Blood flukes (Digenea: Sanguinicolidae) of common freshwater fishes in Europe: A new genus, two new species, and a morphological and molecular characterisation of Sanguinicola armatus

James Omondi Outa; Olena Kudlai

doi:10.1016/j.crpvbd.2026.100436

. 2026 Sep 12;10:100436. doi: 10.1016/j.crpvbd.2026.100436

Blood flukes (Digenea: Sanguinicolidae) of common freshwater fishes in Europe: A new genus, two new species, and a morphological and molecular characterisation of Sanguinicola armatus

James Omondi Outa ^1,^⁎, Olena Kudlai ¹

PMCID: PMC13636018 PMID: 42835913

Abstract

Fish blood flukes are important parasites that can cause mortalities of their hosts. Yet, blood flukes of freshwater fishes are understudied worldwide. Herein, we provide new information on the family Sanguinicolidae using specimens from freshwater fishes in Lithuania. The sanguinicolids were identified using molecular (nuclear 28S rDNA and mitochondrial cox1 DNA) and morphological data. Within the type-genus Sanguinicola, we provide the first genetic characterisation of the type-species Sanguinicola armatus using newly obtained specimens from the type-host Tinca tinca (Tincidae), report new host and geographical records for Sanguinicola volgensis, and describe a new species Sanguinicola bakeri n. sp. from Blicca bjoerkna (Leuciscidae). Sanguinicola bakeri n. sp. is distinguished from congeneric species by the possession of small lateral testicular lobes, an elongate ovary (occupying 16–18% of the body length) and a narrow oviducal seminal receptacle. Phylogenetic analysis based on 28S rDNA sequences confirmed the distinct status of S. bakeri n. sp. within the genus Sanguinicola. We propose a new genus, Ejsmontellus n. g., comprising Ejsmontellus akicola n. sp. (type-species) infecting B. bjoerkna and unidentified Ejsmontellus sp. from Leuciscus leuciscus (Leuciscidae). Ejsmontellus n. g. is diagnosed by the possession of single columns of ventrolateral tegumental spines, a tubular swelling at the anterior end of the oesophagus, a compact heart-shaped ovary, and the lack of posterior oesophageal swelling. Phylogenetically, Ejsmontellus n. g. is distant from Sanguinicola and closest to a clade comprising cercarial isolates from pulmonate snails from Czechia, Poland and South Africa. The discovery of new sanguinicolids from commonly occurring fishes and availability of DNA sequences of undescribed species show that fish blood flukes remain underexplored.

Keywords: Trematoda, Ejsmontellus n. g., Morphological description, 28S rDNA, cox1 mtDNA, Host range, Lithuania

Graphical abstract

Highlights

•
A new genus of the Sanguinicolidae with two species infecting fishes of the Leuciscidae is erected.
•
A new species of Sanguinicola infecting Blicca bjoerkna (Leuciscidae) is described.
•
First genetic data and supplemental description of the type-species of Sanguinicola, Sanguinicola armatus are provided.
•
Phylogenetic relationships between most of the known sanguinicolids remain unresolved.
•
The diversity of blood flukes in freshwater fishes is currently underestimated.

1. Introduction

Fish blood flukes (Platyhelminthes: Digenea) have two-host life cycles that include gastropods, bivalves or polychaetes as intermediate hosts (Orélis-Ribeiro et al., 2014; Cutmore et al., 2023). Adult blood flukes infect the cardiovascular system of their hosts and are known to cause mortalities of infected fish (Li, 1980; Bullard and Overstreet, 2008; Shimazu, 2013). Blood flukes of fish, especially from freshwater ecosystems, remain underreported worldwide (Zhokhov et al., 2021; Warren et al., 2023). The scarcity of information on freshwater fish blood flukes has been attributed to methodological challenges that are associated with isolating and studying them. The flukes are difficult to detect due to their small size and transparent nature and are easily lost through the breakage of blood vessels during parasitological examination of fish (Bullard and Overstreet, 2008; Shimazu, 2013; Zhokhov et al., 2021). Moreover, they tend to deteriorate rapidly after euthanisation of the host and are difficult to stain, leading to poor-quality specimens and inaccuracies in species diagnoses (Plehn, 1905; Warren et al., 2023).

Blood flukes of freshwater fishes are grouped into two families: Acipensericolidae Warren & Bullard, 2023 and Sanguinicolidae Poche, 1926. The Acipensericolidae is represented by a single genus (Acipensericola Bullard & Overstreet, 2008) whose species infect fishes of the order Acipenseriformes in North America. In contrast, members of the family Sanguinicolidae infect fishes belonging to eight orders (Centrarchiformes, Characiformes, Cypriniformes, Esociformes, Labriformes, Perciformes, Salmoniformes and Siluriformes) and have been reported in Africa, Asia, Australia, Europe, and the Americas (Kirk and Lewis, 1994; Nolan and Cribb, 2005; Truong and Bullard, 2013; Orélis-Ribeiro and Bullard, 2015; Zhokhov et al., 2021; Warren and Bullard, 2023). The Sanguinicolidae consists of seven genera: Sanguinicola Plehn, 1905 (type-genus), Cladocaecum Orélis-Ribeiro & Bullard, 2016, Kritsky Orélis-Ribeiro & Bullard, 2016, Nomasanguinicola Truong & Bullard, 2013, Parasanguinicola Herbert & Shaharom, 1995, Plehniella Szidat, 1951, and Pseudosanguinicola Warren & Bullard, 2023. Knowledge of the phylogenetic relationships of sanguinicolids is currently fragmented due to incomplete morphological descriptions of some species, the lack of new collections of adult worms and a paucity of DNA sequence data. Among the described species, only nuclear 28S rDNA sequences are available for Nomasanguinicola canthoensis Truong & Bullard, 2013, Nomasanguinicola dentata (Paperna, 1964), Pseudosanguinicola occidentalis (Van Cleave & Mueller, 1932), Sanguinicola volgensis (Rašín, 1929), and Sanguinicola plehnae Warren & Bullard, 2023 (Warren et al., 2023; Warren et al., 2024). Additionally, sequences of adult sanguinicolids are available for three unidentified species (Warren and Bullard, 2023). Also, the identities of several sanguinicolid cercarial isolates remain uncertain since they have not been matched to any known adult stages (Olson et al., 2003; Brant et al., 2006; Cutmore et al., 2023; Outa and Avenant-Oldewage, 2024; Kundid et al., 2025).

In Europe, since the discovery of blood flukes in freshwater fishes by Plehn (1905), sanguinicolids have been reported from 26 species of fish representing nine families, i.e. Balitoridae, Cobitidae, Cyprinidae, Esocidae, Leuciscidae, Percidae, Salmonidae, Siluridae and Tincidae (Zhokhov et al., 2021). Until now, only six blood fluke species belonging to the family Sanguinicolidae have been described and reported: Sanguinicola armatus Plehn, 1905; Sanguinicola inermis Plehn, 1905; Sanguinicola intermedius Ejsmont, 1926; S. plehnae; Sanguinicola rutili Simon-Martın, Rojo-Vasquez & Simon-Vicente, 1987; and S. volgensis (Rašín, 1929). However, taxonomic uncertainties have been raised regarding Sanguinicola since the genetic relationships of its members remain largely unknown (Orélis-Ribeiro and Bullard, 2016; Warren et al., 2023). In fact, aside from 28S rDNA sequences of S. volgensis and S. plehnae, genetic data are lacking for the other recognised species, including the type-species S. armatus.

The present study reports on sanguinicolids from Lithuania, based on morphological descriptions and analyses of partial sequences of the nuclear 28S rRNA and mitochondrial cox1 genes. In addition to three previously reported Sanguinicola spp. in freshwater fishes in Lithuania, S. armatus, S. inermis and S. intermedius (Krotas, 1968; Rauckis, 1988), we describe a new species, report the first record of S. volgensis, and supplement the known descriptions of S. armatus with additional details on the morphology and morphometric data. We further propose a new genus to accommodate two additional species recorded in this study. We discuss the host range and distribution of freshwater fish blood flukes and present phylogenetic analyses of the interrelationships between the European sanguinicolids and other members of the family Sanguinicolidae.

2. Materials and methods

2.1. Fish sampling and isolation of blood flukes

Fishes were sampled from 10 waterbodies in Lithuania between May 2022 and July 2026 (Fig. 1) under permits obtained from the Environment Protection Agency, Lithuania (license Nos.: 018 and 019 valid for 2022; 016 and 017 valid for 2023; 013, 025, and 032 valid for 2024, 007 valid for 2025 and 010 valid for 2026). Fishes were transported alive to a laboratory aquarium where they were identified based on the key by Kottelat and Freyhof (2007), and their taxonomic status was confirmed on FishBase (Froese and Pauly, 2025). A total of 190 fish specimens representing 6 species were examined. All procedures were performed following the guidelines outlined by the Council for International Organizations of Medical Sciences (CIOMS) and the International Council for Laboratory Animal Science (ICLAS) (CIOMS and ICLAS, 2012). Euthanisation, dissection and examination of fish organs (e.g. heart, gills, liver, kidney, mesenteries, and eyes) under a dissecting microscope followed standard procedures (Cribb and Bray, 2010; Cutmore et al., 2025). To maximise detection of worms that might be lost through breakage of blood vessels during dissection, saline used for washing the body cavity and cranial cavity was examined following Snyder and Clopton (2005). When detected, blood flukes were isolated, rinsed, fixed in hot saline and preserved in 80% ethanol.

2.2. Morphological analyses

Whole worms and specimens whose anterior part had been excised for DNA extraction (hologenophore sensu Pleijel et al., 2008) were prepared for morphological analyses. The specimens were stained in acetocarmine or Mayer’s hydrochloric carmine, destained in acid ethanol, dehydrated in a graded ethanol series, cleared in methyl salicylate and mounted in dammar gum. Morphology of the specimens was studied, and drawings were made on a compound Olympus BX51 microscope with the aid of a drawing tube. Measurements were made using an Olympus BX53 light microscope connected to ZEISS Axiocam camera and Labscope 4.4.0 imaging software. Morphometric measurements are in micrometres (μm), and where applicable, provided as the range followed by the mean in parentheses. The length-by-width measurements are indicated by “×”. The type-specimens, hologenophores and voucher specimens have been deposited in the Platyhelminthes Collection of the Natural History Museum, Geneva, Switzerland (MHNG-PLAT) under the accession numbers MHNG-PLAT-160749-MHNG-PLAT-160759.

2.3. Molecular analyses

2.3.1. DNA extraction, amplification and sequencing

Genomic DNA was isolated from whole specimens (photogenophores sensu Andrade-Gómez and García-Varela, 2021) or hologenophores using the KAPA Express Extract Kit (KAPA Biosystems, Cape Town, South Africa) following the manufacturer’s protocol. Partial sequences of the nuclear 28S rRNA gene (domains D1-D3) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) were amplified. The 28S rRNA gene was amplified using the primers ZX-1 (Scholz et al., 2013) and 1500R (Tkach et al., 2003), with the following PCR amplification profile: denaturation for 3 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C, with a final extension step for 10 min at 72 °C.

Partial fragments of the cox1 gene were amplified using primer combinations Dice1F and Dice14R (Van Steenkiste et al., 2015), with the following PCR amplification profile: denaturation for 3 min at 94 °C, followed by 40 cycles of 40 s at 94 °C, 40 s at 51 °C, 60 s at 72 °C, with a final extension step for 10 min at 72 °C; or Dig_cox1Fa and Dig_cox1R (Wee et al., 2017) and PCR amplification profile: denaturation for 3 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 50 °C, 30 s at 72 °C, with a final extension step for 10 min at 72 °C. Amplified DNA was purified with ExoSAP-IT PCR Cleanup enzymatic kit (ThermoFisher Scientific, Inc., Waltham, MA, USA). Sanger sequencing was completed at Macrogen Europe (Amsterdam, Netherlands) using the same primers as for the PCR amplification. The newly obtained sequences were assembled and edited using Geneious Prime software v.2026.0.2 (Biomatters, Auckland, New Zealand). Selected novel sequences were deposited in the GenBank database under the accession numbers PZ765040-PZ765051 and PZ770260 (28S rRNA gene) and PZ766176-PZ766202 (cox1 gene).

2.3.2. Phylogenetic analyses and species delimitation

Of the 34 newly generated partial 28S rDNA sequences, those that were either non-identical or identical but originating from different fish host species were aligned using MAFFT implemented in Geneious Prime, with selected representative sequences from GenBank, particularly those belonging to the family Sanguinicolidae and those within Lineage II of Cutmore et al. (2023). Sequences of Chimaerohemecus trondheimensis van der Land, 1967 and Hyperandrotrema walterboegeri Orélis-Ribeiro & Bullard, 2013 (Chimaerohemecidae) were used as the outgroup in the 28S rDNA analysis based on the topology in the phylogenetic trees of Cutmore et al. (2023) and Warren et al. (2023). The final dataset was used to estimate the best-fitting nucleotide substitution model by applying the Akaike information criterion (AIC) in jModelTest v.2.1.2 (Darriba et al., 2012). Accordingly, the general time-reversible model incorporating invariant sites and gamma-distributed among-site rate variations (GTR+I+G) was used. Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted using MrBayes software v.3.2.3 (Ronquist et al., 2012) and PhyML v.3.0 (Guindon et al., 2010), respectively, run on the ATGC bioinformatic platform (http://www.atgc-montpellier.fr/phyml/). Bayesian inference analysis was run over 10,000,000 generations. The “burn-in” was set to the first 25% of trees sampled, and consensus topology and nodal support were estimated from the remaining 75% of the trees. Nodal support for the ML analysis was generated by performing 100 bootstrap pseudoreplicates. Tracer v.1.6 (Rambaut et al., 2014) was used to ensure that effective sample size values for all parameters were above 200. Tree topologies were visualised using FigTree v.1.4 software (Rambaut, 2012). In addition, species delimitation analysis was performed with the Bayesian version of the Poisson Tree Processes model (bPTP) (Zhang et al., 2013) using the 28S rDNA tree estimated via BI analysis. This analysis was performed using the bPTP web server (Zhang et al., 2013; available at http://species.h-its.org/ptp) with default parameters.

Newly generated cox1 (n = 27) sequences were aligned with a single previously published sanguinicolid sequence (Sanguinicola sp.). The closest blood fluke species Neospirorchis chapmanae Corner, Cribb & Cutmore, 2023 (Unicaecidae), with a sequence available in GenBank, was used as the outgroup. Alignment was built using MAFFT implemented in Geneious Prime, with reference to the amino acid translation under the trematode mitochondrial code (translation table 21; https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi#SG21) (Garey and Wolstenholme, 1989; Ohama et al., 1990). Neighbour-joining analysis based on Kimura-2-parameter distances was conducted on the cox1 dataset in MEGA 11 (Tamura et al., 2021) to determine species identities; nodal support was estimated from 1000 bootstrap replicates. Pairwise genetic distances (uncorrected p-distance model and number of differences) were calculated in MEGA 11 (Tamura et al., 2021) using the following conditions: “Variance estimation method = none”, “Model/Method = no. of differences” or “p-distance”, and “Substitutions to include = d: transitions + transversions” and “Gaps/Missing data treatment = pairwise deletion”.

3. Results

3.1. Infection patterns

Overall, 20.5% of the 190 examined fish were infected with blood flukes. Both prevalence and intensity showed variations between fish species and sampling sites. The highest prevalence and intensity were recorded in Tinca tinca (L.) (Tincidae) from the Curonian Lagoon (Table 1). Five species were identified based on morphological and/or molecular data: S. armatus, S. volgensis, two new species (Sanguinicola bakeri n. sp. and Ejsmontellus akicola n. g., n. sp.) and an undescribed species, Ejsmontellus sp. Tinca tinca was infected with one species, S. armatus, which was recovered in the heart, bulbus arteriosus, gills and body cavity wash (Table 2). Infections with S. armatus were also recorded in the heart and gills of Carassius gibelio (Bloch) (Cyprinidae), albeit at lower intensity compared with T. tinca (Table 2). Sanguinicola volgensis was recovered at low prevalence and intensity in the heart, gills and cranial cavity wash of Alburnoides bipunctatus (Bloch), Leuciscus leuciscus (L.) and Rutilus rutilus (L.), respectively, all belonging to the Leuciscidae. Leuciscus leuciscus was also infected in the heart and gills with an additional species, Ejsmontellus sp. The leuciscid Blicca bjoerkna (L.) was infected with Sanguinicola bakeri n. sp. and Ejsmontellus akicola n. sp. Sanguinicola bakeri n. sp. was recovered predominantly from the bulbus arteriosus and body cavity wash. Ejsmontellus akicola n. sp. was recorded only in the eye vitreous humour (Table 2). Co-infections of B. bjoerkna with the two species were detected in 9 (47.4%) of the infected fish specimens. Data on prevalence and intensities of individual blood fluke species in the organs of different fish species are provided in Table 2.

Table 1.

Summary data for fish species collected from different localities in Lithuania and examined (n) for blood flukes, with data on infection rates. Prevalence was not calculated for small sample sizes (n < 10).

Fish species	Water body, locality	Coordinates	n	Length (cm)	Weight (g)	Prevalence	Intensity^a
Alburnoides bipunctatus	River Merkys, Trasninkas	54.107813°N, 24.276172°E	1	12	14	–	–
	River Neris, Skirgiškės	54.831178°N, 25.372453°E	11	10–12 (11)	6–12 (10)	–	–
	River Žeimena, Pažeimenė	55.028723°N, 25.847220°E	23	9–11 (10)	4–12 (7)	4.4%	1
Blicca bjoerkna	Curonian Lagoon, Ventė	55.343487°N, 21.174823°E	34	13–33 (19)	23–151 (88)	55.9%	1–17 (5.4)
	Kaunas Reservoir, Lašiniai	54.841848°N, 24.200916°E	2	15–24	48–158	–	–
	River Neris, Paliepė	54.863363°N, 25.745182°E	1	10	14	–	–
Leuciscus leuciscus	River Grūda, Puvočiai	54.119233°N, 24.304610°E	1	20	64	–	–
	River Merkys, Trasninkas	54.107813°N, 24.276172°E	3	11–16	32	1 out of 3	4
	River Neris, Paliepė	54.863363°N, 25.745182°E	5	14–16 (15)	26–36 (31)	1 out of 5	3
	River Neris, Skirgiškės	54.837704°N, 25.375939°E	1	18	50	1 out of 1	1
Rutilus rutilus	Curonian Lagoon, Ventė	55.346735°N, 21.175857°E	7	20–32 (25)	168–481 (231)	1 out of 7	1
	Kaunas Reservoir, Lašiniai	54.803688°N, 24.206013°E	4	15–24 (19)	46–129 (87)	–	–
	River Nemunas, Lipliunai	53.987219°N, 23.914091°E	2	18–24	60–159	–	–
	River Nemunas, Vilkija	55.031036°N, 23.587498°E	10	11–23 (18)	13–139 (76)	–	–
	River Neris, Paliepė	54.863363°N, 25.745182°E	17	13–18 (15)	14–58 (35)	–	–
	River Merkys, Trasninkas	54.107813°N, 24.276172°E	3	15–19	42–68	–	–
	Lake Dusia, Metelyte	54.328986°N, 23.671156°E	2	19–22	61–102	–	–
Carassius gibelio	Curonian Lagoon, Ventė	55.343487°N, 21.174823°E	25	17–33 (23)	61–500 (252)	12.0%	1–2 (1.7)
Tinca tinca	Curonian lagoon, Ventė	55.343487°N, 21.174823°E	14	19–39 (28)	98–962 (378)	71.4%	1–167 (26.0)
	Lake Druksiai, Raipolė	55.649338°N, 26.583776°E	1	35	511	–	–
	Lake Dusia, Metelyte	54.328986°N, 23.671156°E	6	18–26 (22)	78–311 (177)	1 out of 6	1
	Lake Maušelis, Statiškė	54.269453°N, 23.828898°E	6	31–64 (41)	442–848 (674)	–	–
	River Nemunas, Lipliunai	53.987219°N, 23.914091°E	2	19–24	100–275	–	–
	River Nemunas, Vilkija	55.029848°N, 23.588790°E	1	15	56	–	–
	River Neris, Paliepė	54.863363°N, 25.745182°E	8	9–28 (17)	11–458 (106)	1 out of 8	1

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Intensity range (Mean intensity).

Table 2.

Prevalence (%) and intensity range (in parentheses) of sanguinicolids recovered from organs and the body and cranial cavity washes of the fish species examined.

Species	Host	Heart	Bulbus arteriosus	Gills	Eyes	Body cavity wash	Cranial cavity wash
Sanguinicola armatus	Carassius gibelio	20 (1–2)	–	4 (2)	–	–	–
Sanguinicola armatus	Tinca tinca	23.1 (1–15)	15.4 (1–115)	5.1 (1–10)	–	12.8 (1–25)	–
Sanguinicola volgensis	Alburnoides bipunctatus	2.9 (1)	–	–	–	–	–
	Leuciscus leuciscus	–	–	10 (1)	–	–	–
	Rutilus rutilus	–	–	–	–	–	2.2 (1)
Sanguinicola bakeri n. sp.	Blicca bjoerkna	8.1 (1–2)	22.2 (1–3)	2.8 (1)	–	16.2 (1–2)	8.3 (1)
Ejsmontellus akicola n. sp.	B. bjoerkna	–	–	–	37.8 (1–16)	–	–
Ejsmontellus sp.	L. leuciscus	10 (4)	–	10 (3)	–	–	–

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Here, we provide the definition of a new sanguinicolid genus, Ejsmontellus, and the descriptions of Ejsmontellus akicola n. sp., Sanguinicola bakeri n. sp., and Sanguinicola armatus. Morphological description of S. volgensis recorded in the present study is not provided since the newly recorded specimens agree well morphologically with the existing descriptions of the species by Rašín (1929) and Warren et al. (2023). The newly generated 28S rDNA and cox1 sequences of S. volgensis were included in the phylogenetic analyses (Section 3.6).

3.2. Ejsmontellus n. g

Diagnosis: Digenea: Sanguinicolidae. Body dorsoventrally flattened, tapered more posteriorly than anteriorly. Tegument spinose. Tegumental spines straight, deeply rooted in tegument, oriented mostly posterolaterally, arranged in single ventrolateral columns from anterior extremity to level of terminal genitalia. Mouth subterminal. Pharynx absent. Oesophagus straight or slightly sinuous; tubular swelling at anterior end present, posterior oesophageal swelling absent. Intestine median, X-shaped, comprising 4–5 irregular, dendritic caeca. Lateral nerve cords present, nerve commissure indistinct. Testis single, postcaecal, comprising prominent central column flanked by two tandem rows of lobes. Vas deferens median, straight. Cirrus-sac elongate, slightly curved. Seminal vesicle tubular. Cirrus tubular, aspinous. Male genital pore sinistral, opens dorsally. Ovary single, median, post-testicular, compact, with irregular margins, heart-shaped. Oviducal seminal receptacle present. Oötype median, oblong, posterior to uterus. Uterus oblong. Metraterm absent. Mehlis’ gland absent. Female genital pore median, anterior to male genital pore, opens dorsally. Vitellarium follicular, extending from level of anterior oesophageal swelling to level of terminal genitalia. Excretory vesicle Y-shaped. Parasitic in leuciscids.

Type-species: Ejsmontellus akicola n. sp.

Etymology: The genus, with the Latin diminutive suffix ‘ellus’ is named in honor of Leopold Ejsmont (Warsaw, Poland) in recognition of his contribution to the study of the Sanguinicolidae.

ZooBank registration: LSIDurn:lsid:zoobank.org:act:E6AD4DE2-B552-41F4-8A00-3AA57C609146.

Differential diagnosis: The present specimens of Ejsmontellus conform to the diagnosis of the family Sanguinicolidae by Warren and Bullard (2023) in having an X-shaped intestine with short radial blind caeca, a single testis comprising laterally directed lobes, a post-testicular median ovary, genital pores opening separately dorsally, presence of an oviducal seminal receptacle, a single primary vitelline duct connecting with the oviduct proximal to the oötype, and a diffuse vitellarium. It resembles members of Sanguinicola (sensu stricto) and the monotypic Parasanguinicola in having straight, deeply rooted lateral tegumental spines, arranged in single ventrolateral columns. However, Ejsmontellus n. g. can be differentiated from Sanguinicola in the shape and position of the anterior oesophageal swelling (tubular, at the anterior extremity vs fusiform, at some distance from the anterior extremity), the lack of posterior oesophageal swelling, and the shape of the ovary (compact, heart-shaped vs bi-lobed, butterfly-wing-shaped). Ejsmontellus n. g. differs from Parasanguinicola in the shape and position of the anterior oesophageal swelling (tubular, at the anterior extremity vs fusiform, at some distance from the anterior extremity), the lack of posterior oesophageal swelling, the presence of lateral testicular lobes, the shape of the ovary (compact, heart-shaped vs bi-lobed, butterfly-wing-shaped) and the shorter uterus (4–5% vs 33% of body length).

The new genus differs from the monotypic Pseudosanguinicola in the nature and arrangement of the lateral tegumental spines (single column vs densely compacted transverse rows), the lack of an anterior sucker, the longer oesophagus (28–30% vs 24% of body length), the shape and position of the anterior oesophageal swelling (tubular, at the anterior extremity vs fusiform, at some distance from the anterior extremity), the lack of posterior oesophageal swelling and the shape of the ovary (compact, heart-shaped vs bi-lobed, butterfly-wing-shaped). Ejsmontellus n. g. differs from the monotypic Cladocaecum in the presence of lateral tegumental spines, the lack of pharynx, the position of the anterior oesophageal swelling (at the anterior extremity vs at some distance from the anterior extremity), the lack of posterior oesophageal swelling, the morphology and length of intestine (X-shaped with 4–5 short caeca in anterior half of body vs highly branched comprising long central cecum with numerous laterally directed diverticula that terminate in posterior quarter of body), the position of the testis in relation to intestine (post-caecal vs dorsal to intestine), and the shape of the uterus (oblong, compact vs inverse U-shaped, with ascending and descending portions). The new genus differs from the monotypic Kritsky in having lateral tegumental spines and lacking a pharynx and posterior oesophageal swelling, in the shape and position of the anterior oesophageal swelling (tubular, at the anterior extremity vs fusiform, at some distance from the anterior extremity), the fewer intestinal caeca (4–5 vs 6), and the shape of the ovary (compact, heart-shaped vs bi-lobed, butterfly-wing-shaped with finger-like lateral projections). Ejsmontellus n. g. is distinguished from Nomasanguinicola in the presence of lateral tegumental spines, the lack of an anterior sucker, the shape and position of the anterior oesophageal swelling (tubular, at the anterior extremity vs fusiform, at some distance from the anterior extremity), the lack of posterior oesophageal swelling, the shape of the ovary (compact, heart-shaped vs bi-lobed, butterfly-wing-shaped), the lack of Mehlis’ gland, and the shape of the uterus (oblong, compact vs inverse U-shaped, with ascending and descending portions). The new genus differs from Plehniella in having lateral tegumental spines, the lack of an anterior sucker and pharynx, the position of the anterior oesophageal swelling (at the anterior extremity vs some distance from the anterior extremity), the lack of posterior oesophageal swelling, fewer caeca (4–5 vs 6), the shape of the ovary (compact, heart-shaped vs butterfly-wing-shaped with deeply lobed margins), the shape of the uterus (oblong, compact vs inverse U-shaped, with ascending and descending portions) and in having separate genital pores.

3.3. Ejsmontellus akicola n. sp.

3.3.1. Taxonomic summary

Type-host: Blicca bjoerkna (L.) (Cypriniformes: Leuciscidae).

Type-locality: Curonian Lagoon (55.343487°N, 21.174823°E), Ventė, Lithuania.

Type-material: Holotype: MHNG-PLAT-160749. Paratypes: MHNG-PLAT-160750 (3 whole-mounted). Hologenophore: MHNG-PLAT-160751 (1 mounted).

Site in host: Eye vitreous humour.

Representative DNA sequences: 2 out of 9 generated 28S rDNA sequences were unique and therefore only these were deposited in GenBank (PZ765048 and PZ765049). The cox1 sequences were deposited under the accession numbers PZ766192-PZ766200.

ZooBank registration: To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species and the new genus have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:59C77811-B829-4A3C-8512-938A6127D0DA. The LSIDs for the new names are as follows: Ejsmontellus n. g. (urn:lsid:zoobank.org:act:E6AD4DE2-B552-41F4-8A00-3AA57C609146); and Ejsmontellus akicola n. sp. (urn:lsid:zoobank.org:act:7BCB22F6-317F-46E7-8B4A-FDA7D2E65F42).

Etymology: The specific epithet refers to the site of infection: derived by combining ‘akis’, meaning ‘eye’ in Lithuanian and the Latin suffix ‘cola’, meaning ‘inhabitant of’.

3.3.2. Description

[Based on 7 fixed adult specimens; measurements of the holotype are given in the text; measurements of the entire series are provided in Table 3; Fig. 2]. Body flat, oblanceolate, 678 × 125, widest at level of testis, 5.4 times longer than maximum width, tapering unequally; post-ovarian field significantly narrower than preceding body part, body margins appear crenate. Tegument spined. Tegumental spines straight, deeply rooted in tegument, encased in tegumental protrusions or projecting slightly beyond tegumental surface; arranged in single ventrolateral columns, 37 per column, starting at short distance (12) from anterior extremity, reaching level of midpoint between ovary and uterus, 153 from posterior end; arranged more densely in anterior body, less dense posteriorly. Anterior body spines small, 5.3–8.8 × 0.9–1.4 (7.2 × 1.3), 5.0–6.5 (5.8) times longer than wide; mid-body spines (from posterior of anterior oesophageal swelling to level of ovary), 11–15 × 1.2–1.8 (13 × 1.5), 7.7–12.0 (9.0) times longer than wide; posteriormost spines, 12–14 × 1.3–1.5 (13 × 1.4), 9–10 (9.0) times longer than wide. Tegumental protrusions in anterior body small, about 2 times smaller than in mid- and posterior body part. Lateral nerve cords extend posteriorly, nerve commissure indistinct. Mouth subterminal; pharynx absent. Oesophagus 190 long, 28% of body length; with tubular anterior swelling, located short distance (7) from anterior extremity of body, 38 × 6, 20% of oesophageal length; subsequent part of oesophagus narrow (2), widens slightly (3) at the junction with intestine. Intestine 42 × 38, 6% of body length, 30% of body width, median, X-shaped, comprising 5 (4 in paratypes) unequal, dendritic caeca; 3 anterior caeca (2 large, 23 × 13, 21 × 11 and 1 small appearing secondarily formed, 8 × 5); 2 posterior caeca (13 × 10, 12 × 6).

Table 3.

Morphometric data for Sanguinicola armatus, Sanguinicola bakeri n. sp. and Ejsmontellus akicola n. sp. Measurements are given as the range followed by the mean in parentheses.

Species	Sanguinicola armatus	Sanguinicola bakeri n. sp.	Ejsmontellus akicola n. sp.
Host	Tinca tinca, Carassius gibelio	Blicca bjoerkna	Blicca bjoerkna
Character	Range (n = 12)	Range (n = 9)	Range (n = 7)
Body	601–863 × 98–181 (718 × 133)	613–1073 × 100–175 (807 × 138)	576–706 × 86–125 (660 × 103)
BL/BW	4.7–6.6 (5.4)	5.0–6.6 (5.7)	5.4–7.1 (6.5)
Total number of lateral tegumental spines	158–174 (164)	172–200 (185)	70–76 (72)
Distance from anterior extremity to 1st spine	8–12 (10)	8–14 (11)	12–17 (14)
Distance from last spine to posterior extremity	60–112 (86)	56–90 (72)	138–165 (149)
Length of lateral column of spines/BL (%)	85–89 (86)	89–91 (90)	73–78 (76)
Anterior body spines	9–12 × 1.2–2.2 (11 × 1.5)	9.3–34 × 0.9–2.5 (17 × 1.4)	5.3–8.8 × 0.9–1.4 (7.2 × 1.3)
Mid-body spines	9.8–15 × 0.9–1.9 (12 × 1.4)	21–39 × 1.1–3.2 (29 × 2.2)	11–15 × 1.2–1.8 (13 × 1.5)
Posterior body spines	10–14 × 0.9–1.7 (12 × 1.2)	11.4–34 × 1.1–3.1 (23 × 2)	12–14 × 1.3–1.5 (13 × 1.4)
Distance from anterior extremity to NC	95–127 (104)	103–146 (117)	–
Distance from anterior extremity to NC/BL (%)	13–17 (15)	12–15 (14)	–
Oesophagus length	184–264 (222)	209–303 (244)	181–212 (199)
Oesophagus length/BL (%)	28–35 (31)	28–31 (29)	28–32 (30)
Distance from anterior extremity to AOS	42–53 (47)	34–48 (41)	6.5–7.2 (6.8)
AOS	24–37 × 7–12 (31 × 10)	19–28 × 8–10 (23 × 9.6)	31–57 × 5–7 (43 × 6)
AOS length/oesophagus length (%)	13–16 (15)	9–11 (10)	17–27 (22)
POS	22–36 × 7–20 (27 × 14)	17–28 × 7–11 (23 × 9.2)	–
POS length/oesophagus length (%)	10–15 (12)	8–11 (9)	–
Intestine	46–75 × 39–66 (61 × 50)	44–65 × 36–45 (51 × 42)	30–42 × 27–48 (36 × 35)
Intestine length/BL (%)	7–11 (8.8)	5–8 (6.3)	4–6 (5.3)
Intestine width/BW (%)	33–46 (38)	26–38 (28)	23–43 (32)
Anterior caeca	10–25 × 6–16 (18 × 10)	11–23 × 6–18 (19 × 13)	17–23 × 11–17 (21 × 14)
Posterior caeca	13–43 × 6–29 (25 × 17)	20–38 × 9–25 (26 × 16)	12–17 × 6–13 (15 × 10)
Testis	125–230 × 54–89 (177 × 66)	215–378 × 39–78 (279 × 56)	120–238 × 41–68 (190 × 50)
Testis length/BL (%)	21–27 (25)	30–35 (33)	21–34 (29)
Testis width/BW (%)	40–59 (50)	33–48 (38)	38–54 (48)
Number of testicular lobes	8–12 pairs	16–19 pairs	14–17 pairs
CTC width	14–22 (27)	11–17 (13)	12–28 (18)
CTC width/testis width (%)	21–33 (27)	19–34 (24)	27–41 (35)
Post-testicular field length	218–310 (252)	197–322 (259)	211–250 (232)
Post-testicular field length/BL (%)	33–37 (35)	28–34 (30)	33–37 (34)
Cirrus-sac	88–125 × 16–25 (109 × 22)	83–101 × 17–29 (95 × 22)	74–86 × 8–17 (82 × 13)
Seminal vesicle	88–125 × 11–16 (109 × 14)	83–99 × 6–12 (94 × 9)	74–86 × 4.2–12 (82 × 8.3)
Ovary	57–99 × 43–88 (76 × 63)	114–175 × 53–92 (142 × 73)	52–65 × 34–55 (58 × 47)
Ovary length/BL (%)	10–12 (11)	16–18 (17)	7.6–9.2 (8.6)
Ovary width/BW (%)	42–59 (48)	47–52 (50)	38–55 (44)
Post-ovarian field length	151–195 (172)	86–158 (124)	152–189 (166)
Post-ovarian field length/BL (%)	21–27 (24)	12–16 (14)	22–28 (25)
Oviducal seminal receptacle	101–138 × 16–28 (121 × 22)	51–89 × 6–10 (67 × 8)	54–92 × 10–15 (71 × 12)
Oötype	21–27 × 16–24 (23 × 19)	23–32 × 17–25 (28 × 22)	25–29 × 11–15 (26 × 13)
Thickness of oötype wall	3–5 (4)	5–9 (6)	4–6 (5)
Uterus	16–24 × 13–22 (20 × 17)	20–26 × 17–22 (23 × 19)	27–37 × 9–14 (31 × 12)
Thickness of uterus wall	3–4 (3)	4–6 (5)	3–5 (4)
Uterus length/oötype length	0.7–1 (0.9)	0.7–0.9 (0.8)	1.1–1.3 (1.2)

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Abbreviations: AOS, anterior oesophageal swelling; BL, body length; BW, body width; CTC, central testicular column; NC, nerve commissure; POS, posterior, oesophageal swelling.

Fig. 2 — *Ejsmontellus akicola* n. sp. ex *Blicca bjoerkna*. A Holotype, whole specimen (MHNG-PLAT-160749), dorsal view. B Terminal genitalia of the holotype, dorsal view. *Abbreviations*: c, cirrus; cs, cirrus-sac; fgp, female genital pore; mgp, male genital pore; oö, oötype; o, ovary; osr, oviducal seminal receptacle; ov, oviduct; sv, seminal vesicle; u, uterus; vd, vas deferens; vit, vitelline duct.

Testis single, 208 × 68, 3 times longer than wide, 31% of body length, 54% of body width, postcaecal, median, occupying much of middle body; comprising central column, 28 wide, 41% of testis width, flanked by two tandem rows of irregular lateral lobes, 14 pairs measuring 8–23 × 9–25 (15 × 18). Post-testicular space 250 long, 37% of body length. Vas deferens, 58 × 3, at least 8 times narrower than central testicular column, straight, emanates from posterior margin of central column, enters cirrus-sac near posterior to ovary. Cirrus-sac lanceolate, thin-walled, slightly curved distally, oriented anteroposteriorly, 85 × 17, 5 times longer than wide. Seminal vesicle tubular, 12 wide, occupying entire length of cirrus-sac. Male genital pore sinistral, at level of uterus-oötype junction, opens dorsally.

Ovary single, compact, heart-shaped, with irregular margins, median, small, 60 × 55, 8.8% of body length, 44% of body width, post-testicular. Post-ovarian field, 160 long (24% of body length), at least 2 times narrower than preceding part. Oviduct emanates medially from ovary, passes posteriorly, extends sinistrally along posterior margin of ovary, curves sharply before entering oviducal seminal receptacle. Oviducal seminal receptacle lanceolate, 92 × 13, 7 times longer than wide, thin-walled (1), oriented anteroposteriorly, slightly curved dextrally, narrowing at level of uterus, unites with vitelline duct before entering oötype posteriorly. Oötype median, oblong, 29 × 13, oötype wall 4 thick. Uterus anterior to oötype, slightly longer than oötype, oblong, 34 × 11, uterus wall 5 thick. Female genital pore median, anterior to male genital pore, opens dorsally. Vitellarium follicular, extends from anterior extremity to posterior to ovary.

Excretory vesicle Y-shaped; excretory pore subterminal.

3.3.3. Remarks

See the differential diagnosis of the new genus above.

3.4. Sanguinicola armatus Plehn, 1905

3.4.1. Taxonomic summary

Hosts: Tinca tinca (L.) (Cypriniformes: Tincidae) (type-host) and Carassius gibelio (Bloch) (Cypriniformes: Cyprinidae).

Localities: Curonian Lagoon, Ventė (55.343487°N, 21.174823°E), Lake Dusia (54.328986°N, 23.671156°E) and River Neris, Paliepė (54.863363°N, 25.745182°E), Lithuania.

Voucher material: 8 whole-mounted specimens ex T. tinca (MHNG-PLAT-160753; MHNG-PLAT-160754) and 1 whole-mounted specimen ex C. gibelio (MHNG-PLAT-160752).

Site in host: Recovered from the heart, bulbus arteriosus, gills and body cavity wash.

Representative DNA sequences: 3 out of 11 generated 28S rDNA sequences were unique and therefore only these were deposited in GenBank (PZ765040-PZ765042). The cox1 sequences were deposited under the accession numbers PZ766176-PZ766183.

3.4.2. Description

[Based on 12 mounted adult specimens; measurements are provided in Table 3; Fig. 3A and B]. Body flat, lanceolate, widest at level just anterior to testis. Anterior extremity with proboscis-like tip, more prominent in live specimens. Posterior extremity bluntly rounded. Tegument spined. Tegumental spines straight, deeply rooted in tegument, oriented mostly posterolaterally, arranged in single ventrolateral columns starting at short distance from anterior extremity, reaching level of oötype posteriorly, 79–89 (84) per column, not projecting beyond tegumental surface; arranged more densely in anterior body, less dense posteriorly. Nerve commissure in anterior fifth of body, lateral nerve cords extend from anterior extremity to posterior of terminal genitalia. Mouth subterminal, possessing two tegumental elevations into mouth cavity. Pharynx absent. Oesophagus straight, with 2 fusiform swellings; first anterior to nerve commissure, second at posterior end of oesophagus. Intestine median, located in anterior half of body, comprising 5 unequal radial caeca (2 anterior and 3 posterior), with posterior medial cecum larger than others.

Testis single, postcaecal, occupying much of middle body, comprising central column flanked by two tandem rows of irregularly shaped lobes, 8–12 pairs, slightly longer than wide. Vas deferens emanates from posterior margin of central testicular column, extends sinistrally near posterior margin of ovary, enters cirrus-sac. Cirrus-sac S-shaped, oriented anteroposteriorly, curving dextrally towards midline, then sinistrally; cirrus-sac wall thin, indistinct in some specimens. Seminal vesicle tubular. Male genital pore sinistral, opens dorsally.

Ovary bi-lobed, butterfly-wing-shaped, with irregular margins, median, anterior third encompassing posterior part of testis. Oviduct appears to join oviducal seminal receptacle at posterior end of ovary; receptacle oriented anteroposteriorly, extending dextrally posteriorly, narrowing at level of uterus, unites with vitelline duct before entering oötype posteriorly. Oötype median, subtriangular to oval, slightly larger than uterus. Uterus anterior to oötype, ovoid. Female genital pore median. Vitellarium follicular, extending from anterior of nerve commissure to posterior margin of ovary.

Excretory vesicle V-shaped; excretory pore subterminal.

3.4.3. Remarks

Sanguinicola armatus was described as S. armata from T. tinca in Germany (Plehn, 1905). Although Plehn (1905) treated Sanguinicola as feminine, the Latin suffix -cola (lives in) is neither feminine nor variable, but masculine. Hence, we concur with Yamaguti (1971) and Nolan and Cribb (2005), who noted that the specific epithet ‘armata’ is grammatically incongruent with the genus.

The original description of S. armatus was largely incomplete, including only information on body size and the presence of a proboscis-like anterior extremity tip, tegumental spines, and body parenchyma. To the best of our knowledge, there is no type-material available. The only detailed description of the species with its illustrations was provided by Ejsmont (1926) using specimens collected from the type-host in Poland. The morphology of the newly-collected material of S. armatus in the present study agrees well with the description of Ejsmont (1926) particularly in the presence of the proboscis-like anterior tip, the shape and distribution of tegumental spines, and the possession of oesophagus with two fusiform swellings, intestine with five radial caeca, testis with lateral paired testicular lobes, cirrus-sac making a dextral and sinistral curve posteriorly, bi-lobed butterfly-wing-shaped ovary encompassing posterior part of the testis, triangular shaped thick-walled oötype, uterus that is smaller than oötype, sinistral male genital pore and median female genital pore.

The present specimens differ from the material described by Ejsmont (1926) in the minima and maxima of body dimensions (601–863 × 98–181 vs 720–1350 × 160–230) and the number of tegumental spines (79–89 vs 60–100 per column). Plehn (1905) recorded a body length of 1500 based on a single observation and did not provide the number of tegumental spines. Ejsmont (1926) attributed the large variations in body size and the number of tegumental spines to differences in the growth stages of the specimens. Also, it appears that the body dimensions provided by Plehn (1905) and Ejsmont (1926) were from live specimens, while the present measurements were taken from specimens that had been preserved in 80% ethanol, which might explain the differences. Although the oviducal seminal receptacle was not mentioned in the previous works, illustrations by Ejsmont (1926) showed an enlargement of the oviduct, which corresponds to the oviducal seminal receptacle. Overall, the present specimens conform well to the description of S. armatus. The identification is further supported by the fact that they were obtained from the type-host (except for a few specimens from C. gibelio).

Sanguinicola armatus is distinguished from the congeneric species considered valid (Warren et al., 2023) by a combination of morphological features. Compared with the species that have been reported in Europe, it resembles S. intermedius and S. volgensis, whose marginal spines do not project beyond the tegumental surface. However, S. armatus differs from S. intermedius in the lack of tegumental setae and intestine morphology (comprising 5 non-dendritic caeca vs 4–5 dendritic caeca, with the 5th cecum secondarily formed). It differs from S. volgensis in intestine morphology (comprising 5 non-dendritic caeca vs 4–5 dendritic caeca), in having a smaller testis (21–27% vs 29–35% of body length), and in the arrangement of the lateral testicular lobes (paired vs non-paired), the size and position of the ovary in relation to the testis (encompassing posterior part of the testis vs immediately post-testicular) and uterus length (shorter than oötype vs longer than oötype). Sanguinicola armatus is distinguished from S. plehnae by the number and nature of the tegumental spines (60–100 per column, not projected beyond tegumental surface vs 118–122 per column, distal part projecting beyond tegumental surface), the number of intestinal caeca (5 vs 4), in having a smaller testis (21–27% vs 41% of body length), longer post-testicular field (33–39% vs 26% of body length) and in the position of the ovary in relation to the testis (encompassing posterior part of the testis vs immediately post-testicular). Sanguinicola armatus is differentiated from S. rutili by the number of intestinal caeca (5 vs 4), by having a more elongate testis (21–27% vs 15% of body length), and by the position of the ovary in relation to the testis (encompassing posterior part of the testis vs short distance from the testis). It differs from S. inermis (incertae sedis according to Warren et al., 2023) by the presence of tegumental spines, the absence of tegumental setae, and the course of vas deferens (straight vs looped ventrally and dorsally before entering the cirrus-sac).

Sanguinicola armatus differs from Sanguinicola lophophorus Erickson & Wallace, 1959 from North America in the distribution of the tegumental spines (reaching the level of oötype vs level of ovary), the course of oesophagus (straight vs sinuous), the number of intestinal caeca (5 vs 4), and the course of vas deferens (straight vs sinuous). It differs from the Asian species Sanguinicola skrjabini Akhmerov, 1960 in having fewer tegumental spines (60–100 vs > 300 per column) and in the number of intestinal caeca (5 vs 4). It is distinguished from Sanguinicola magnus Hu, Long & Lee, 1965 by body proportion (4.7–6.6 vs 3.2–3.4 times longer than wide), by having fewer testicular lobes (8–12 vs 27–29 pairs) and by the position of the ovary in relation to the testis (encompassing the posterior part of the testis vs immediately post-testicular). Sanguinicola armatus differs from both Sanguinicola lungensis Tang and Ling, 1975 and Sanguinicola rhodei Wang, 1983 by the number of intestinal caeca (5 vs 4), the course of vas deferens (straight vs sinuous), and the position of the ovary in relation to the testis (encompassing the posterior part of the testis vs immediately post-testicular). It is differentiated from Sanguinicola hasegawai Shimazu, 2013 by the lack of tegumental setae, the number of intestinal caeca (5 vs 4), the position of the ovary in relation to the testis (encompassing the posterior part of the testis vs immediately post-testicular), and the shape of the oötype (subtriangular to oval vs spherical).

3.5. Sanguinicola bakeri n. sp.

3.5.1. Taxonomic summary

Type-host: Blicca bjoerkna (L.) (Cypriniformes: Leuciscidae).

Type-locality: Curonian Lagoon (55.343487°N, 21.174823°E), Ventė, Lithuania.

Type-material: Holotype: MHNG-PLAT-160755. Paratypes: MHNG-PLAT-160756; MHNG-PLAT-160757 (2 whole-mounted). Hologenophores: MHNG-PLAT-160758; MHNG-PLAT-160759 (2 mounted).

Site in host: Recovered from heart, bulbus arteriosus, gills, body cavity wash and cranial cavity wash.

Representative DNA sequences: 3 out of 9 generated 28S rDNA sequences were unique and therefore only these were deposited in the GenBank database (PZ765043, PZ765044 and PZ770260). The cox1 sequences were deposited under the accession numbers PZ766184-PZ766189.

ZooBank registration: The LSID for the new name Sanguinicola bakeri n. sp. is urn:lsid:zoobank.org:act:DFEF4BB7-6FB8-4CEA-B470-967182C5AACD.

Etymology: The species is named after Dr Nathan Jay Baker (State Scientific Research Institute Nature Research Centre, Vilnius, Lithuania) in recognition of his assistance with fieldwork and collection of blood flukes.

3.5.2. Description

[Based on 9 fixed adult specimens; measurements of the holotype are given in the text; measurements of the entire series are provided in Table 3; Fig. 3C and D]. Body flat, lanceolate, 724 × 133, widest at level of posterior margin of intestine, 5.4 times longer than maximum width, body margins appear serrated. Anterior extremity with proboscis-like tip, more prominent in live specimens. Posterior extremity bluntly rounded. Tegument spined. Tegumental spines straight, deeply rooted in tegument, encased in conical protrusions, oriented mostly posterolaterally, arranged in single ventrolateral columns, 91 per column, starting at short distance (11) from anterior extremity, reaching level of oötype, at 58 from posterior extremity; arranged more densely in anterior body, less dense posteriorly. Spines anterior to nerve commissure, 9.7–17 × 0.9–1.4 (14 × 1.2), 9–14 (12) times longer than wide, mid-body spines (posterior of nerve commissure to level of ovary), 22–25 × 1.1–1.5 (23 × 1.3), 15–21 (18) times longer than wide, posteriormost spines 11–17 × 1.1–1.6 (16 × 1.3), 10–15 (12) times longer than wide. Nerve commissure at 103 from anterior extremity of body; lateral nerve cords extend posteriorly to level of uterus.

Mouth subterminal. Pharynx absent. Oesophagus curves slightly posteriorly, 209 × 2, 29% of body length, with two fusiform swellings; first swelling anterior to nerve commissure, 21 × 8, at 41 from anterior extremity, second swelling at posterior end of oesophagus, 22 × 7. Intestine median, 44 × 36, 6% of body length, 27% of body width, located in anterior half of body; X-shaped, deeply lobed, comprising 4 unequal caeca, 2 anterior caeca (13 × 6; 18 × 17) smaller than 2 posterior caeca (21 × 9; 26 × 9).

Testis single, 215 × 45, 4.8 times longer than wide, 30% of body length, 34% of body width, postcaecal, median, occupying much of middle body; comprising central column, 11 wide, 24% of testis width, flanked by two tandem rows of spherical to subspherical lateral lobes, 18 pairs, 8–22 × 8–20 (14 × 14). Post-testicular space 208, 29% of body length. Vas deferens narrower than central testicular column, emanates from posterior margin of central testicular column, enters cirrus-sac near posterior part of ovary. Cirrus-sac thin-walled, 83 × 17, S-shaped, oriented anteroposteriorly, curving towards mid-line, then sinistrally; encloses tubular seminal vesicle. Male genital pore level with oötype, sinistral, opens dorsally.

Ovary bi-lobed, butterfly-wing-shaped, with irregular margins, 115 × 69, 1.7 times longer than wide, 16% of body length, 52% of maximum body width, post-testicular, at short distance from testis. Post-ovarian field short, 97 (13% of body length). Oviduct emanates medially from ovary, joins oviducal seminal receptacle near posterior to ovary. Oviducal seminal receptacle tubular, 56 × 6, nearly 3 times narrower than cirrus-sac, oriented anteroposteriorly, extending dextrally posteriorly, narrowing at level of uterus, before entering oötype posteriorly. Vitelline duct indistinct or unites with oviduct before entering oötype in paratype (Fig. 3D). Oötype median, ovoid, 23 × 18, oötype wall 5 thick. Uterus spherical, 21 × 21, anterior to oötype, uterus wall 3.7 thick. Female genital pore median, anterior to male genital pore. Vitellarium follicular, extending from level of anterior oesophageal swelling to posterior to ovary.

Excretory vesicle Y-shaped; excretory pore subterminal.

3.5.3. Remarks

The present specimens conform morphologically to the generic diagnosis of Sanguinicola by Warren et al. (2023) in the body < 9 times longer than wide, the presence of straight lateral body spines, deeply rooted in the tegument, occurring in a single column on each lateral side, and in having an X-shaped medial intestine, terminating in anterior half of body, single postcaecal testis having an array of lateral lobes, bi-lobed butterfly-wing-shaped ovary, lateral male genital pore, medial female genital pore, post-ovarian oötype and straight uterus.

Sanguinicola bakeri n. sp. is distinguished from the congeneric species by its short testicular lobes that appear spherical, a more elongate ovary (16–18% of body length) and a narrow oviducal seminal receptacle (about 3 times narrower than the cirrus-sac). It resembles S. volgensis and S. rutili, whose margins also appear serrated due to prominent tegumental conical protrusions. However, S. bakeri n. sp. differs from S. volgensis in having a smaller uterus (shorter than oötype vs longer than oötype). It is differentiated from S. rutili in having a more elongate testis (30–35% vs 15% of body length) and in the shape of the oötype (ovoid vs triangular). The new species differs from S. armatus in having a more elongate testis (30–35% vs 21–27% of body length), a more elongate ovary (16–18% vs 10–12% of body length), a shorter post-ovarian field (12–16% vs 21–27% of body length), and in the position of the ovary in relation to the testis (short distance from testis vs encompassing the posterior part of testis). Sanguinicola bakeri n. sp. is distinguished from S. intermedius by the lack of tegumental setae and in having spines that are encased in prominent conical tegumental protrusions and a greater number of testicular lobes (16–19 vs 10 pairs). The new species differs from S. plehnae by possessing fewer tegumental spines (86–100 vs 118–122 per column), a smaller testis (30–35% vs 41% of body length) and by the shape of testicular lobes (nearly spherical vs elongate). It is differentiated from S. inermis (incertae sedis according to Warren et al., 2023) by the presence of tegumental spines and the lack of tegumental setae, and by the course of vas deferens (straight vs looped ventrally and dorsally).

Sanguinicola bakeri n. sp. differs from S. lophophorus in the distribution of the tegumental spines (reaching the level of oötype vs the level of ovary). It differs from S. skrjabini in having fewer tegumental spines (86–100 vs > 300 per column). It is distinguished from S. magnus by having fewer testicular lobes (16–19 vs 27–29 pairs) and body proportion (5–6.6 vs 3.2–3.4 times longer than wide). The new species is differentiated from both S. lungensis and S. rhodei by the course of the oviduct and vas deferens (straight vs sinuous). It differs from S. hasegawai in having a smaller uterus (shorter than oötype vs longer than oötype) and in lacking tegumental setae.

3.6. Molecular identification

A total of 61 partial sequences (28S, n = 34; cox1, n = 27) were generated from isolates of adult sanguinicolid specimens (Table 4). Comparative sequence and phylogenetic analyses confirmed the identification of the isolates based on morphology as members of the family Sanguinicolidae.

Table 4.

Summary data for sanguinicolid isolates from fish in Lithuania. Asterisks (*) indicate hologenophores.

Species	Host	Site in host	Locality	Isolate	GenBank ID
Species	Host	Site in host	Locality	Isolate	28S rDNA	cox1 mtDNA
Sanguinicola armatus	Carassius gibelio	Gills	Curonian Lagoon	BT10	PZ765041	–
Sanguinicola armatus	Tinca tinca	Heart	Curonian Lagoon	BT2	PZ765040	PZ766176
Sanguinicola armatus	Tinca tinca	Body cavity wash	Curonian Lagoon	BT3	ns	–
Sanguinicola armatus	Tinca tinca	Bulbus arteriosus	Curonian Lagoon	BT4*	ns	PZ766177
Sanguinicola armatus	Tinca tinca	Bulbus arteriosus	Curonian Lagoon	BT5*	ns	PZ766178
Sanguinicola armatus	Tinca tinca	Heart	Curonian Lagoon	BT6	ns	PZ766179
Sanguinicola armatus	Tinca tinca	Heart	Curonian Lagoon	BT7	ns	–
Sanguinicola armatus	Tinca tinca	Heart	Curonian Lagoon	BT8	ns	PZ766180
Sanguinicola armatus	Tinca tinca	Body cavity wash	Curonian Lagoon	BT9	ns	PZ766181
Sanguinicola armatus	Tinca tinca	Heart	Curonian Lagoon	FP763	ns	PZ766182
Sanguinicola armatus	Tinca tinca	Gills	Lake Dusia	FP1074	PZ765042	PZ766183
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Body cavity wash	Curonian Lagoon	BT1	PZ765043	PZ766184
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Heart	Curonian Lagoon	BT38	ns	PZ766185
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Cranial wash	Curonian Lagoon	BT39*	ns	–
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Body cavity wash	Curonian Lagoon	BT41	PZ770260	–
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Body cavity wash	Curonian Lagoon	BT42	ns	PZ766186
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Heart	Curonian Lagoon	BT44	ns	PZ766187
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Heart	Curonian Lagoon	BT45	PZ765044	–
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Bulbus arteriosus	Curonian Lagoon	BT46	–	PZ766188
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Bulbus arteriosus	Curonian Lagoon	BT51*	ns	PZ766189
Sanguinicola bakeri n. sp.	Blicca bjoerkna	Body cavity wash	Curonian Lagoon	BT58	ns	–
Sanguinicola volgensis	Alburnoides bipunctatus	Heart	River Žeimena	FP30	PZ765045	–
Sanguinicola volgensis	Leuciscus leuciscus	Gills	River Neris	FP522	PZ765047	PZ766190
Sanguinicola volgensis	Rutilus rutilus	Cranial wash	Curonian Lagoon	BT31	PZ765046	PZ766191
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT36	ns	PZ766192
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT37*	ns	PZ766193
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT40	ns	PZ766194
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT43	ns	PZ766195
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT47	ns	PZ766196
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT48	ns	PZ766197
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT49	ns	PZ766198
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	BT50	PZ765048	PZ766199
Ejsmontellus akicola n. sp.	Blicca bjoerkna	Eye vitreous humour	Curonian Lagoon	FP675	PZ765049	PZ766200
Ejsmontellus sp.	Leuciscus leuciscus	Gills	River Neris	FP29	PZ765050	PZ766201
Ejsmontellus sp.	Leuciscus leuciscus	Heart	River Merkys	FP31	PZ765051	PZ766202

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Abbreviation: ns, identical sanguinicolid sequences from the same host species that were generated but not submitted to the GenBank database.

The 28S rDNA phylogenetic trees resulting from both BI and ML analyses were identical and demonstrated the position of isolates obtained in the present study in three strongly supported clades (Fig. 4). The first clade (Clade A) comprised isolates belonging to the genus Sanguinicola, including its type-species S. armatus. Within this clade, novel isolates from A. bipunctatus, L. leuciscus and R. rutilus were similar or identical to previously reported sequences of S. volgensis ex Leuciscus idus and Pelecus cultratus. Isolates from T. tinca and C. gibelio, identified as S. armatus formed a strongly supported branch within Clade A, while isolates of the new species S. bakeri n. sp. from Blicca bjoerkna formed another strongly supported branch. The intraspecific divergence was low and ranged from 0.1 to 0.3% (1–4 bp) for S. armatus, 0.1–0.2% (1–2 bp) for S. bakeri n. sp., and 0–0.1% (0–1 bp) for S. volgensis. Interspecific divergence ranged from the lowest between S. bakeri n. sp. and S. volgensis (1.3–1.5%, 15–16 bp) to the highest between S. armatus and S. plehnae (7.5%, 87–88 bp). The representative sequence of S. plehnae was the most distant from others within the genus Sanguinicola (6.3–7.5%, 73–88 bp).

Fig. 4 — Bayesian inference (BI) phylogram reconstructed using the 28S rDNA sequences for species of the family Sanguinicolidae and unidentified blood flukes from freshwater ecosystems. BI posterior probability values and maximum likelihood (ML) bootstrap values associated with the branches are shown as BI/ML; support values lower than 0.90 (BI) and 70 (ML) are not shown. The scale-bar indicates the expected number of substitutions per site. Sequences of the species generated in the present study are coloured in different colours and presented as the GenBank accession number, followed by trematode name, fish host names, site in host, and country of origin. Species boundaries of isolates of *Sanguinicola* spp. and *Ejsmontellus* spp. inferred with the bPTP approach are plotted on the right-hand side. Shaded rectangles indicate clades A-C. *Abbreviations*: AUS, Australia; CZ, Czechia; LT, Lithuania; NAM, Namibia; POL, Poland; RUS, Russia; SA, South Africa; VIET, Vietnam; USA, United States of America.

The second clade (Clade B) consisted of sequences obtained exclusively from molluscs in South Africa, Poland and the Czechia. The interspecific divergence within Clade B (Sanguinicolidae gen. spp.) ranged from the lowest between two isolates from Europe, Sanguinicola cf. inermis ex Lymnaea stagnalis and Sanguinicola sp. ex Radix auricularia (2.4%, 27 bp) to the highest between the latter and Sanguinicolidae gen. sp. 4 ex Burnupia trapezoidea from South Africa (9.1%, 77 bp). Within Clade B, the sequence of Sanguinicola sp. ex R. auricularia was the most distant from the South African species (6.1–9.1%, 67–77 bp).

The third clade (Clade C) was resolved as a sister clade to Clade B and consisted of sequences representing the new genus, Ejsmontellus n. g., namely E. akicola n. sp. obtained from the eye vitreous humour of B. bjoerkna, and an unidentified species from the gills and heart of L. leuciscus. The intraspecific divergence was low for E. akicola n. sp. 0.1% (1 bp) and absent for Ejsmontellus sp. Divergence between these two species of Ejsmontellus ranged from 2.2 to 2.3% (25–26 bp).

The member of the genus Pseudosanguinicola, P. occidentalis, formed a clade with an isolate of an unidentified sanguinicolid cercaria, which was resolved as sister to the clades discussed above. Both isolates were obtained from fish and mollusc hosts in North America and differed by 7.1% (82 bp).

Except for two species belonging to the genus Nomasanguinicola, N. canthoensis and N. dentata, the taxonomic position of the remaining isolates in the 28S rDNA phylogenetic tree (Fig. 4) remains unresolved, as most sequences were derived from cercarial isolates or adult material insufficient for identification or formal description (Brant et al., 2006; Cutmore et al., 2023; Warren and Bullard, 2023).

Genetic divergence between the clades representing the genus Sanguinicola (Clade A), Clade B consisting of cercarial isolates, and the genera Ejsmontellus (Clade C) and Pseudosanguinicola ranged from the lowest between Clades B and C (7.3–10.8%, 81–101 bp) to the highest between Clade B and Pseudosanguinicola (14.6–17.9%, 151–175 bp). Sanguinicola was closest to Pseudosanguinicola (11.1–13.4%, 129–156 bp). The divergence between these clades was largely higher than interspecific divergence observed between isolates within Clade A (1.4–7.5%, 16–88 bp), Clade B (2.4–9.1%, 27–77 bp) and Clade C (2.2–2.3%, 25–26 bp), with a single exception – the lower minima between closely related Clades B and C (7.3–10.8%, 81–101 bp). A summary of nucleotide comparison of 28S rDNA sequences of the sanguinicolids is provided in Supplementary Table S1.

Species boundaries of the isolates of Sanguinicola spp. and Ejsmontellus spp. inferred from the bPTP analysis were congruent with the results of BI and ML phylogenetic analyses (Fig. 4, Supplementary Fig. S1).

Phylogenetic analysis of the cox1 dataset (Fig. 5) recovered six species, five of which were obtained in the present study. The intraspecific divergence within the cox1 dataset was low for S. bakeri n. sp. (0–0.2%, 0–1 bp), S. volgensis (0.2%, 1 bp), E. akicola n. sp. (0–0.4%, 0–2 bp), or absent for Ejsmontellus sp., whereas divergence was higher for S. armatus (0–5.3%, 0–25 bp). Isolates collected from the same waterbody, the Curonian Lagoon, were closely related (0–0.2%, 0–1 bp), whereas a single isolate from another waterbody, Lake Dusia, showed a divergence of 5.1–5.3% (24–25 bp) (Supplementary Table S2). The genetic divergence between Sanguinicola, Ejsmontellus and a cercaria designated as Sanguinicola sp. ex R. auricularia, that appears to belong to an unknown genus, ranged from 14.3 to 21.9% (68–104 bp), with the lowest values between Sanguinicola sp. ex R. auricularia and Ejsmontellus (14.3–15%, 68–71 bp) and the highest divergence between Sanguinicola and Ejsmontellus (16.2–21.9%, 75–104 bp).

4. Discussion

The results of the present study demonstrate that screening freshwater fish for blood flukes requires more effort than is often applied during routine parasitological surveys. Researchers have commonly examined the heart and gills, the main organs from which most freshwater fish blood flukes have been detected and described, with limited attention to other organs or to collecting those that escape when the blood vessels are severed. As a result, most blood flukes of freshwater fish have been recovered only occasionally and at low prevalence and intensity of infection (Cribb and Bray, 2010; Zhokhov et al., 2021). This may explain the scarcity of information on this group of trematodes in freshwater environments.

Three species (S. armatus, S. bakeri n. sp. and S. volgensis) from the present study were recovered not only from the bulbus arteriosus, heart and gills, but also from the body and cranial cavity washes. Notably, S. bakeri n. sp. was found more frequently in the body wash than in the gills and heart of B. bjoerkna. These findings show that following the approach described for blood flukes in turtles by Snyder and Clopton (2005), it is important to collect and screen the saline used for washing fish body cavities for the presence of blood flukes. It should be noted that most fish blood flukes inhabit the blood and vascularised organs, and rarely the body cavity. In the family Sanguinicolidae, only members of the genera Kritsky and Plehniella are known to mature in the body cavity of their hosts (Orélis-Ribeiro and Bullard, 2015). Hence, specimens that were recovered from the body cavity wash in the present study might have escaped from severed vessels during fish dissection.

Beyond the organs of the cardiovascular system, studies on marine fish have shown that blood flukes inhabit cranial cavity and eyes of their hosts (Alama-Bermejo et al., 2011; Cutmore et al., 2025; Yong et al., 2025). In freshwater fish, metacercariae of Tylodelphys Diesing, 1850 and Diplostomum von Nordmann, 1832 have been widely reported as the predominant trematodes that infect the cranial cavity and eyes (Blasco-Costa et al., 2014, 2017; Chaudhary et al., 2017; Kudlai et al., 2026). The present study shows that adult sanguinicolids also inhabit the cranial cavity and eyes of fish. Ejsmontellus akicola n. sp. appears to be site-specific since it was recovered only from the eye vitreous humour while the recovery of S. armatus, S. bakeri n. sp. and S. volgensis from multiple sites and body wash suggests that they are not site-specific. Since most of the existing records are from surveys that focussed on isolating blood flukes from the heart and gills (Cutmore et al., 2025), site specificity of sanguinicolids remains to be explored.

Taxonomy of the Sanguinicolidae has been problematic for many years due to incomplete morphological descriptions of some species, inconsistencies in diagnostic morphological features, lack of molecular data and uncertainties regarding host ranges (Ejsmont, 1926; Stunkard, 1983; Kirk and Lewis, 1993; Smith, 2002). In addition, detailed morphological assessments of known species are hindered by the absence of some type-specimens and the poor condition of the available vouchers (Orélis-Ribeiro and Bullard, 2016; Warren et al., 2023). These problems continue to persist due to the difficulties associated with collecting and studying blood flukes of freshwater fishes and the paucity of new researchers who are dedicated to this group of trematodes. Although there has been an increase in the availability of sanguinicolid genetic sequences recently, most isolates have not been formally described morphologically (Cutmore et al., 2023; Warren and Bullard, 2023; Outa and Avenant-Oldewage, 2024; Kundid et al., 2025).

Most sanguinicolids have historically been assigned to the type-genus Sanguinicola. Considering the remarkably distinct morphological features and diverse host ranges among species that were assigned to Sanguinicola, it was suggested that members of the genus represented several genera (Orélis-Ribeiro and Bullard, 2016). Consequently, the diagnostic features of Sanguinicola by Ejsmont (1926) and Smith (2002) were emended by Warren et al. (2023) to include only species having a single column of lateral tegumental spines. This reduced the number of valid Sanguinicola species from 26 to 11. However, genetic data were available for only two species, S. plehnae and S. volgensis. In this study, we provided the first genetic characterisation of the type-species S. armatus, evaluated its relationship with congeneric species, and described a new species, S. bakeri n. sp. Availability of these novel sequence data confirms that the clade comprising S. armatus, S. bakeri n. sp., S. plehnae and S. volgensis represent Sanguinicola (sensu stricto). All four species are consistent with the diagnostic features of Sanguinicola. Notably, S. plehnae is genetically distant from the remaining Sanguinicola spp. for which DNA sequences are available, which may reflect its host association. Sanguinicola plehnae parasitises a fish species of the order Esociformes, whereas the other species infect mainly Cypriniformes. Genetic data are still missing for seven other species of Sanguinicola considered valid: S. hasegawai, S. intermedius, S. lophophorus, S. lungensis, S. magnus, S. rutili and S. skrjabini. Thus, their phylogenetic relationships with other sanguinicolids are currently unknown.

Of the known sanguinicolid species from Europe, S. inermis requires further study to ascertain its taxonomic position. This species lacks tegumental spines, a feature that is considered diagnostic for the genus Sanguinicola. Currently, there are no sequences of adults of S. inermis. Although there is a single 28S rDNA sequence from Poland that is designated as Sanguinicola cf. inermis on GenBank, its identity is questionable since it was obtained from a cercaria whose morphological description was not provided and there are no sequences of corresponding adults for comparison (Orélis-Ribeiro et al., 2014; Outa and Avenant-Oldewage, 2024). The sequence of S. cf. inermis showed close phylogenetic affinities with another cercarial isolate from Czechia, designated as Sanguinicola sp., and four species from South Africa. The phylogenetic position of the cercarial isolates tentatively assigned to Sanguinicola indicates that they belong to an unknown sanguinicolid genus spanning two continents, Europe and Africa.

Based on the available genetic data, Sanguinicola (s. s.) is closest to the monotypic Pseudosanguinicola. This close phylogenetic relationship is also reflected in some similar morphological characteristics such as the presence of two oesophageal swellings, morphology and location of the intestine, a butterfly-wing-shaped ovary, a medial female genital pore, and a lateral male genital pore. Also, P. occidentalis and one species of the genus Sanguinicola (S. volgensis) both appear to infect fishes of the genus Perca (Percidae). Sanguinicola and Pseudosanguinicola are differentiated principally by the arrangement of lateral tegumental spines and 28S rDNA sequence divergence of 11.1–13.4% (129–156 bp). Other sanguinicolids which closely resemble P. occidentalis in having tegumental spines that are arranged in transverse rows rather than a single column include Sanguinicola fontinalis Hoffman, Fried & Harvey, 1985, Sanguinicola idahoensis Schell, 1974, Sanguinicola maritimus Nolan & Cribb, 2005 and Sanguinicola ugui Shimazu, 2007, which are regarded as incertae sedis by Warren et al. (2023). Although the arrangement of spines provides a practical basis for intergeneric distinction, it remains to be seen whether this feature is fully consistent with phylogenetic data.

Following the key to fish blood flukes provided by Warren and Bullard (2023), Ejsmontellus n. g. exhibits morphological characteristics that are consistent with the Sanguinicolidae. However, the new genus is distinguished from other sanguinicolid genera by a combination of morphological features. The type-species, E. akicola n. sp., differs from known sanguinicolids in having a significantly narrow post-ovarian body field compared with the preceding body part, few widely spaced tegumental spines, a tubular swelling that is located at the anterior end of the oesophagus, a small compact, heart-shaped ovary, and in the lack of posterior oesophageal swelling. Nonetheless, the placement of E. akicola n. sp. and Ejsmontellus sp. in the family Sanguinicolidae is supported by the phylogenetic results. The new genus is genetically closer to the clade comprising cercarial isolates from Czechia, Poland and South Africa (7.3–10.8%, 81–101 bp) than to Sanguinicola (13.9–14.7%, 155–165 bp) and Pseudosanguinicola (15.2%, 169–170 bp), based on 28S rDNA. It remains to be seen if the close genetic relationship between Ejsmontellus n. g. and the clade comprising cercarial isolates will be reflected in their morphological features and fish host affinities. The phylogenetic results further indicate that the gap in identities between known adults and larval isolates remains wide, as nine other cercarial isolates from Australia and the USA are representatives of unidentified genera that are closely related to Pseudosanguinicola.

Nomasanguinicola, which is represented by N. canthoensis and N. dentata, was phylogenetically distant (> 23% genetic divergence for 28S rDNA) from Ejsmontellus n. g., Pseudosanguinicola and Sanguinicola. Nomasanguinicola differs markedly from the three by the presence of denticles around the mouth, a Mehlis’ gland and an inverted U-shaped uterus, and the lack of lateral tegumental spines. Currently, there are no DNA sequences for members of the genera Cladocaecum, Kritsky, Parasanguinicola, and Plehniella. Hence, their phylogenetic relationships with other genera within the Sanguinicolidae remain unknown.

Patterns of host specificity and geographical distribution in freshwater fish blood flukes remain poorly resolved, as many species are still undiscovered and earlier records based on morphology alone require molecular verification. In Europe, only three species of Sanguinicola (S. rutili, S. plehnae and S. bakeri n. sp.) are oioxenous (sensu Euzet and Combes, 1980), each restricted to a single host species. Sanguinicola rutili and S. plehnae were described from Achondrostoma arcasii (Leuciscidae) and Esox lucius (Esocidae), respectively, and have not been recorded since, while S. bakeri n. sp. infects B. bjoerkna. The remaining species are largely euryxenous, infecting fish from several families (Zhokhov et al., 2021). Sanguinicola armatus and S. inermis have both been reported from five fish families, i.e. Cobitidae, Cyprinidae, Esocidae, Leuciscidae and Tincidae (Zhokhov et al., 2021).

To our knowledge, this is the first record of S. volgensis in the Baltics, specifically Lithuania and from A. bipunctatus as a host. The occurrence of S. volgensis outside its native range (River Volga Basin) and its occurrence in different host species is consistent with records from England, Hungary and Poland, where it was reported from fish belonging to the Esocidae, Leuciscidae and Percidae (Kirk and Lewis, 1994; Zhokhov et al., 2021). Sanguinicola intermedius is known from cyprinids in Lithuania, Poland, Russia and Ukraine, with a single report from Cobitidae in Czechia (Zhokhov et al., 2021). It should be noted that most of the reports of S. armatus, S. inermis, S. intermedius and S. volgensis were not accompanied by illustrations or molecular data. Considering the historical taxonomic problems within the Sanguinicolidae, it is possible that some of those records were for misidentified species. Beyond Europe, species of the genus Sanguinicola (s.s.) occur predominantly in Asia and infect cypriniforms of the families Acheilognathidae, Cyprinidae, Nemacheilidae and Xenocyprididae, and only a single species is known from a leuciscid fish in North America (Erickson and Wallace, 1959; Shimazu, 2013).

Apart from Nomasanguinicola, which shows a comparable transcontinental distribution, with species recorded from catfishes (Siluriformes) of the family Clariidae in both Asia and Africa (Warren et al., 2024), the remaining sanguinicolid genera are not widely distributed. The monotypic Parasanguinicola has been reported from a species of the family Latidae in Malaysia, the monotypic Pseudosanguinicola is restricted to species belonging to Percidae in North America, the monotypic Cladocaecum, Kritsky and Plehniella have been recorded from catfishes in South America (Herbert and Shaharom, 1995; Orélis-Ribeiro and Bullard, 2016; Warren et al., 2023). Ejsmontellus n. g. is now known only from leuciscids in Europe, specifically Lithuania. The absence of new records of Cladocaecum, Kritsky, Parasanguinicola and Plehniella within and outside of their native ranges is probably a reflection of the paucity of studies on blood flukes of freshwater fishes. The biogeographical factors which regulate the distributions and host associations of the Sanguinicolidae remain to be explored.

5. Conclusions

The description of a new genus and two new species from common European freshwater fishes, a fauna that has been investigated for parasites for over a century, demonstrates that the diversity of freshwater blood flukes is substantially greater than currently recognised. These records also reflect the difficulties associated with isolating and studying these flukes and suggest they have been overlooked in previous studies. Further efforts are likely to show that the freshwater blood fluke diversity rivals that of marine systems, which are currently far better characterised. This will require combining targeted sampling, morphological and molecular characterisation of species, and taxonomic revision of records currently placed in Sanguinicola. In agreement with Cutmore et al. (2025), we conclude that successful screening for blood flukes requires additional time and, ideally, a dedicated researcher focused solely on these worms. During parasitological examination, the search for blood flukes should be prioritised and must begin immediately after fish euthanisation. The vascularised organs such as heart and gills should be examined first, but it is equally important to collect and screen the saline used for washing the different body cavities for the presence of blood flukes. This is especially critical when adult specimens are required in sufficient numbers for morphological characterisation.

Ethical approval

This study received approval for compliance with Animal Welfare and Ethical Requirements (Reference No. GGT-12) from the Animal Welfare Committee of the State Scientific Research Institute Nature Research Centre, Lithuania. Fish specimens were collected under permits obtained from the Environment Protection Agency, Lithuania (license Nos.: 018 and 019 valid for 2022; 016 and 017 valid for 2023; 013, 025, and 032 valid for 2024, 007 valid for 2025, and 010 valid for 2026). All procedures were performed according to International Guiding Principles for Biomedical Research Involving Animals outlined by the Council for International Organizations of Medical Sciences (CIOMS) and the International Council for Laboratory Animal Science (ICLAS).

CRediT authorship contribution statement

James O. Outa: Conceptualization, Investigation, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Writing - original draft, Writing - review & editing. Olena Kudlai: Conceptualization, Investigation, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Writing - original draft, Writing - review & editing.

Funding

This research received funding from the Research Council of Lithuania (LMTLT) (S-PD-24-132).

Declaration of competing interests

The authors declare that they have no known competing interests that could have appeared to influence the work reported in this paper.

Acknowledgements

We gratefully acknowledge the following colleagues who assisted in the collection of fishes: Vytautas Rakauskas and Žilvinas Pūtys, and in fish examination: Camila Pantoja, Rasa Binkienė, Nathan Jay Baker, and Ivanna Dmytrieva. We thank Dr David Gibson of the Natural History Museum in London for his help with Latin.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crpvbd.2026.100436.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1

mmc1.pdf^{(467.9KB, pdf)}

Multimedia component 2

mmc2.xls^{(78.5KB, xls)}

Data availability

All data generated in this study are included in this article and its supplementary files. The type-specimens, hologenophores and voucher specimens have been deposited in the Platyhelminthes Collection of the Natural History Museum, Geneva, Switzerland (MHNG-PLAT) under the accession numbers MHNG-PLAT-160749 to MHNG-PLAT-160759. The novel sequences are deposited in the GenBank database under the accession numbers PZ765040-PZ765051 and PZ770260 (28S rDNA) and PZ766176-PZ766202 (cox1).

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Blood flukes (Digenea: Sanguinicolidae) of common freshwater fishes in Europe: A new genus, two new species, and a morphological and molecular characterisation of Sanguinicola armatus

James Omondi Outa

Olena Kudlai

Abstract

Graphical abstract

Highlights

1. Introduction

2. Materials and methods

2.1. Fish sampling and isolation of blood flukes

Fig. 1.

2.2. Morphological analyses

2.3. Molecular analyses

2.3.1. DNA extraction, amplification and sequencing

2.3.2. Phylogenetic analyses and species delimitation

3. Results

3.1. Infection patterns

Table 1.

Table 2.

3.2. Ejsmontellus n. g

3.3. Ejsmontellus akicola n. sp.

3.3.1. Taxonomic summary

3.3.2. Description

Table 3.

Fig. 2.

3.3.3. Remarks

3.4. Sanguinicola armatus Plehn, 1905

3.4.1. Taxonomic summary

3.4.2. Description

Fig. 3.

3.4.3. Remarks

3.5. Sanguinicola bakeri n. sp.

3.5.1. Taxonomic summary

3.5.2. Description

3.5.3. Remarks

3.6. Molecular identification

Table 4.

Fig. 4.

Fig. 5.

4. Discussion

5. Conclusions

Ethical approval

CRediT authorship contribution statement

Funding

Declaration of competing interests

Acknowledgements

Footnotes

Appendix A. Supplementary data

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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