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All issues Volume 32 (2025) Parasite, 32 (2025) 27 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 27
Number of page(s) 18
DOI https://doi.org/10.1051/parasite/2025020
Published online 23 April 2025

© K. Gulyás et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Employing Wright’s concept of “isolation by distance” [115], we can expect that genetic differentiation will increase with increasing geographic distance between two groups of organisms. Geographical isolation fundamentally prevents gene flow between two groups, which has a pivotal role in the formation of demes (e.g., [20, 52, 78, 80, 95]). However, distance is not the only factor affecting genetic differentiation. The other important factor is landscape isolation, strongly affecting population connectivity, which promotes population diversification even in smaller geographical regions (e.g., [27, 63, 76, 101]). Therefore, the concept of “isolation by environment” was formed [108, 109], summarizing the correlation between environmental heterogeneity and spatial variation in gene flow. At first sight, the assessment of demographic structure appears to be a rather straightforward and logical task. However, a more complicated process may be recognizing demes among parasites. While geographical factors undoubtedly affect genetic structure in the parasites on a large spatial scale, we can expect that on a local scale, the structure will be more affected by ecological and environmental factors (as in vertebrates: e.g., [2, 62]). In addition, the host specificity and ecology of the associated hosts also appear to be key factors, introducing an additional layer into the spatial structure of parasites [9].

Amphibians play a key role in ecosystems by regulating insect populations, serving as prey, and hosting a diverse range of parasites (trematodes, nematodes, cestodes, and acanthocephalans), contributing to helminth transmission as both intermediate and definitive hosts [16, 48]. The life cycles of these parasites are closely linked to their amphibian hosts, with potential implications for amphibian health and population stability [16]. Host mobility is a major determinant of parasite gene flow (e.g., see [60]) in parasites that lack free-living stages or have free-living stages with limited dispersal. However, in parasites with free-living stages, the genetic structures of populations are primarily determined by the dispersal abilities of these stages, and also by the mode of reproduction of the parasite (sexual versus asexual) [57]. Larval dispersal and survival will play an important role in parasites with a direct life cycle, as exhibited by many nematode species.

Nematodes are among the most common parasites of amphibians (together with trematodes), particularly of the genera Cosmocercoides Wilkie, 1930, Gyrinicola Yamaguti, 1938, Falcaustra Lane, 1915, Oswaldocruzia Travassos, 1917, and Rhabdias Stiles & Hassall, 1905 [48]. These nematodes infect their hosts either through direct skin penetration or by the ingestion of larval stages with food [35, 36, 48]. A significant number of nematode species associated with amphibians belong to the cosmopolitan genus Oswaldocruzia [23], which is predominantly found in the intestines of amphibians and reptiles [31, 86, 100]. It is a taxonomically diverse group, whose representatives are often difficult to identify due to its morphological uniformity and low host specificity [23]. The genus comprises approximately 92 species [31, 51, 100, 103, 114] and is characterized by a strong speciation potential, leading to the existence of numerous closely related species [22].

The first species of the genus Oswaldocruzia described in the Palaearctic was Oswaldocruzia filiformis Goeze, 1782. This nematode is a common and widely distributed parasite of amphibians, particularly those of the genera Bufo and Rana [29, 30, 44]. It has a direct life cycle, with invasive larvae occurring on the surface of soil and vegetation, from where they usually infect host orally during its feeding [35, 44]. The variability in the size and morphology of O. filiformis across different host species and regions has been well-documented. The adaptability of O. filiformis to diverse environmental conditions is reflected by its phenotypic plasticity, enabling the species to develop distinct ecomorphs across regions with varying ecological characteristics [45]. Despite an increasing level of research on amphibian nematodes [17, 44, 50, 100, 114], molecular genetic studies on Oswaldocruzia remain limited [46, 47, 113], underscoring the need for further investigation.

Previous studies on the parasite fauna of amphibians in Czechia and Slovakia revealed the presence of 30 different nematode species, including seven species of Oswaldocruzia: O. molgeta Lewis, 1928, O. goezei Skrjabin et Schulz, 1952, O. filiformis (Goeze, 1782), O. iwanitzkyi Sudarikov, 1951, O. ukrainae Iwanitzky, 1928, O. lenteixerai Pérez Vigueras, 1939, and O. subauricularis (Rudolphi, 1819) [104–108]. These studies were conducted nearly half a century ago, primarily relying on the morphological identification of parasites without accounting for their genetic characterization. The aim of our study was therefore to use molecular markers to refine the distribution of species in the genus Oswaldocruzia, to determine their genetic variation and population-genetic structure, and to relate these to the host specificity of individual species. Because of the southward shift in amphibian species richness and endemism within Europe [93], we hypothesize that the genetic variation in Oswaldocruzia nematodes will only correspond weakly to the population structure of their amphibian hosts. Additionally, due to the low host specificity of O. filiformis [44, 45], we expect that different host species will harbor genetically closely related nematode populations.

Material and methods

Ethics statement

Scientific permits for material collection and processing were provided by the Directorate of Forest Management, the Ministry for the Environment and Energy of the Hellenic Republic (181012/807/28-3-2019); the National Agency of Protected Areas, the Ministry of Tourism and the Environment of the Albanian Republic (No. 480/2019), Romanian Administratia Rezervati ei Bios Ferei Delta Dunarii (no. 362/2023), and the Ministry of the Environment of the Slovak Republic (No. 2963/2013-2.2 and no. 519/2022-6.3). All live captured animals used in this study were humanely euthanized and processed in accordance with ethical guidelines and legal regulations in the respective countries where the research was conducted.

Material collection

A total of 719 frogs (either captured live or collected as cadavers and subsequently frozen) from 74 localities across the Balkans (Albania, Bulgaria, Greece, and Romania), and Central Europe (only Czechia and Slovakia) were analyzed for parasite presence (Fig. 1, Table 1, Supplementary Table S2). Nematodes were found in 11 host species: Pelophylax ridibundus (Pallas, 1771) (n = 21), Pelophylax esculentus (Linnaeus, 1758) (n = 15), Rana temporaria Linnaeus, 1758 (n = 10), Rana dalmatina Fitzinger, 1839 (n = 9), Bufo bufo (Linnaeus, 1758) (n = 224) and Bufotes viridis (Laurenti, 1768) (n = 12) and further unidentifiable Rana sp. (n = 1) in Slovakia, and Pelophylax epeiroticus (Schneider, Sofianidou & Kyriakopoulou-Sklavounou, 1984) (n = 2), Pelophylax kurtmuelleri (Gayda, 1940) (n = 7), and Pelophylax shqipericus (Hotz, Uzzell, Guenther, Tunner & Heppich, 1987) (n = 1) and further unidentified Pelophylax sp. (n = 22) in the Balkans. The identification of Pelophylax water frog species was based on morphological characteristics [32, 67, 72] and molecular markers, specifically sequences of the mitochondrial ND2 fragment and microsatellites. Further details regarding the ND2 and microsatellite laboratory analyses, along with the genetic identification of water frogs, can be found in studies by Plötner et al. [71], Hoffmann et al. [37], and Papežík et al. [67, 68]. In northeastern Greece and southern Bulgaria, where the ranges of three species that hybridize meet, water frogs have been assigned only to the genus.

thumbnail Figure 1

Map of sampling localities in Central Europe and the Balkans. Green markers indicate positive records of Oswaldocruzia spp. and white markers represent localities where no Oswaldocruzia spp. were recorded. Localities where fewer than three host specimens were examined are circled in grey. A = Central Europe and the Balkan peninsula; B = Central Europe (Czechia and Slovakia); C = the Balkan peninsula; AL = Albania; BG = Bulgaria; CZ = Czechia; GR = Greece; RO = Romania; SK = Slovakia.

Table 1

List of collection localities with coordinates, and number of examined frog individuals per each present species.

Prior to the examination, frozen frogs were thawed, and live frogs were euthanized using clove oil extract and subsequently sacrificed by cutting the spinal cord. The internal organs were then placed in a saline solution and examined under a stereomicroscope. All the helminths, including nematodes, were removed from the intestines, killed with hot saline solution (0.85% NaCl) (excluding those already killed by freezing), and preserved in 70% or 96% ethanol for subsequent molecular analyses. A total of 2,563 Oswaldocruzia nematodes were extracted from the frogs’ small intestines. Basic quantitative descriptors of the parasite populations, such as prevalence, mean abundance, and the minimum and maximum intensities of infection, were calculated for each parasite species, as outlined by Bush et al. [15]. Prevalence, defined as the percentage of frogs infected by a specific parasite species, and mean abundance, defined as the average number of parasite specimens per individual host (including both infected and uninfected hosts), were calculated. To interpret the quantitative data, a 95% confidence interval was calculated for the mean abundance, following the recommendation by Rózsa et al. [81]. Due to the relatively small sample size of hosts per population, the bias-corrected and accelerated bootstrap (BCa) method was employed, using QPweb [77], to calculate the confidence interval for mean abundances. Selected non-damaged specimens, representing paragenophores, were deposited in the Helminthological Collection of the Institute of Parasitology of the Czech Academy of Sciences (IPCAS), Czechia, under the accession number IPCAS N-1291.

Genomic DNA extraction, amplification, sequencing

Prior to DNA extraction, the nematodes were identified to genus level by their localization in the intestine of the hosts and general morphology. Then, they were randomly selected from each locality for DNA extraction. Total nematode genomic DNA was extracted using either a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) or NucleoSpin® Tissue kit (Macherey-Nagel, Düren, Germany), following the respective manufacturer’s protocol. The fragment of the mitochondrial cytochrome oxidase c subunit I gene (COI) was amplified for each Oswaldocruzia specimen – from at least three host specimens from each host population or all of them, if fewer specimens per population were available. For amplification, the primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′) [11] were used, and polymerase chain reactions (PCR) were carried out in a total volume of 15 μL containing 1U of DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 1× Taq Buffer, 1.5 mM MgCl2, 300 μM of each dNTP, 0.5 μM of each primer, 2 μL of DNA template (corresponding to 20 ng/μL) and nuclease-free water. The PCR conditions followed the protocol optimized by Kirillova et al. [44]. PCR products were detected by electrophoresis in 1.5% agarose gels stained with GoodView (SBS Genetech, Beijing, China). Amplified products were purified using EPPiC Fast (A&A Biotechnology, Gdansk, Poland), following the manufacturer’s protocol. Sequencing was performed in both directions using the PCR primers. Commercial services provided by Macrogen Europe (the Netherlands) were used for sequencing.

Sequence dataset assembly and phylogenetic analyses

Prior to sequence alignment, GenBank was screened to obtain homologous COI sequences of Oswaldocruzia spp. A total of 21 O. filiformis and 6 O. ukrainae sequences originating from Russia, and one Oswaldocruzia sp. sequence from a specimen from Italy were retrieved from GenBank (Supplementary Table S1). To assess whether all conspecific sequences formed a monophyletic group, the sequence alignment was built using the Fast Fourier transform algorithm implemented in MAFFT software [43] and included all available and newly generated Oswaldocruzia COI sequences; two Ancylostoma sequences [A. ceylanicum (MW549613) and A. tubaeforme (NC_034289)] were used as an outgroup for rooting of the phylogram. The alignment was then manually trimmed to unify the length of all sequences, and translated into amino acids to avoid any signal misreads using Universal Invertebrate Mitochondrial Code (transl_table=5). Identical sequences were then removed for phylogenetic analyses. The sequence data were treated as codon partitioned, and a GTR model was selected independently for each position within the codon, including both a gamma distribution and the proportion of invariable sites. Phylogenetic trees were constructed using Bayesian inference (BI) and maximum likelihood (ML) approaches in MrBayes v. 3.2. [79] and RAxML v. 8.1.12 [98, 99], respectively. BI analysis used the Metropolis-coupled Markov chain Monte Carlo algorithm with two parallel runs of one cold and five hot chains and was run for 107 generations, sampling trees every 100 generations. The initial 30% of all saved trees were discarded as “burn-in” after checking that the standard deviation split frequency fell below 0.01. The convergence of the runs and the parameters of individual runs were checked using Tracer v. 1.7.1 [75]. Posterior probabilities for each tree node were calculated as the frequency of samples recovering a given clade. The clade bootstrap support for ML trees was assessed by simulating 103 pseudoreplicates.

A subsequent dataset was built from all available O. filiformis sequences, including the newly generated ones, and was used to assess intraspecific genetic variability on the wide geographical range level. The level of DNA polymorphism in COI sequences, i.e., haplotype diversity (Hd), nucleotide diversity (π), the number of unique haplotypes, and the number of variable sites, was assessed using DnaSP 5 [55]. To examine genetic diversity, the following statistical analyses were performed in R v. 4.1.3 [74]. A pairwise matrix of uncorrected p-distances was generated using the dist.dna() function from the ape package [69]. This matrix was used as input for principal coordinates analysis (PCoA) conducted with the vegan package [65] to visualize genetic diversity in multivariate space. Based on the position of each individual in the multivariate space, 95% confidence interval (CI) ellipses were drawn around the individuals to facilitate easier interpretation. The results were visualized using the ggplot2 package [112]. Uncorrected p-distance calculations and PCoA were performed in R v. 4.1.3.

Lastly, a median-joining haplotype network constructed in PopART [53] was used to assess the population genetic structure of O. filiformis based on COI haplotypes from individuals in Slovakia with respect to individual major river basins and host associations. The dataset included all O. filiformis sequences originating from individuals from the Central European amphibians. All sequences used in the present study, including those newly generated, are presented in Supplementary Table S1. New sequences were deposited in GenBank with accession numbers PV168566–PV168633.

Results

Diversity and distribution of Oswaldocruzia spp.

A total of 330 anurans (out of 719) were infected by nematodes of the genus Oswaldocruzia, out of which we identified two species: Oswaldocruzia filiformis and O. ukrainae. Oswaldocruzia ukrainae was found only in B. viridis from three Slovak localities (KVP, MP, PH) (see Supplementary Figs. 1 and 2 for localities with abbreviated IDs). We confirmed the occurrences of Oswaldocruzia filiformis specimens both in Central Europe (Czechia and Slovakia), and the Balkans (Albania, Bulgaria, Greece, and Romania) (Fig. 1, Figs. S1, S2). By excluding the negative localities, the prevalence of Oswaldocruzia samples in the studied frog populations varied from 9.1% (VLO, Slovakia – P. esculentus) to 100% [BR; BU (Rana sp. only); HS; HD; JD; KS; MV; MH; PZ; SH (R. temporaria only); SR – each from Slovakia and IO (P. kurtmuelleri only) – from Greece] (Table 2, abbreviations for localities are in Table 1). From all the populations with a 100% prevalence of Oswaldocruzia specimens, only the BR, MV, and MH populations had more than one host sample examined. From the localities where more than one host individual was examined, the highest mean abundance (21) and intensity of infection (7–48) was recorded among Bufo bufo at MH (Slovakia).

Table 2

Epidemiologic characteristics of the Oswaldocruzia parasites calculated for each host population.

Phylogenetic relationships and intraspecific genetic variability of Oswaldocruzia spp.

Based on the quality and lengths of the sequences, a total of 174 newly generated Oswaldocruzia spp. COI sequences were selected for subsequent phylogenetic analyses. A total of 76 unique COI haplotypes were recognized in O. filiformis. All O. ukrainae specimens carried the same genetic variant of the COI gene. Identical O. filiformis sequences were removed from the dataset for phylogenetic tree reconciliation (i.e., each haplotype is represented only by a single sequence, see Supplementary Table S1 for selected sequences), and the final alignment was built on 91 sequences (also including two Ancylostoma orthologous sequences as an outgroup for rooting the phylogenetic tree and 28 Oswaldocruzia sequences retrieved from GenBank) and spanned 370 unambiguously aligned nucleotide positions. Both phylogenetic analyses (BI and ML) generated trees with congruent topologies, and therefore only the BI tree is presented with posterior probabilities and bootstrap support values (Fig. 2). The phylogenetic analyses congruently confirmed the monophyly of all conspecific sequences. The dataset also included the sequence of Oswaldocruzia sp. from Elaphe quatuorlineata from Italy, which was in a sister position to the cluster encompassing all O. ukrainae sequences.

thumbnail Figure 2

Phylogenetic tree of 90 COI sequences of three Oswaldocruzia species reconstructed by Bayesian inference. The tree is based on a 370 bp-long alignment and rooted using Ancylostoma tubaeforme and A. ceylanicum as the outgroup. Each Oswaldocruzia filiformis represents a unique haplotype. Haplotype numbers correspond to those in Figure 4 and Supplementary Table S1. Values at the nodes indicate posterior probabilities (>70) from the Bayesian inference, and bootstrap values (>50) from the maximum likelihood analysis. Lower values are shown as dashes (–). The length of branches represents the number of substitutions per site. The sequences retrieved from GenBank are greyed.

Within 76 unique haplotypes recognized among 189 O. filiformis COI sequences (including those retrieved from GenBank), 53 polymorphic sites were identified. Out of all haplotypes, 19 were recorded from more than a single Oswaldocruzia individual. These were also almost exclusively present in more than one locality. The uncorrected p-distances between haplotypes ranged between 0.3 and 3.5%. The overall haplotype diversity (Hd) was 0.954, and nucleotide diversity (π) reached 1.1%. The translated sequence alignment spanned 123 amino acid positions and only three sites were identified as polymorphic, i.e., the majority of the nucleotide substitutions were recognized as synonymic. The results of PCoA divided all haplotypes into four relatively well-separated, but artificially defined clusters (Fig. 3). No apparent pattern was observed based on host associations. A weak degree of geographic structuring was recognized among the four clusters. Cluster A encompassed the majority of haplotypes carried by O. filiformis individuals from the Volga/ and Ural basins and southern Balkans. The majority of haplotypes within cluster B were from individuals from the Danube basin; however, this cluster also encompassed the single haplotype from the southern Balkans [OF_9, carried by a single individual from the LR locality, and also by an individual in BM (Romania)]. Cluster C again encompassed mainly haplotypes from the Danube basin. However, it also included the widespread haplotype OF_1, which was recognized among individuals in the Danube basin (Slovakia – various localities), the Southern Balkans (Albania – QP, Bulgaria – KR, and Greece – GR, LR, and AR), and the Volga basin (Russia – Uzola floodplains), and haplotype OF_10 from the Volga and from the Danube basin. This cluster also included the unique haplotype OF_40, found in only one individual of O. filiformis at LR. The last cluster, labelled D, contained only haplotypes from O. filiformis specimens in the Danube basin, and three unique haplotypes from Greece, specifically from IO and LR. Notably, the unique haplotype OF_28, found in an O. filiformis specimen from Ioannina Lake (Greece), was distinct from all four clusters on the first two dimensions. The river basins and the localities related to each basin are shown in Supplementary Figure S3.

thumbnail Figure 3

Principal coordinates analysis (PCoA) showing genetic diversity among the analysed Oswaldocruzia filiformis COI sequences obtained in Central Europe and the Balkans. The colors and shapes of the marks are associated with the major geographical district and host taxon, respectively.

Genetic variability and distribution of O. filiformis in Central Europe

The haplotype network, with trait groups based on the major river basins in Central Europe (Fig. 4), showed no associations across the distribution of the haplotypes in Central Europe with local river basins. Oswaldocruzia filiformis specimens from Central Europe carried 51 COI haplotypes (out of 76 in total), out of which 46 were not recognized among the individuals from other countries. The highest haplotype diversity was recognized among specimens collected from B. bufo (Fig. 5), with 22 being unique for the O. filiformis specimens from this host. Six haplotypes were unique for the individuals from P. ridibundus. A total of 14 haplotypes were identified in multiple O. filiformis specimens (i.e., OF_1, OF_3, OF_4, OF_5, OF_6, OF_7, OF_10, OF_13, OF_16, OF_20 OF_25, OF_34, OF_37, OF_62). The most common haplotypes among O. filiformis in Central Europe were OF_1 (recognized in 16 specimens) and OF_6 (in 15 specimens). The overall haplotype diversity of O. filiformis in Central Europe was 0.950, and nucleotide diversity reached a value of 1.2%.

thumbnail Figure 4

Population-genetic structure of Oswaldocruzia filiformis found in Slovak populations of frogs, based on COI haplotypes presented as a median-joining haplotype network. The sizes of the circles in the network are proportional to the relative frequencies of the haplotypes; small black circles represent missing haplotypes. The vertical lines represent the number of substitutions between individual haplotypes. Different colors represent major river basins according to the legend. The haplotype numbers correspond to those in Figure 1 and Supplementary Table S1.

thumbnail Figure 5

Population-genetic structure of Oswaldocruzia filiformis found in Slovak populations of frogs, based on COI haplotypes presented as a median-joining haplotype network. The sizes of the circles in the network are proportional to the relative frequencies of the haplotypes; small black circles represent missing haplotypes. The vertical lines represent the number of substitutions between individual haplotypes. Different colors represent specific host taxa for O. filiformis according to the legend. The haplotype numbers correspond to those in Figure 1 and Supplementary Table S1.

Discussion

While parasites of economically important animals, such as fish and livestock, as well as those affecting humans are being extensively studied [1, 4, 83, 121], parasites of amphibians are receiving comparatively less attention. Although their direct impact on human health is limited, understanding host-parasite relationships is essential for recognizing their ecological roles in, and contributions to overall ecosystem dynamics. This study investigated the genetic variability of Oswaldocruzia species collected from various amphibian hosts across several Central European and Balkan countries and compared these findings with existing COI sequences of Oswaldocruzia obtained by Kirillova et al. [44] from European Russia and by Mendoza-Roldan et al. [58] from Italy.

The occurrence of Oswaldocruzia spp. in the Palearctic

The genus Oswaldocruzia has been reported worldwide, with studies documenting its occurrence across various continents such as South America [31, 96, 114], Central America [13, 14], North America [34], Europe [23, 97, 106], Africa [5], Australia [6], and Asia [25, 56, 119]. Here, we report the occurrence of the genus Oswaldocruzia in nine anuran species from the genera Pelophylax, Rana, Bufo, and Bufotes across two Central European countries (Czechia and Slovakia) and the Balkan Peninsula, including Albania, Romania, Bulgaria, and Greece (Table 1). Two species of Oswaldocruzia were identified: O. filiformis and O. ukrainae. The first species, O. filiformis, is considered to have a widespread distribution and broad host range [29, 44]. A previous study in Bulgaria [12] recorded O. filiformis in various anurans, such as Bombina bombina, Bombina variegata, B. bufo, B. viridis, Hyla arborea, Rana graeca, R. temporaria, and R. dalmatina. In Türkiye, its occurrence was documented in B. bufo [24], B. viridis, and Pelophylax sp. [24, 42], Hyla arborea [42], as well as in Rana macrocnemis [117] and Pelodytes caucasicus [118]. Additionally, O. filiformis has been identified in caudate amphibians such as Salamandra salamandra from Bulgaria [12], Lissotriton vulgaris from Belarus [92] and Germany [94], and Ommamotriton vittatus from Türkiye [120]. Reports from reptiles include occurrences in Lacerta trilineata (Türkiye [116]), Lacerta viridis (Czechia [10]), Zootoca vivipara (Spain [85]; Belarus [90]), Anguis fragilis, Natrix natrix, and Vipera berus (Belarus [91]). Other European reports span Russia [64], Poland [66], France [97], Ireland [30], Hungary [36], Ukraine [50], Austria [87], and Czechia and Slovakia [106]. Our study expands the known distribution of O. filiformis to other Balkan countries, such as Romania, Greece, and Albania, and increases its host range to 30 amphibian and reptile species. Previous studies on O. ukrainae indicate its occurrence within a limited range of hosts, including Bombina bombina (Linnaeus, 1758), B. variegata (Linnaeus, 1758), B. bufo, B. viridis, and Rana arvalis [46], with the highest number of reports observed in B. viridis [5, 106], which may suggest a potential host association with B. viridis. Our results further support this possibility, as the only host species of O. ukrainae in this study was B. viridis. Nevertheless, in certain populations, B. viridis was also found to be infected with O. filiformis (Slovak localities PS and KVP) in this study. However, no cases of co-infection with other Oswaldocruzia species were identified. This finding is not unique, as previous studies [106, 116] also reported B. viridis as a host of O. filiformis.

Epidemiology of O. filiformis in the Palearctic

Chikhlyaev et al. [18] and Griffin [30] documented fluctuations in the prevalence and abundance of O. filiformis in bufonid and ranid frogs across studied localities in Russia and Ireland, respectively. These observations align with our findings, where O. filiformis in B. bufo hosts showed variability in prevalence and abundance across the studied localities. Kuzmin et al. [50] also reported a fluctuating trend in Oswaldocruzia infections across different Pelophylax species in northern Ukraine. They found a substantially lower prevalence and abundance of Oswaldocruzia spp. in P. esculentus compared to the investigated populations of P. ridibundus. However, this does not align with our observations, where the two Pelophylax species exhibited similar epidemiological indices across the investigated populations. These contrasting and highly variable results suggest a complex infection landscape for Oswaldocruzia spp. within different anuran populations and localities. This variability may be associated with factors such as the ecology of host species, host population densities, and parasite transmission dynamics, or may reflect the sampling effort, which varies among studies (e.g., [70, 73, 82]).

Population-genetic structure of Oswaldocruzia spp.

To the best of our knowledge, only three studies [46, 47, 94] have specifically focused on the genetic variability of Oswaldocruzia species in Europe so far. Sinsch et al. [94] identified O. filiformis from Lissotriton vulgaris on the basis of morphology and DNA sequences. Initially, Kirillova et al. [44] investigated the genetic variability of O. filiformis in correlation with morphological characteristics across the Volga and Ural basins. Later, Kirillova et al. [46] analyzed O. ukrainae COI sequences in B. viridis. By analyzing 174 newly-obtained COI sequences, we confirmed the presence of two Oswaldocruzia species in this study. Congruently with Kirillova et al. [46], all O. ukrainae specimens possessed a single COI variant. In contrast to O. ukrainae, our analyses revealed substantial genetic variability within O. filiformis, as 76 different COI haplotypes were recognized. Oswaldocruzia filiformis was the first species of the genus Oswaldocruzia described in the Palaearctic. This nematode is a common and widely distributed parasite of amphibians, particularly those of the genera Bufo and Rana [29, 30, 44]. The contrasting level of genetic variability is not unusual among parasites (e.g., [3, 26, 33, 41, 59, 61, 102]. Host-specific nematodes are limited to a narrower range of hosts, which reduces opportunities for gene flow among populations. This can lead to a smaller effective population size and a decrease in genetic diversity over time. Moreover, due to host specialization, specialist species tend to evolve traits or genetic forms tightly suited to their host, potentially limiting their ability to adapt to other environments or hosts and further reinforce certain traits within the populations. On the other hand, generalist nematodes exploit multiple host species and a broader range of ecological niches, further promoting higher genetic variability as a result of broader population dynamics and opportunities to adapt to more diverse environments (see [19, 38, 60]). In the generalist O. filiformis, we assumed that the haplotype variation would be associated with either host species (similarly as in Shaw et al. [89] and Shaw et al. [88]) or geographical distribution (as in [54]); however, our analysis revealed only limited spatial genetic structuring of its populations across the investigated area.

The results of the PCoA (Fig. 3) split the haplotypes of O. filiformis into four distinct clusters with notable separation. Among the 76 unique haplotypes identified, 19 were detected in multiple Oswaldocruzia specimens; however, there was no noticeable pattern regarding host association across these clusters, indicating a weak host-specific component in shaping the genetic variation of O. filiformis. Despite the lack of host-driven patterns, some geographic structure was observed, aligning with findings from previous studies [3, 7, 59, 84], further promoting the notion that nematodes exhibiting host generalist behavior are likely to display a weak population-genetic structure. Cluster A included almost all haplotypes from the Volga and Ural basins, but also encompassed haplotypes from the other two defined basins. Contrastingly, clusters B, C, and D were primarily composed of haplotypes from specimens collected within the Danube basin, suggesting a certain level of genetic differentiation within this vast geographical area. Additionally, three unique haplotypes from Greece were included in these clusters (the Ioannina Lake and Loutros River), suggesting potential interregional gene flow or historical connectivity between these locations and the Danube basin populations. Surprisingly, a unique haplotype (OF_28) was identified in the O. filiformis specimen from Ioannina Lake (Greece) which was distinctly separated from all four observed clusters (Supplementary Table S1). Significant divergence of the haplotype OF_28 may indicate the existence of a cryptic species in this region. As Gómez et al. [28] noted, cryptic species complexes often arise from recent, and sometimes rapid, speciation events. Such events might be ongoing in the Ioannina Lake, and it is possible that the new endemic haplotype (or cryptic species) emerged locally. All the other investigated specimens from this population carried OF_13, also present within cluster A. Further genetic and morphological analyses are needed to confirm whether the separation observed in OF_28 is associated with the existence of a distinct species or just reflects an intraspecific variation within O. filiformis.

Parasite distribution is directly influenced by the distribution of hosts [49]. However, to some extent, environmental factors may also contribute to the shaping of genetic differentiation among O. filiformis populations, although our results, concurrently with previous studies [44], suggest non-specific population-genetic patterns in this nematode species. Also, genetic diversity in amphibians, and potentially their parasites, is to some extent shaped by postglacial dispersal and lineage mixing as species recolonized deglaciated regions. The observed haplotype heterogeneity among the studied localities likely reflects these processes, highlighting the interconnected and continuous distribution of O. filiformis and its hosts from the Balkans. Its lifestyle, comprising both free-living and parasitic stages, offers multiple potential mechanisms for distribution. The nematode’s free-living stage enables independent movement (either on its own or long-range human-mediated [111]), while its parasitic stage relies on host species for dispersal. Furthermore, its wide host range, which includes amphibians, reptiles, and fish, enhances its dispersal potential across diverse ecological niches. The combination of a free-living life strategy, opportunistic parasitism, and a broad host range has likely allowed O. filiformis to utilize various dispersal routes and further facilitated its widespread distribution, and thus contributed to the observed genetic heterogeneity across the studied localities.

Population genetic structure of Oswaldocruzia filiformis in Central Europe

Our comprehensive sampling campaign in Central Europe allowed us to study the population-genetic pattern on a small regional scale. Among the seven distribution groups defined by the major river basins in Slovakia, none of the haplotypes showed exclusive affiliation to a specific basin, instead appearing scattered randomly across all basins (Fig. 4). We would assume that at least in a region as geographically confined as Slovakia (and Czechia), there would be minimal genetic variation within the populations of O. filiformis. However, our results yet again revealed no associations between the obtained sequences and the potential distribution routes for amphibians associated with river basins. The observed distribution pattern of O. filiformis throughout Europe may be explained by the phylogeography of its host species. Previous studies have emphasized the importance of glacial and interglacial periods, as well as the subsequent recolonization of Central and northern Europe, in shaping species and genetic diversity in amphibians and reptiles (e.g., [8, 40, 68, 110]). Southern Europe primarily served as a region of numerous microrefugia for amphibians and reptiles, promoting genetic diversification and the formation of unique and endemic genetic variants [21, 39]. While these major geoclimatic events shaped genetic variation in the hosts, the weak population-genetic structure observed in adult stages of Oswaldocruzia suggests the existence of additional drivers contributing to diversification in their nematode parasites.

Out of all 74 haplotypes identified in our dataset, Oswaldocruzia from Central Europe carried 46 haplotypes occurring only in this region (Table 2). However, this might be mainly due to sample bias, as a significantly larger number of frogs were examined from Central Europe compared to other regions, given that the frogs in Central Europe harbored more nematode individuals. This sample bias is also partially reflected in the distribution of haplotypes among host taxa, as the highest diversity was recorded among specimens collected from B. bufo. Most populations from Central Europe shared the haplotypes OF_1 and OF_6. The OF_1 haplotype was recorded not only from Central Europe but also from Albania, Bulgaria, and Greece. In contrast, the OF_6 haplotype was recorded only among populations in Central Europe. The widespread presence of haplotype OF_1 suggests historical or ongoing genetic flow between the mentioned regions, indicating a shared evolutionary history or dispersal events that connected these populations. In contrast, haplotype OF_6 represents localized genetic variation, likely shaped by regional ecological or environmental factors, which created this unique haplotype, probably thanks to limited dispersal or specific adaptation to the local environment. Further investigations of the ecological and evolutionary dynamics of O. filiformis and its interactions with host species are crucial for gaining deeper insights into the factors that have driven its distribution and genetic diversity. Additional genetic data on O. filiformis from a wider range of hosts and locations, together with sequence data on other species of the genus Oswaldocruzia, might shed more light on the diversification processes within this genus and elucidate the parasite’s shaping of host-parasite relationships in dynamic landscapes.

Conclusion

Oswaldocruzia nematodes are common parasites of poikilothermic vertebrates. Although their species diversity is relatively well-documented, limited information is available regarding their host relationships at the genetic level and their phylogeography. These parasites exhibit varying levels of host specificity, ranging from strictly host-specific species, parasitizing a single host species, to true generalists such as O. filiformis. Our molecular data reveal that the specialist species O. ukrainae, which is closely associated with B. viridis, exhibits low genetic variability in the mitochondrial COI gene. In contrast, populations of O. filiformis in the western Palearctic exhibit an extremely high number of haplotypes. The distribution of these haplotypes indicates only weak population genetic structuring; however, their abundance highlights the elevated mutation rate in generalist nematode parasites. Notably, this high genetic variability is more pronounced at the nucleotide level compared to protein sequences, a fact that should be taken into account in future research.

Acknowledgments

We are grateful to Adam Javorčík, Jana Christophoryová, Simona Papežíková, Jessica Hriňáková, Miloš Halán, Romana Gašparovičová, Lucie Seidlová, Eva Čisovská Bazsalovicsová, and Ľudmila Juhásová for assistance during parasite collection and processing. We are indebted to Martin Danilák, Jaroslav Brndiar, Tomáš Flajs, Peter Drengubiak, Mária Apfelová and Gréta Nusová from the State Nature Conservancy of the Slovak Republic for providing amphibian cadavers for the purposes of this study. We kindly thank Matthew Nicholls for the English revision of the final draft.

Funding

This study was financially supported by the Scientific Grant Agency of the Slovak Republic project no. VEGA 1/0583/22. Additionally, the collection of material was partially funded by VEGA 2/0052/24 and the Slovak Research and Development Agency under contracts APVV DS-FR-22-0006 and APVV-23-0015.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Supplementary material

Supplementary Table S1: A list of analyzed Oswaldocruzia spp. individuals with respective hosts and collection localities and COI GenBank accession numbers for each haplotype representative sequence.

ID = specific code used for the respective Oswaldocruzia specimen; Haplotype = respective haplotype corresponding to Figures 2 and 4 and further discussed in the Results and Discussion sections; Cluster = position within multivariate space resulting from PCoA, as shown in Figure 3. Sequences retrieved from GenBank are marked by asterisks (*), sequences used for construction of phylogenetic tree are marked by a dagger (†).

Access here

Supplementary Table S2: Individual host infections of Oswaldocruzia parasites at each collection site. Access here

thumbnail Supplementary Figure S1:

Regional map showing in detail the collection sites from Central Europe (Czechia and Slovakia).

Localities where O. ukrainae was confirmed are marked with a black dot inside the circle. A = Localities from Western Slovakia and Czechia; B = Localities from Central Slovakia; C = Localities from Eastern Slovakia; CZ = Czechia; AT = Austria; SK = Slovakia; PL = Poland; UA = Ukraine; HU = Hungary. The locality abbreviations correspond to those in the other tables and text.

thumbnail Supplementary Figure S2:

Regional map showing in detail the collection sites from the Balkan peninsula.

A = Localities from Romania; B = Localities from the remaining Balkan countries; HU = Hungary; RO = Romania; MD = Moldova; UA = Ukraine; HR = Croatia; BA = Bosnia and Herzegovina; RS = Serbia; BG = Bulgaria; RKS = Kosovo; AL = Albania; MK = Macedonia; GR = Greece; TR = Türkiye. The locality abbreviations correspond to those in the other tables and text.

thumbnail Supplementary Figure S3:

Defined river basins used in the PcoA analyses.

Dots on the map represent all sequences used in the PcoA analyses obtained during this study or from the GenBank database. Green dots represent localities with novel data from Central Europe, yellow dots represent localities with novel data from the Balkan peninsula, pink dots represent the Italian locality of Oswaldocruzia sp., for which sequence data were obtained from the GenBank database from Italy, and brown dots represent the Russian localities of Oswaldocruzia spp., for which sequence data were retrieved from GenBank. A = South-eastern European River basins; B = Danube River basin; C = Volga and Ural River basins.

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Cite this article as: Gulyás K, Balogová M, Pipová N, Papežík P, Uhrovič D, Mikulíček P, Brázová T & Benovics M. 2025. Insights into the genetic diversity and species distribution of Oswaldocruzia nematodes (Trichostrongylida: Molineidae) in Europe: apparent absence of geographic and population structuring in amphibians. Parasite 32, 27. https://doi.org/10.1051/parasite/2025020.

All Tables

Table 1

List of collection localities with coordinates, and number of examined frog individuals per each present species.

In the text
Table 2

Epidemiologic characteristics of the Oswaldocruzia parasites calculated for each host population.

In the text

All Figures

thumbnail Figure 1

Map of sampling localities in Central Europe and the Balkans. Green markers indicate positive records of Oswaldocruzia spp. and white markers represent localities where no Oswaldocruzia spp. were recorded. Localities where fewer than three host specimens were examined are circled in grey. A = Central Europe and the Balkan peninsula; B = Central Europe (Czechia and Slovakia); C = the Balkan peninsula; AL = Albania; BG = Bulgaria; CZ = Czechia; GR = Greece; RO = Romania; SK = Slovakia.
In the text
thumbnail Figure 2

Phylogenetic tree of 90 COI sequences of three Oswaldocruzia species reconstructed by Bayesian inference. The tree is based on a 370 bp-long alignment and rooted using Ancylostoma tubaeforme and A. ceylanicum as the outgroup. Each Oswaldocruzia filiformis represents a unique haplotype. Haplotype numbers correspond to those in Figure 4 and Supplementary Table S1. Values at the nodes indicate posterior probabilities (>70) from the Bayesian inference, and bootstrap values (>50) from the maximum likelihood analysis. Lower values are shown as dashes (–). The length of branches represents the number of substitutions per site. The sequences retrieved from GenBank are greyed.
In the text
thumbnail Figure 3

Principal coordinates analysis (PCoA) showing genetic diversity among the analysed Oswaldocruzia filiformis COI sequences obtained in Central Europe and the Balkans. The colors and shapes of the marks are associated with the major geographical district and host taxon, respectively.
In the text
thumbnail Figure 4

Population-genetic structure of Oswaldocruzia filiformis found in Slovak populations of frogs, based on COI haplotypes presented as a median-joining haplotype network. The sizes of the circles in the network are proportional to the relative frequencies of the haplotypes; small black circles represent missing haplotypes. The vertical lines represent the number of substitutions between individual haplotypes. Different colors represent major river basins according to the legend. The haplotype numbers correspond to those in Figure 1 and Supplementary Table S1.
In the text
thumbnail Figure 5

Population-genetic structure of Oswaldocruzia filiformis found in Slovak populations of frogs, based on COI haplotypes presented as a median-joining haplotype network. The sizes of the circles in the network are proportional to the relative frequencies of the haplotypes; small black circles represent missing haplotypes. The vertical lines represent the number of substitutions between individual haplotypes. Different colors represent specific host taxa for O. filiformis according to the legend. The haplotype numbers correspond to those in Figure 1 and Supplementary Table S1.
In the text
thumbnail Supplementary Figure S1:

Regional map showing in detail the collection sites from Central Europe (Czechia and Slovakia).

Localities where O. ukrainae was confirmed are marked with a black dot inside the circle. A = Localities from Western Slovakia and Czechia; B = Localities from Central Slovakia; C = Localities from Eastern Slovakia; CZ = Czechia; AT = Austria; SK = Slovakia; PL = Poland; UA = Ukraine; HU = Hungary. The locality abbreviations correspond to those in the other tables and text.
In the text
thumbnail Supplementary Figure S2:

Regional map showing in detail the collection sites from the Balkan peninsula.

A = Localities from Romania; B = Localities from the remaining Balkan countries; HU = Hungary; RO = Romania; MD = Moldova; UA = Ukraine; HR = Croatia; BA = Bosnia and Herzegovina; RS = Serbia; BG = Bulgaria; RKS = Kosovo; AL = Albania; MK = Macedonia; GR = Greece; TR = Türkiye. The locality abbreviations correspond to those in the other tables and text.
In the text
thumbnail Supplementary Figure S3:

Defined river basins used in the PcoA analyses.

Dots on the map represent all sequences used in the PcoA analyses obtained during this study or from the GenBank database. Green dots represent localities with novel data from Central Europe, yellow dots represent localities with novel data from the Balkan peninsula, pink dots represent the Italian locality of Oswaldocruzia sp., for which sequence data were obtained from the GenBank database from Italy, and brown dots represent the Russian localities of Oswaldocruzia spp., for which sequence data were retrieved from GenBank. A = South-eastern European River basins; B = Danube River basin; C = Volga and Ural River basins.
In the text

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