Metacercariae of trematodes (Digenea) in the flesh of the common carp in Europe: Random occurrence of an unknown parasite or an issue of epidemiological concern?

Abstract

In fillets of cultured common carp intended for human consumption, large cysts containing larvae (metacercariae) of trematodes (Digenea) were found. These larvae belong to the family Diplostomidae, whose adults most often mature in birds and mammals. Although humans may acquire diplostomid infection only incidentally and the number of reported human cases of diplostomosis is not very high, except for alariosis, the presence of metacercariae in carp fillets is an epidemiological and economic concern due to the popularity of carp as a food source in Central and Eastern Europe. Morphological and molecular data (sequencing of three genetic markers) did not allow the assignment of the larvae to any known diplostomid genus.

Graphical abstract

Highlights

• •Large cysts were found in fillets of carp, most important freshwater fish farmed in Central and Eastern Europe.
• •The larvae in cysts belong to the family Diplostomidae, which most often mature in birds and mammals.
• •Molecular data did not assign the larvae to any known diplostomid genus.
• •This finding may have epidemiological and economic consequences.

1. Introduction

Common carp (Cyprinus carpio Linnaeus, 1758) is the most important freshwater fish cultured in fishponds in Central and Eastern Europe. In the Czech Republic, it accounts for 85–90% of farmed fish. It is popular for human consumption, especially at Christmas, and is also a favoured sport fish among anglers. The health of cultured carp can be affected by numerous pathogens, including parasitic worms (helminths). These are usually found in the circulatory system (Sanguinicola blood flukes) or in the gastrointestinal tract, such as adult tapeworms (Cestoda), roundworms (Nematoda), and thorny-headed worms (Acanthocephala), but larvae of trematodes (metacercariae) can be present in other organs, including muscle (Williams and Jones, 1994; Moravec, 2001). Cyprinoid fish, including common carp, can harbour metacercariae of trematodes such as Opisthorchis felineus and Metagonimus romanicus in Europe, which are potential causative agents of human fish-borne disease (Pozio et al., 2013; Scholz et al., 2024; Oros et al., 2025).

Recently, large cysts were found in the fillets of a carp (leather carp, i.e., form without scales) intended for human consumption from a fishpond in eastern Bohemia (Pardubice Region), Czech Republic. The fish was 4 years old, weight of 3.5 kg and it was F1 crossbred of Hungarian mirror carp M2 and Northern mirror carp M72. Fish was in good condition and did not differ from other market fish, except for the mentioned presence of cysts with parasites in the muscle and subcutaneous tissue.

Examination of these cysts revealed that they contained metacercariae (second-stage larvae) of unknown trematodes. As records of trematode metacercariae located in the musculature of common carp in Europe are very scarce, and given the potential epidemiological significance of this finding (carp is often eaten smoked, which does not necessarily kill encysted helminth larvae, or raw as steak tartare), the metacercariae found in the fillets of the common carp are genetically characterised, and their possible identification and epidemiological importance are discussed in this account.

2. Results

Metacercariae were found in a carp sold in a fish shop for human consumption. When the fish was cut into large pieces (fillets), spherical whitish structures (capsules) resembling blisters were observed in the musculature (Fig. 1). The total number of these capsules could not be reliably counted, but it is estimated that there were more than a dozen per fillet (Fig. 1). The cysts containing metacercariae were then fixed in 80% ethanol and sent to the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences in České Budějovice (IPCAS) for identification, including genetic characterisation.

Fig. 1.

Fillets of common carp (Cyprinus carpio) (A, B, D, E) with cysts containing metacercariae of an unknown diplostomid trematode (C).

The capsules measure 2.50–3.25 mm in length and 1.75–2.50 mm in width (Fig. 1). They contain metacercariae folded within elongate to ovoid, thin hyaline cysts measuring 1.00–1.23 mm in length and 0.76–0.83 mm in width. The folded metacercariae (maximum length occupied within the cyst) are 0.78–1.03 mm long and 0.45–0.65 mm wide (Fig. 1; Suppl. file – video). Their body is divided into widely oval to the elongate anterior part (prosoma) containing large holdfast organ (Fig. 1C) and the much shorter posterior part (opisthosoma), obdeltoid in shape (see Clopton, 2004 for shape terminology). As only fixed metacercariae were available to the present authors, their more detailed morphological characterisation, including observation of pseudosuckers, was not possible (attempts to excyst fixed metacercariae were unsuccessful).

Partial 28S (primers ZX-1/1500R), ITS1–5.8S-ITS2 (BD1/BD2), and cox1 (Dice-1F/Dice-14R) regions were PCR-amplified following Tkach et al. (2003), Morgan and Blair (1995), and Van Steenkiste et al. (2015), respectively. Sanger sequencing was performed by Eurofins Genomics (Germany), and contigs were assembled in Geneious Prime® 2025.0.2. Sequences were compared with GenBank via BLAST (accessed 29 April 2026); five new sequences (PZ694486–PZ694488, PZ696928, PZ696929) were generated. A 28S-based Maximum Likelihood (ML) tree was constructed using IQ-TREE (Minh et al., 2020), implemented on The European Galaxy server (usegalaxy.eu). Nodal support was assessed using the ultrafast bootstrap test with 1000 replicates (Hoang et al., 2018). The analysis included one new and 109 Diplostomidae sequences (alignment: 975 nt), with Cyathocothylidae gen. sp. (MH521249) and Cyathocotyle sp. (MN726943) as the outgroups (Achatz and Tkach, 2025). For the dataset, the TVM + F + R4 model was selected in ModelFinder under the Akaike Information Criterion (Kalyaanamoorthy et al., 2017). Two ITS1–5.8S-ITS2 sequences and two cox1 sequences were used to calculate pairwise distances in MEGA11 (Tamura et al., 2021).

Genetic data placed the metacercariae within Diplostomidae, but more precise identification is not possible with the available sequences. In the 28S analysis (Fig. 2), the metacercaria from common carp (PZ694486) emerged as a novel lineage. It did not cluster with any species with significant nodal support and was genetically closest to the metacercariae designated as BCM (= black cysts in muscle) or Diplostomidae gen. sp. (MK604826) by Hoogendoorn et al. (2020), with a genetic divergence of 2.6% (25 nt). These BCM metacercariae were also found encysted in muscle, but in banded tilapia (Tilapia sparrmanii; Cichlidae) from the North West Province of South Africa. Comparisons based on the ITS1–5.8S–ITS2 revealed an 11.7% (114 nt) divergence between our specimens (PZ694487 and PZ694488) and Diplostomidae gen. sp. (BCM; MK604886). Furthermore, in the analysis of the cox1 region, our sequences (PZ696928 and PZ696929) exhibited a 16.8% (59 nt) divergence from the Diplostomidae gen. sp. (BCM; MK605692) sequence of Hoogendoorn et al. (2020).

Fig. 2.

Phylogenetic tree from Maximum Likelihood analysis of the 28S rDNA sequence dataset of the Diplostomidae. Support values below 70 (ML) are not shown. The scale bar indicates the expected number of substitutions per site. The sequence generated in this study is shown in bold.

3. Discussion

The Diplostomidae Poirier, 1886 (Digenea) is a large and diverse family of widely distributed digeneans that most often mature in mammals and birds. The life cycle typically involves snails, fish or amphibians as intermediate hosts, and mammals and fish-eating birds as definitive hosts (Niewiadomska, 2002). The metacercarial stages of some species in several genera are known to be pathogenic to their fish intermediate hosts and can cause severe morbidity and, occasionally, mortality in cases of high infection intensity (Williams and Jones, 1994; Blasco-Costa and Locke, 2017). In contrast, the number of human cases caused by diplostomids is low, and they are considered occasional or rare human-parasitic trematodes (Muller, 2002).

The present finding of metacercariae of an unknown diplostomid has three distinct consequences: (i) from an epidemiological perspective, it indicates that some potential foodborne parasites can occur in the muscle of the most commonly consumed freshwater fish in Central and Eastern Europe, which is sometimes eaten raw as steak tartare; (ii) from an economic perspective, the presence of relatively large cysts containing parasite larvae in fish fillets can have serious economic consequences as following the EU regulation, no visible parasites are allowed on sold fish; and (iii) from a biological perspective, the occurrence of a parasite in one of the two most studied freshwater fish in Europe, which cannot be identified even to the genus level, is challenging and highlights insufficient knowledge of the diversity of helminth parasites on this continent.

Regarding European regulations on food safety, the European Food Safety Authority (EFSA) panel concluded that all wild-caught fish species may be exposed to and infected with zoonotic parasites due to the relatively low fish host specificity of these parasites. The same applies to freshwater fishing areas, with many regions having multiple studies reporting the presence of zoonotic parasites in wild-caught fish species (https://doi.org/10.2903/j.efsa.2024.9090).

In conclusion, this unexpected finding provides further evidence of the need to monitor new parasites, including those in commercially important fish and other aquatic animals. Although parasites of the common carp have been intensively studied in the Czech Republic, diplostomid metacercariae of unknown genus have never been found in the muscle of this fish (Moravec, 2001). Integrating molecular phylogenetic and morphological data to characterise the parasites found is also essential, in contrast to the previous unsatisfactory practice of fixing every suspicious structure or animal in formalin, which makes genotyping impossible. This finding also highlights the potential risk of consuming raw or undercooked (smoked) fish products and the need to prevent human infection with fish-borne parasites.

CRediT authorship contribution statement

Tomáš Scholz: Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization, Supervision, Writing – original draft. Camila Pantoja: Investigation, Formal analysis, Data curation, Writing – review & editing. Jakub Adámek: Investigation, Writing – review & editing. Jiří Hronek: Investigation, Writing – review & editing.

Declaration of competing interest

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