Infection of the Carpathian brook lamprey ( Eudontomyzon danfordi Regan, 1911) with a dermocystid parasite in the Tisza River Basin, Hungary

The Carpathian brook lamprey ( Eudontomyzon danfordi Regan, 1911) is an endemic protected species of Cephalaspidomorphi in the Carpathian Basin. No parasites have become known from these jawless vertebrates to date. Here, the authors describe an infection from a single specimen manifesting in protuberant skin cysts 7–10 mm in diameter, scattered on the body surface. Similar dermal infection was observed in 25 of the 274 lampreys recorded in the population survey. Skin cysts filled with round spore-like structures of a dermocystid parasite were found. These particles measured 8–14 µm in diameter and had an about 0.5-µm-thick wall, and containing

In fishes, the occurrence of Dermocystidium spp. is the most common. The systematic position of the genus Dermocystidium Perez, 1907 was a subject of debate for a long time. Some authors, like Dyková and Lom (1992), argued that due to formation of hyphae, this organism is in phylogenetic relation with fungi. By phylogenetic analysis, Ragan et al. (1996) stated that this clade diverges near the animal-fungal dichotomy, and it is a type organism from which metazoa and fungi may have evolved. Mendoza et al. (2002) classified  (Csaba & Láng, 1991;Molnár, 1979;Molnár, Müller, Lefler, & Csorbai, 2008;Molnár & Sövényi, 1984).
In this paper, we report infection by a dermocystid parasite causing cysts on the skin of the Carpathian brook lamprey, which was collected from a small stream in the Tisza River basin.

| Sampling
Lampreys were collected with electric sampling equipment (Hans Grassl GmbH) as part of a sampling for a phylogenetic study on 18 April 2017 from the Kemence stream (48.429306°N 21.447389°E) in the Zemplén Mountains on the territory of Aggtelek National Park, Hungary. During the fishing, some specimens with skin cysts were noticed. Two hundred and seventy-four lampreys of a length from 15 to 20 cm were caught. Each lamprey was observed for a while in a portable fish tank and then released back into the stream. As the Carpathian brook lamprey is a strictly protected species in Hungary, we have killed only a single 16-cm-long (at least 4 years old) mature animal. The specimen was killed by severing the spinal cord (Addis et al., 2012) then cut into two pieces, and the anterior portion of the body was preserved for further investigations in 70% ethanol, and the posterior ones was in 10% buffered formalin.

| Morphological and histological methods
Pathological studies were performed in the laboratory of the Fish Parasitological and Pathological Team of the Institute for Veterinary Medical Research, Centre for Agricultural Research. A lamprey specimen having protuberant cysts in the skin was investigated under a preparation microscope, including examination of inner organs. A single cyst from the posterior part fixed in 10% buffered formalin was opened from which a small amount of white, tiny granular material was extracted. Thousands of spore-like particles found in this smear were studied under a light microscope.
Photographs and digital images were taken using an Olympus BX53 research microscope equipped with cellSens Entry image archiving software. The spore-like particles were measured on the basis of digital images.
A cross-sectional slice from the portion of the body segment fixed in 10% buffered formalin was routinely processed for histopathology. 4-to 5-μm-thick sections were cut and stained with haematoxylin and eosin.
Histological description of tissues, where cysts were formed, was based upon the guidelines of Elliott (2011).

| Molecular methods
Genomic DNA was extracted from 80% ethanol fixed material obtained from the cysts using the Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan) according to the manufacturer's instructions. Amplification and sequencing of the 18S rDNA were The sequence fragments were assembled using MEGA 6.06 software (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). The contiguous 18S rDNA sequences and the most similar dermocystid sequences from the GenBank based on BLAST matches were aligned with the CLUSTAL W software (Thompson, Higgins, & Gibson, 1994).
DNA pairwise distances were calculated with the MEGA 6.06 software using the p-distance model. Phylogenetic analysis was performed via maximum likelihood (ML), and Sphaerothecum destruens and S. caipira were used as out-group. The data set was tested using MEGA 6.06 for the nucleotide substitution model of best fit, and the model shown by the Akaike information criterion (AIC) as the best fitting one was chosen (GTR + G + I model). Bootstrap values based on 1,000 resampled data sets were generated.

| RE SULTS
Twenty-five out of the collected 274 Carpathian brook lamprey specimens (9.1%) showed protuberant cysts on the skin. Scattered on the body surface of the infected specimens, 1-10 (3.5 ± 3.4) cysts were found in the skin (Figure 1). On the single animal killed for further examination, 10 protuberant cysts 7-10 mm in diameter were situated in different parts of the body. In the smear excreted from the ruptured cyst, thousands of round spore-like particles were observed and 50 of them were measured 8-14 (10.49 ± 2.41) µm in diameter based on digital images (Figure 2). They had an approximately 0.5-µm-thick wall and a relatively scarce cytoplasm filled with granular mass. In some spore-like particles, the nucleus (arrows) could be identified among the granules.
In histological sections, the cyst emerges over the body surface, located in the hypodermis of the skin (Figures 3 and 4). The

| D ISCUSS I ON
Numerous different species of dermocystid parasites have been described worldwide, which infect freshwater and anadromous fishes produce gill infections, skin lesions, visceral diseases and eye infections (Feist, Longshaw, Hurrell, & Mander, 2004;Hassan, Osman, & Mahmoud, 2014;Mahboub & Shaheen, 2020;Molnár et al., 2008;Zhang & Wang, 2005). However, our study is the first finding of a dermocystid species as the first parasite ever recorded in the Carpathian brook lamprey belonging to the class of Cephalaspidomorphi.
Dermocystid infections are generally manifested as small round, oval or elongate cysts sometimes stuffed with long spore-producing hyphae (Dyková & Lom, 2007), with different locations and morphology of the spores, depending on the parasite species (Novotny & Smolova, 2006).
In the developmental cycle of several Dermocystidium sp., a small multicellular plasmodium grows and becomes confined within a distinct hyaline cyst wall, and then, the multinucleate cytoplasmic contents become segmented into uninucleate cells that are eventually transformed into a large number of spores (Bruno, 2001;Pekkarinen, Lom, Murphy, Ragan, & Dyková, 2003;Pekkarinen & Lotman, 2003).
Mature spores contain a large central vacuole or refractile body the cytoplasm with the nucleus being restricted to a narrow peripheral layer (Dyková & Lom, 1992). The skin cyst that developed on the trunk of the Carpathian brook lamprey in the hypodermis differed from dermocystid infections found earlier in eel, common carp and crucian carp in Hungary (Csaba & Láng, 1991;Molnár et al., 2008;Molnár & Sövényi, 1984).
The wall of the cyst of this species was very thin, in contrast to the cyst walls of other species described by Molnár and Sövényi (1984) and Feist etal. (2004) from the eel and the bullhead Cottus gobio Linnaeus, 1758, respectively.
Within the cyst, only separate spore-like particles were found that inside structures did not resemble to mature Dermocystidium spores and hyphal forms were not present.
A possible reason for this might be that the infection was only in an early stage of infection. It is known from the work of Červinka et al. (1974) that in the early stages of development, the spores show a much more granular structure than the more mature ones that are characterized by a ring shape. Pekkarinen and Lotman (2003) and  who studied the development of Dermocystidium percae and D. fennicum found that developing spores have granulated structure similarly to the spore-like particles in present study. The lack of mature spores does not make well-grounded to identify the species found by us as a Dermosporidium sp.; therefore, we referring it as dermocystid parasite.
Sampling further lamprey specimens and conducting a more detailed study for finding the expected mature stages of the parasite is problematic. Carpathian brook lamprey species is a strictly protected animal, and due to its hidden lifestyle in the mud, the collection is limited to a few days during the spawning period.

F I G U R E 4
Histological section of the dermocystid cyst stained with H&E.; epidermis (e); dermis (white arrow); pigmented layer (blank arrow); hypodermis (black arrow); wall of the cyst (small arrow); spore-like particles in cyst (S); adipose tissue of the hypodermis (a); skeletal muscle (m) [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 5 Enlarged section of Figure 4 showing the cyst and the skin layers of the fish. Spore-like particles in cyst (S); cyst wall (small arrow); space (SP) between the detached cyst wall and the hypodermis (black arrow); pigmented layer (white arrow); dermis (d); epidermis (e) [Colour figure can be viewed at wileyonlinelibrary. com] Despite these morphological differences, the molecular results clearly proved that our species is related to the Dermocystidium species known from salmonid and percid fishes, but the phylogenetic distance is remarkable. No other species could be regarded as a close However, tetrapods are phylogenetically much closer relatives to bony fish than to lampreys and their relatives. Considering this fact, it should be emphasized that this is the first record of a dermocystid parasite from a separate and phylogenetically basal vertebrate taxon, the Cephalaspidomorphi. Their presence in lampreys can be explained by the fact that pathogens in Mesomycetozoea are true generalists (Gozlan et al., 2014), and therefore, it is possible that dermocystid species can parasitize a broad range of taxa including Petromyzontiformes in addition to bony fishes.

ACK N OWLED G EM ENTS
The authors express their thanks to László Lontay, ranger of F I G U R E 6 Phylogenetic tree of the partial 18S rDNA sequences of the dermocystid parasite detected in Carpathian brook lamprey (GenBank accession no.: MT373572) compared with reference isolates (with accession numbers) obtained from GenBank. The tree was derived by maximum-likelihood analysis (MEGA 6.06) of a 1411-bp region. Bootstrap support values (>75%), indicated at the nodes, were obtained from 1,000 bootstrap replicates and are reported as percentages. Scale bar indicates the number of nucleotide substitutions per site.