HKY+I model. For Bayesian Inference, two simultaneous Markov Chain Monte Carlo runs were deployed for 5,000,000 generations. Phylogenetic trees were reconstructed based on haplotypes of newly obtained sequences, along with a number of sequences from several Diplostomum species retrieved from GenBank. The sequences of Alaria alata (Goeze 1782) (Diplostomidae) with accession nos. OL439156 (cox1 gene tree) and MZ057614 (ITS cluster tree) from the GenBank database were included in the phylogenetic analyses as an outgroup. FigTree v.1.4.4 was used to visualize the final trees. Popart 1.7 (Leigh and Bryant Reference Leigh and Bryant2015) was utilized to assess haplotype network using the Median-Joining method for novel haplotypes together with sequences retrieved from previous studies trimmed to their shortest length (353 for cox1). The distribution of genetic variation was examined using analysis of molecular variance (AMOVA) as implemented in Arlequin v3.5.2.2 (Excoffier and Lischer Reference Excoffier and Lischer2010) with significance tests based on 1000 permutations. Spatial genetic structure was assessed using Mantel tests between pairwise genetic and geographic distance matrices conducted in R v4.1.2 (R Core Team 2021). Genetic p-distances were calculated using the ape package (Paradis and Schliep Reference Paradis and Schliep2019), while geographic distances were computed from latitude and longitude coordinates using the geodist package (Padgham and Sumner Reference Padgham and Sumner2025). Mantel tests were performed with 10,000 permutations using the vegan package (Oksanen et al. Reference Oksanen, Blanchet, Friendly, Kindt, Legendre, McGlinn, Minchin, O’Hara, Simpson, Solymos, Stevens, Szoecs and Wagner2020).
Results
Lens metacercariae of Diplostomum were found only in parrs of Atlantic salmon caught from the Kola and Pecha rivers (Table 1). Non-lens Diplostomum spp. metacercariae were observed in the retina and vitreous humor of parrs from both fish species (Table 1). The lowest level of prevalence of these parasites was observed in Nolleshjoki and Kutujoki Rivers (6.7% for both water bodies), and the highest level in Sojgjoki River (66.7%). Overall, for non-lens metacercariae, 22 and 35 specimens were encountered in the retina and vitreous humor, respectively.
Both Maximum Likelihood and Bayesian Inference analyses, based on partial cox1 gene sequences, produced trees with identical topology. In these phylogenetic reconstructions, lens metacercariae newly collected in rivers of Murmansk Region and Teletskoye Lake clustered within the clade of ‘Diplostomum mergi’ Lineage 3, while non-lens metacercariae from both localities and site of infection (retina and vitreous humor) – within the clade of Diplostomum numericum Niewiadomska, Reference Niewiadomska1988. Analysis of genetic distances revealed intraspecific variation of 0–1.84% among D. numericum sequences and 0–0.86% among ‘D. mergi’ Lineage 3 sequences. The maximum interspecific distance in the analyzed dataset was observed between D. numericum and ‘D. mergi’ Lineage 3 (16.4% ± 1.9), while the minimum distance was found among different lineages within the D. mergi clade (2.6% ± 0.9). Mean interspecific distances ranged from 2.9% ± 0.86 to 15.2% ± 1.9. Phylogenetic analyses of ITS cluster sequences placed the lens and non-lens metacercariae from the examined salmonids within the ‘D. mergi’ Lineage 3 and the D. numericum + D. petromyzifluviatilis clade, respectively (Fig. 1).
Bayesian Inference and Maximum Likelihood consensus tree for Diplostomum spp. haplotypes based on: A) cox1 partial region; B) complete ITS1-5.8S-ITS2 cluster. Nodal support for BI and ML is indicated as BI/ML; values >75 are shown. The newly generated sequences highlighted in bold. Host designations: Ss: Salmo salar, St: Salmo trutta. Sample localization codes: H: vitreous humor, R: retina, L: lens.
The cox1 haplotype networks revealed complex mutational relationships. These networks comprised 28 haplotypes for D. numericum and 14 haplotypes for ‘D. mergi’ Lineage 3, with no formation of distinct haplogroups in either case (Fig. 2).
Haplotype distribution map of Diplostomum spp. and median-joining haplotype network with cox1-haplotypes (novel obtained haplotypes highlighted in bold and by a star) confined to their locality: (А and А1) D. numericum; (B and B1) ‘Diplostomum mergi’ Lineage 3. Unique sequences highlighted by a star and coloured white. Shorebird flyways were mapped according to Galbraith et al. (Reference Galbraith, Jones, Kirby and Mundkur2014). Sampling locations are indicated on the map: 1: Murmansk Region, 2: Lake Teletskoye.
Sequences belonging to the D. numericum clade, which shared a haplotype with our specimens, were recorded in several northern European localities: Iceland, Norway, and Germany. Notably, D. numericum and ‘D. mergi’ Lineage 3 haplotypes identical to those documented in the Far North of European Russia were also detected in Teletskoye Lake, Siberia (Fig. 2).
Results of AMOVA revealed that the majority of genetic variation in D. numericum and D. mergi (89.46% and 76.35%, respectively) was distributed within sampling areas, while only a small proportion (10.54% and 23.65%) was attributed to differences among areas (Table 2). Mantel tests revealed no significant correlation between genetic and geographic distances for D. numericum (r = 0.20, p = 0.032) or ‘D. mergi’ Lineage 3 (r = 0.16, p = 0.105).
Analysis of molecular variance (AMOVA) of cox1 gene fragments Diplostomum spp. isolates considering their differentiation among sampling localities
Discussion
Although non-lens metacercariae were isolated from distinct ocular sites, including the retina and vitreous humor, phylogenetic analyses confirmed that all collected larvae were conspecific and molecularly identifiable as D. numericum. This study provides the first documented occurrence of D. numericum metacercariae in the ocular fundus, expanding their known tropism beyond the vitreous humor, where they were exclusively reported previously (Sokolov et al. Reference Sokolov, Ieshko and Lebedeva2023).
The second species, ‘Diplostomum mergi’ Lineage 3, was found exclusively in the lens. This digenean species belongs to the large clade of D. mergi species complex, which also includes D. parviventosum Dubois, 1932, and several other species-level taxa, currently referred to as different D. mergi lineages (e.g., Schwelm et al. Reference Schwelm, Georgieva, Grabner, Kostadinova and Sures2021; Selbach et al. Reference Selbach, Soldanova, Georgieva, Kostadinova and Sures2015). This large clade received its name from the species affiliation of cercariae identified and molecularly characterized by Niewiadomska and Laskovsky (Reference Niewiadomska and Laskowski2002). All isolates of the D. mergi complex clade genotyped to date were either cercariae or metacercariae. Although the morphology of Diplostomum cercariae is relatively informative and has resolved certain taxonomic issues (e.g., Niewiadomska Reference Niewiadomska1986), reliable species-level identification remains challenging. This difficulty arises due to the incompleteness of data on this larval stage in a significant number of Diplostomum spp. recognized both within the framework of traditional models of their species composition and, especially, given the modern concept of hidden diversity within this digenean genus. Identification of metacercariae of Diplostomum is possible within the framework of the taxonomic model of this genus proposed by Shigin (Reference Shigin1986). However, none of the isolates of metacercariae of the D. mergi complex clade was described on samples fixed in strict accordance with A. Shigin’s method, i.e., fixation of live specimens in acetocarmine (Komatsu et al. Reference Komatsu, Itoh and Ogawa2019; Kudlai et al. Reference Kudlai, Oros, Kostadinova and Georgieva2017). Thus, to date there is not a single reliable evidence of conspecificity of any member of the D. mergi complex clade with either D. mergi sensu Shigin (Reference Shigin1986, Reference Shigin and Freze2002) or D. parviventosum sensu Shigin (Reference Shigin1986, Reference Shigin and Freze2002). In this regard, the name of all isolates of the D. mergi complex clade is largely arbitrary.
D. numericum was not differentiated from D. petromyzifluviatilis on the tree based on the ITS cluster dataset. Such a pattern has been repeatedly demonstrated in previous studies (Faltýnková et al. Reference Faltýnková, Georgieva, Kostadinova, Blasco-Costa, Scholz and Skírnisson2014; Lebedeva et al. Reference Lebedeva, Chrisanfova, Ieshko, Guliaev, Yakovleva, Mendsaikhan and Semyenova2021, Reference Lebedeva, Popov, Yakovleva, Zaicev, Bugmyrin and Makhrov2022). This marker is probably not suitable for analysing evolutionary events at the species level in Diplostomum.
To date, limited data are available on parasite infections in salmonid fishes under Arctic conditions. Previously, only 13 isolates from Salvelinus alpinus (Linnaeus 1758) and 25 isolates from S. trutta from Hafravatn Lake in Iceland have been analyzed (Blasco-Costa et al. Reference Blasco-Costa, Faltýnkova, Georgieva, Skírnisson, Scholz and Kostadinova2014), revealing just four Diplostomum species, with three species found in each host species. In comparison, the species richness of diplostomids in fish sampled from rivers of the Murmansk region appears to be somewhat lower, although this difference is not pronounced. These differences in species richness are most likely determined by the limited overlap between habitats of pulmonate mollusks (the first intermediate hosts of these digeneans) and fish in fast-flowing river conditions, coupled with the restricted diversity of piscivorous birds foraging in these rivers.
Notably, we observed no geographically associated structure in haplotype diversity for either D. numericum or ‘D. mergi’ Lineage 3. AMOVA revealed extensive genetic connectivity and weak spatial structuring across the distribution of both Diplostomum species, with the majority of genetic variation partitioned within rather than among sampling locations. Consistently, Mantel tests showed no significant isolation by distance, supporting the absence of geographically associated genetic differentiation. This distribution pattern likely reflects the high dispersal capacity of Diplostomum spp. via migratory fish-eating birds. It should also be noted that the lack of geographic structure may be attributed to the relative conservatism of cox1 compared to other mitochondrial genes. For instance, in dicrocoeliids, it has been demonstrated that fast-evolving mitochondrial genes atp6, nad5, and nad3 would be more suitable markers than the slow-evolving gene cox1 in population-level studies (Suleman et al. Reference Suleman, Khan, Tkach, Muhammad, Zhang, Zhu and Ma2020).
Most collection sites of D. numericum and ‘D. mergi’ Lineage 3, including pre-molecular era records (Niewiadomska Reference Niewiadomska1988; Shigin and Stanislavetz Reference Shigin and Stanislavetz1989), occur within the East Atlantic flyway of migratory birds – particularly among their potential definitive hosts (Anatidae and Gaviidae) (Galbraith et al. Reference Galbraith, Jones, Kirby and Mundkur2014). However, both species were also detected in Teletskoye Lake in southern Western Siberia, Russia – a location situated within the West Asia flyway (Galbraith et al. Reference Galbraith, Jones, Kirby and Mundkur2014). The available data are currently insufficient to determine whether Teletskoye Lake represents an isolated population fragment of these Diplostomum species, potentially sustained through overlapping East Atlantic, West Asia, and Mediterranean flyways that facilitate parasite exchange between avian host populations, or whether it constitutes part of a broader continuous distribution across Europe and Siberia.
Further study of the distribution of Diplostomum spp. in different regions requires the collection of material from more geographical locations, including the collection of adults from the definitive hosts that are the main dispersers of parasites of this genus. The availability of such data would shed light on how the geographical range of individual Diplostomum species is established and varies, which is still an open question. Most likely, a complex of reasons can explain this variation: (i) the scarcity or abundance of intermediate host species; (ii) the range and flyways of the definitive host species.
Data availability
All the sequences acquired during the study are available in GenBank under accession numbers PP911545–PP911560 (ITS1-5.8s-ITS2), PP916530–PP916545, and PX472836–PX472841 (cox1).
Acknowledgements
The authors acknowledge Dr. Denis Efremov for his contribution to specimen collection.
Financial support
The fieldwork from the Russian Far North was funded by the ENI-CBC Kolarctic project, SALMUS, KO1017 ‘Salmonid Fish and Freshwater Pearl Mussel—Ecosystem Services and Biodiversity in the Green Belt of Fennoscandia’ (EI, SS, AP), and under the state-ordered project number 122032100130-3 (EI, AP). The sequencing of diplostomids from fish inhabiting Teletskoye Lake was paid with founds the Russian Science Foundation (grant number 23-74-10101).
Competing interests
Authors stated no conflict of interest.
Ethical standard
The result of this work has not been published previously and is not under consideration elsewhere.
Ethical approval
All applicable institutional, national, and international guidelines for the care and use of animals were followed.
