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Journal:AVET

Article Number:004.2020.00001

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Queries and/or remarks

[Q1] Please confirm that the forename(s) and surname(s) have been identified correctly and are presented in the desired order, and please carefully verify the spelling of all authors’names.

[Q2] References“Nordmann, 1832; von Nordmann, 1832; McCallum, 1921; Ivashkin, 1964; Hanek and Threlfall, 1969; Müller, 1936”are cited in the text but not provided in the reference list. Please provide them in the reference list or alternatively delete the citations from the text.

[Q3] Please provide the volume number and page range for the bibliography in Ref.‘Czeglédi et al., 2019’.

[Q4] Please provide the volume number and page range for the bibliography in the references‘Moshu (2014)’.

[Q5] Please provide a definition for the significance of“bold”values in Table 1 and Table 2.

[Q6] Please note that as per style, the ORCID information is mandatory for the corresponding author.

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New record of metacercariae of the North American Posthodiplostomum Centrarchi (digenea, diplostomidae) in pumpkinseed ( Lepomis Gibbosus ) in Hungary

G ABOR CECH

¹^p

, DI ANA S ANDOR

^1,2

, K ALM AN MOLN AR

¹

,

^Q6

PETRA PAULUS

³

, MELITTA PAPP

³

, B ALINT PREISZNER

⁴

,

ZOLT AN VIT AL

⁴

, AD AM VARGA

¹

and CSABA SZ EKELY

¹ ^Q1

1Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungaria krt. 21, H-1143, Budapest, Hungary

2E€otv€os Lorand University, Doctoral School of Biology, Programme of Zootaxonomy, Animal Ecology and Hydrobiology, Budapest, Hungary

3National Food Chain Safety Ofﬁce, Veterinary Diagnostic Directorate, Budapest, Hungary

4Centre for Ecological Research, Balaton Limnological Institute, Tihany, Hungary

Received: October 18, 2019 • Accepted: November 21, 2019

ABSTRACT

Two species of the genus Posthodiplostomum (Digenea: Diplostomatidae) (Posthodiplostomum brevicaudatum Nordmann, 1832 and Posthodiplostomum cuticola Nordmann, 1832) are known as parasites of Hungarian nativeﬁshes. Metacercariae ofP. cuticolaare widespread in Europe and cause black spot disease. Several species ofPosthodiplostomumwere described also from North America but none of them has been isolated in Hungary up to now.Posthodiplostomum centrarchiHoffman, 1958 has been detected recently in pumpkinseeds (Lepomis gibbosusL., 1758) in several European countries.

Posthodiplostomum centrarchiwas isolated for theﬁrst time in Hungary from pumpkinseeds caught in the Maconka water reservoir in 2015. Thereafter, several natural waters (e.g. the river Danube, Lake Balaton and the Sio channel) were sampled in order to determine its presence and distribution. Only the native speciesP. cuticolawas detected in Lake Balaton on cyprinids but a relatively high infection rate was observed in the Sio channel close to the lake. Pathological changes were absent, and metacercariae were mostly attached to the surface of the liver, kidney and heart. The phylogenetic analysis of the ITS and COI sequences ofP. centrarchiandP. cuticolaclustered into two distinct branches, which was in agreement with the morphological results.

KEYWORDS

Lepomis gibbosus,Posthodiplostomum, non-native species, first observation in Hungary, histology

INTRODUCTION

PosthodiplostomumDubois, 1936 (Digenea: Diplostomidae) species are distributed worldwide (Lopez-Hernandez et al., 2018;Niewiadomska 2002;Ritossa et al. 2013) and well known asfish and water-bird parasites (Lumsden and Zischke 1963;Miller 1954). The metacercariae of these species, especially P. cuticola (von Nordmann, 1832), cause black spot disease or Post- hodiplostomuminfection that occurs on the body surface,fins, scales, or in the musculature and body cavity offishes (Horak et al. 2014; Ondrackova et al. 2004; Tobler and Schlupp 2008;

Zrncic et al. 2009). These encysted stages often induce developmental disorders of theﬁsh skeleton that may be lethal in the case ofﬁngerlings (Lucky 1970). The most common signs are

Acta Veterinaria Hungarica

DOI:

10.1556/004.2020.00001

ORIGINAL ARTICLE

*Corresponding author.

E-mail:cech.gabor@agrar.mta.hu. Tel.:

þ36 (1) 467 4060; fax:þ36 (1) 467 4076

RESEARCH ARTICLEAVET-2020.00001_proof 21 February 2020 9:20 pm

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weight loss due to appetite decrease, lesions of the liver and kidney, and digestive dysfunction (Iqbal et al. 2014;Lane and Morris 2000; Lopez-Hernandez et al. 2018; Rolbiecki 2004;

Sch€aperclaus 1990).

More than 25 species of Posthodiplostomum have been described in the world (Lopez-Hernandez et al. 2018;Ritossa et al. 2013) but in Europe only four species of this genus have been reported: Posthodiplostomum cuticola (von Nordmann, 1832) and P. brevicaudatum (von Nordmann, 1832) as native species, as well as P. minimum(McCallum, 1921) and P. centrarchi Hoffman (1958), both originating from North America. The occurrence of P. minimum in Europe was based on only three metacercariae (Grabda- Kazubska et al. 1987) and no further conﬁrmations of this record were published. Posthodiplostomum centrachi had not been reported in the European pumpkinseed population before 2017 (Bykhovskaya-Pavlovskaya and Kulakovskaya 1985; Molnar 1969; Moravec 2001; Moshu 2012, 2014;

Roman-Chiriac 1960). In Hungary, the native species P.

cuticola and P. brevicaudatum were documented by Jaczo (1941)andMolnar (1968), whileSzekely and Molnar (1996) reported only P. cuticola. Recently, the presence ofP. centrarchi has been reported published from several European countries: Bulgaria, Slovakia, the Czech Republic, Portugal, Ukraine and Germany (Kvach et al. 2017, 2018a;

Ondrackova et al., 2019;Stoyanov et al. 2017a).

The nomenclature and the fish intermediate host speci- ficity ofP.centrarchiare still under debate.Hoffman (1958, 1999)consideredP. centrarchi a subspecies ofP. minimum and named itP. minimum centrarchibecause he proved that it could infect only centrarchid ﬁshes while P. minimum minimumwas able to infect only cyprinids. The strict host speciﬁcity of theP. minimumsubspecies was supported by Locke et al. (2010a,b), Lane et al. (2015), Stoyanov et al.

(2017a)andBoone et al. (2018). Accordingly,Stoyanov et al.

(2017a) elevatedP. minimum centrarchi to species rank as P. centrarchiand this novel name has become general since then (Kvach et al. 2018a;Ondrackova et al. 2019).

Metacercariae of an unknown Posthodiplostomum species, non-indigenous in Hungary, werefirst recorded in 2015 when pumpkinseeds were sent to the National Food Chain Safety Office (NFCSO) from a North Hungarian water reservoir for a routine survey. These metacercariae were tentatively identified as P. centrarchi, a typical parasite of North American centrarchidfishes. To investigate the extent of this infection, we started a survey on the distribution, development and possible occurrence of these metacercariae in the Hungarian pumpkinseed population and on their pathogenic role in native fishes.

In this study, we report the first finding ofP. centrarchi metacercariae in pumpkinseeds from a small water reservoir in Hungary. We also describe a recent occurrence of this non-native trematode parasite in the Sio channel close to Lake Balaton. In order to obtain adult stages and to study the histological changes induced in the host, we successfully infected chicks with metacercariae under laboratory condi- tions. Both metacercariae and adult stages were studied by morphological and molecular methods.

MATERIALS AND METHODS

Sample collection

Up to 2015, pumpkinseed specimens had been collected randomly from different areas of Hungary and studied for parasitic infections. In 2015, 6 out of 8 pumpkinseeds were found to be parasitised byP. centrarchimetacercariae during a general veterinary survey in the Maconka water reservoir.

After the detection of the first metacercariae in pumpkinseed, our research group started to study the parasite fauna of the pumpkinseeds in Lake Balaton and in its outflow, the Sio channel (Table 1). In the littoral zone of Lake Balaton, the pumpkinseed is the most abundant non-native fish species (Czegledi et al. 2019). Eighteen pumpkinseed specimens derived from the River Danube at Szentendre and a single specimen from the River Ipoly at Ipolyt€olgyes was examined (Table 2). Besides 142 pumpkinseeds, 16 specimens of roach (Rutilus rutilus) and 11 white breams (Blicca bjoerkna) were collected from Lake Balaton and preserved to check for Posthodiplostomum infection in cyprinid fishes.

Moreover, 10 pumpkinseed individuals were caught from the Sio channel. Most of the fish were gathered using an electrofishing gear but several individuals were obtained by a 15-metre-long seine net. The fish were carried to the laboratory alive in oxygenated plastic bags, kept in aerated water tanks and subjected to complete parasitological dissection within 3 days.

Dissection, artificial digestion and microscopy

Before extermination, the pumpkinseeds were sedated with a drop of clove oil added to their water, then killed by a cervical cut. Each fish was investigated under a Zeiss dissecting microscope and the left side of the abdominal wall was cut down. Metacercariae were collected from the surface of the organs, excysted using a fine needle and placed in 0.9% saline solution for approximately 10 min. About 300 live metacercariae were kept alive for experimental infections.

The rest of the parasites were counted and fixed in 80%

ethanol for molecular examinations. The infected tissue samples were fixed in Bouin’s solution, embedded in parafﬁn wax, cut to 4–5

m

m thick sections and stained with haematoxylin and eosin. Live metacercariae and histological sections were studied with an Olympus BH2 compound microscope equipped with Nomarski differential interfer- ence contrast optics, then photographed with an Olympus DP 20 digital camera.

Experimental infection

Two experimental infections were carried out to obtain adult stages ofPosthodiplostomumspp. for detailed morphological analysis and description. In the ﬁrst case (09.02.2016–

22.02.2016), each of two one-day-old unfed domestic chicks (Gallus gallus domesticusL., 1758) (‘A’and ‘B’ individuals) was force-fed with 50 freshly collected P. centrarchi metacercariae from pumpkinseeds in the Maconka water reservoir. After the infection, the chicks were marked with green 115

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colour on their head and wings. Two more individuals were preserved as negative control, which were left unmarked.

Chick ‘A’was killed by a cervical cut on 17.02.2016, while specimen ‘B’ and the negative controls were killed on 22.02.2016.

During the second experiment (03.10.2017–11.10.2017), 3 one-day-old unfed domestic chicks (‘C’, ‘D’, ‘E’) were infected per os with 50 P. cuticola metacercariae collected from white breams, and these birds were marked with red colour on their head. The two negative controls were unmarked. Chick ‘C’ was decapitated on 10.10.2017, then chicks‘D’,‘E’and the negative controls were decapitated on

11.10.2017. All chicks had been purchased from a com- mercial supplier (Hegyhat BR Kft., Szentgotthard-Rabaf€uzes, Hungary). Formal ethical approval was given by the Gov- ernment Ofﬁce of Pest County (permit PEI/001/1004-4/

2015).

After the dissection, the gut system of each bird was isolated, then divided into three regions, namely duodenum plus jejunum, ileum and colon. These parts of the intestine were cut up and immersed in three different sedimentation cones containing 0.9% saline solution. After stirring for 10 min, they were allowed to subside for 30 min, then the pieces of gut and the intestinal content in the sediment were Table 2.Collection sites and dates of pumpkinseed (Lepomis gibbosus)

Collection site Collection date Number/fish

Size of fish (cm)

Infection with P. centrarchi Szentendre (Danube)

47839⁰50.3⁰⁰N, 1984⁰53.5⁰⁰E

04.30–05.04.2009 5 7.8 (±1.8) No

07.20–08.06.2013 12 7.6 (±2.1) No

09.07.2015 1 14 No

Maconka water-reservoir 4881⁰45.9⁰⁰N, 19849⁰8.9⁰⁰E

12.18.2015 8 10.6 (±0.7) 6

Ipoly, Ipolyt€olgyes 47855⁰51.2⁰⁰N, 18846⁰15.3⁰⁰E

05.12.2017 1 11 No

Table 1.Collection sites and dates of pumpkinseed (Lepomis gibbosus) in the Lake Balatonregion ^Q5

Collection site Collection date Number/fish

Size of fish (mean±SE)

Infection with P. centrarchi Balatonmariaf€urd}o

46842⁰34.3⁰⁰N, 17822⁰54.8⁰⁰E

08.02.2017 8 7.9 (±3.4) No

Balatonszemes 46848⁰36.8⁰⁰N, 17845⁰55⁰⁰E

09.04.2008 1 9 No

06.04.2012 6 9.6 (±1) No

06.26.2018 5 8.8 (±1.3) No

Balatonvilagos 46858⁰0.5⁰⁰N, 1889⁰27.8⁰⁰E

07.01.2010 4 7.8 (±3.9) No

Keszthely 46845⁰22.1⁰⁰N, 17814⁰59.6⁰⁰E

07.27.2009 22 9.9 (±2.3) No

05.04.2012 2 9 (±1.4) No

04.11.2013 7 9.6 (±1.3) No

06.20.2016 12 9.9 (±1.7) No

05.17.2017 1 10 No

Siofok

46854⁰43.3⁰⁰N, 1882⁰32.5⁰⁰E

02.29.2015 1 10 No

06.26–07.18.2018 40 9.8 (±3.1) No

Sio channel 46854⁰26.8⁰⁰N, 1882⁰48.5⁰⁰E

07.02.2018 10 12 (±1.9) 4

Tihany

46854⁰51.6⁰⁰N, 17853⁰36.5⁰⁰E

07.30.2008 3 11 (±4.6) No

05.07.2011 2 10.5 (±2.1) No

07.19.2012 3 8.3 (±1.2) No

06.26–07.02.2018 20 8.6 (±3.2) No

Zanka

46852⁰26.2⁰⁰N, 17842⁰17⁰⁰E

05.22.2017 5 8.8 (±2.2) No

Acta Veterinaria Hungarica

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studied for trematodes under a Zeiss stereo microscope. All detected worms were removed and stored in 80% ethanol.

Molecular methods

Samples preserved in 80% ethanol were centrifuged at 8,000g for 5 min to remove the ethanol. The DNA was extracted using a QIAGEN DNeasyTM tissue kit (animal tissue protocol; Qiagen, Hilden, Germany) and eluted in 100

m

^{L AE}

buffer. The ITS region (part of 18S rDNA, ITS1, 5.8S rDNA, ITS2 and part of 28S rDNA) was amplified by a nested PCR as described by Sandor et al. (2017). The amplification of COI was performed using the primers (Plat-diploCOX1F and Plat-diploCOX1R) and protocol described by Moszc- zynska et al. (2009). PCR products were electrophoresed in 1.0% agarose gels in Tris-Acetate-EDTA (TAE) buffer gel, stained with 1% ethidium bromide. Purification was carried out with EZ-10 Spin Column PCR Purification Kit (Bio Basic Inc., Markham, Canada). Purified PCR products of the ITS region and COI were sequenced with the PCR primers and with two additional inner primers 5.8Sr (5⁰-TGTCGAT- GAAGAGCGCAGC-3⁰) and 5.8S2 (5⁰-TAAGCCGACCCTCGG- ACAGG-3⁰) (Tkach et al. 2000) in the case of the ITS region. ABI BigDye Terminator v3.1 Cycle Sequencing Kit was used for sequencing and the sequences read using an ABI 3100 Genetic Analyser.

Phylogenetic analysis

Assembly of the sequenced fragments was done by MEGA version X (Kumar et al. 2018) and ambiguous bases clarified using corresponding ABI chromatograms. Alignments of the genes ITS and COI were done with the software CLUSTAL W (Thompson et al. 1994). The two alignments (ITS region and COI) were corrected manually using the alignment editor of the software MEGA version X. Sequences were deposited in the GenBank under the accession numbers (MN08027492, MN17928090, Table 3). DNA pairwise distances were calculated with the MEGA X software using the p-distance substitution model. Maximum likelihood (ML) analysis was performed for both alignments. The analysed samples are listed inTable 3.Bolbophorus damni- ficus and Clinostomum marginatum (AF470583 and MK426663) were chosen as the outgroup for ITS and COI genes. The dataset was tested using MEGA X 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 for each partition. ML analyses were performed in MEGA X under the HKYþG model for the ITS region and GTRþG for the COI. Bootstrap values based on 1,000 resampled datasets were generated. The phylogenetic trees were visualised using the tree explorer of MEGA X.

RESULTS

The first Posthodiplostomum centrarchi infection of 6 pumpkinseed individuals was accidentally recorded from

the Maconka water reservoir in 2015. Each ﬁsh was parasitised by 35–150 metacercariae located on the surface of internal organs and the mesentery; furthermore, some individuals were found in excysted condition. The excysted specimens clearly showed the morphological features of the genus Posthodiplostomum and corre- sponded to P. centrarchi (Figs 1 and 2) as characterised and depicted byHoffman (1999)andKvach et al. (2018a).

In 2016 and 2017, large numbers of pumpkinseeds were examined from different regions of Lake Balaton and from the rivers Danube and Ipoly but such infection proved to be absent from these samples. Finally, our research isolated metacercariae of P. centrarchi in pumpkinseed specimens from the Sio channel in 2018, at the outﬂow of the water-gate. No P. centrarchi infection was found in roach and white bream dissected as controls; nevertheless, cysts of P. cuticola were recorded in 3 white bream specimens. In contrast, 4 out of the 10 pumpkinseed specimens collected from the Sio channel showed infection with 20–150 metacercariae.

These twoPosthodiplostomumspecies could be identiﬁed and distinguished easily under compound and dissecting microscope based their morphology and predilection site.

All specimens ofP. centrarchiwere found in the abdominal cavity contrary to metacercariae of P. cuticola, which were located in the skin, muscles andﬁns. In histological sections, theP. centrarchicysts were mostly found in the mesenteries and inside the interstitium of the kidney and liver (Fig. 3).

Less frequently they were located in the muscle of the heart’s atrium (Fig. 4). They were never found in the skeletal muscle or in the muscle of the ventricle but they readily adhered to this latter. A special observation is that 3 out of the 4 P.

centrarchi infected specimens from the Sio channel had a heavy co-infection with the nematode Schulmanela petrushewskii (Shulman 1948) Ivashkin 1964 in the liver (Fig. 5).

In the first experimental infection, two chicks were fed withP. centrarchimetacercariae per os. After 8 days, 5 and 4 adult trematodes, respectively, were found in their colon at dissection (Fig. 6). During the second experiment, when three chickens were infected withP. cuticolametacercariae, only 2 adultﬂukes were found in the bursa of Fabricius of a single chick after incubation for 8 days.

NineteenPosthodiplostomumsamples were analysed for the ITS region and COI genes, including metacercarial and adult developmental stages (Table 3). The ampliﬁed ITS region of the samples was more than 1,200 bps, with the alignment being 1,067 bps long after removing poorly aligned positions and divergent regions, and containing 807 conservative and 216 variable (116 of them parsimony- informative) sites. The COI fragments exceeded 460 bps and the alignment consisted of 458 bps, including 230 conservative and 228 variable (158 of them parsimony-informative) sites. The sequences of the ITS region and COI genes of the samples were in agreement with the results from the morphological and experimental studies. The analysed samples were similarly positioned on both phylogenetic trees (Figs 7A and B), one major group constituted by the samples 343

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400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

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Table 3.List of the sequenced metacercariae and adult samples ofPosthodiplostomumspp.

Sample

Morphological

identification Host Developmental stage Date of collection Site of collection ITS sequenc

COI sequence

1 PD1 Posthodiplostomum

centrarchi

Chick (ﬁrst infection)

adult 17 February 2017 Maconka water

reservoir

MN080274 MN179280

dentrarchi

reservoir

MN080275 MN179281

centrarchi

reservoir

MN080276 MN179282

sp.

Pumpkinseed (Lepomis gibbosus)

metacercaria 27 November 2015 Maconka water reservoir

MN080277 MN179283

sp.

27 November 2015 Maconka water reservoir

MN080278 –

sp.

MN080279 –

sp.

MN080280 MN179284

sp.

MN080281 MN179285

sp.

MN080282 –

10 PD10 Posthodiplostomum sp.

MN080283 MN179286 11 PD11 Posthodiplostomum

sp.

Common bream (Abramis brama)

6 April 2016 Lake Balaton (Siofok)

sp.

19 May 2016 Lake Balaton (Balatonszemes)

sp.

19 May 2016 Lake Balaton (Balatonszemes)

MN080287 –

12 December 2015 Maconka water reservoir

MN080288 –

Roach (Rutilus rutilus)

25 October 2016 River Danube (Szentendre)

MN080289 –

White bream (Blicca bjoerkna)

25 February 2016 Lake Balaton (Siofok)

MN080290 –

Rudd (Scardinius erythrophthalmus)

22 May 2017 Lake Balaton (Szigliget)

sp.

Rudd (Scardinius erythrophthalmus)

22 May 2017 Lake Balaton (Szigliget)

MN080292 –

ActaVeterinariaHungarica

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RESEARCHARTICLEAVET-2020.00001_proof21February20209:20pm

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belonging toP. centrarchi, and apart from them another one consisting ofP. cuticolasamples, both groups supported by high bootstrap values (above 97). Between them, several other Posthodiplostomum and Ornithodiplostomum species were placed, most of them described and discussed by Locke et al. (2010a,b).

DISCUSSION

In this study, the first finding of Posthodiplostomum centrarchimetacercariae was reported from pumpkinseeds in a small Hungarian water reservoir (Maconka) in 2015. In 2018, additional data were presented about the current presence of this non-native trematode parasite from Sio channel, which is the outﬂow of Lake Balaton. This was the ﬁrst record of P. centrarchi being in close relation with the waters of Lake Balaton. All samples were examined by morphological, histological and molecular methods;

furthermore, 5 one-day-old chicks were infected with metacercariae in order to obtain adult stages ofP. centrarchi.

The parasite fauna of pumpkinseed in Europe is relatively well studied: for a long time only three monogenean species,Gyrodactylus avalonia Hanek and Threlfall, 1969, Onchocleidus similis M€uller, 1936 and O. dispar M€uller, 1936 were known from Hungary and the neighbouring countries (Gussev et al. 1985;Kvach et al. 2018b;Moravec 2001; Ondrackova et al. 2011; Roman 1953; Roman- Chiriac 1960; Stoyanov et al. 2017b; Vojtek 1958), repre- senting the original American trematode parasite fauna of pumpkinseeds. However, several other protozoan and helminth species common in Europe were also recorded Figure 2.Excysted metacercaria of Posthodiplostomum centrarchi

from the abdominal cavity of a pumpkinseed. Scale bar5100m^m

Figure 3.Histological section of the liver of a pumpkinseed infected with two metacercariae ofPosthodiplostomum centrarchi. M: metacercaria. Haematoxylin and eosin (HE) staining. Scale bar5100m^m

Figure 1.Encysted metacercaria ofPosthodiplostomum centrarchi from the abdominal cavity of a pumpkinseed. Scale bar5100m^m 571

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in thisﬁsh (Moshu 2012,2014;Roman-Chiriac 1960). The pumpkinseed has been present in Hungary since 1905 and it became widespread all over the country during the second half of the last century (Takacs et al. 2017).Molnar (1963,1968)was theﬁrst to study the parasite fauna of the pumpkinseed in Hungary and he reported also O. similis andO. dispar.Recently,Stoyanov et al. (2017a)has proved thatP. centrarchioccurs in several European countries and even in Sturovo (47849⁰ 34⁰⁰ N, 18838⁰ 38⁰⁰ E), Slovakia,

next to the Hungarian border.Kvach et al. (2018a)stated that the infection of the European pumpkinseed population with P. centrarchi might be caused by the repeated imports of largemouth bass (Micropterus salmoides) to Europe. However, the results ofBoone et al. (2018)seem to contradict this by emphasising that there are differences in the host speciﬁcity of Postodiplostomum species towards the ﬁsh species belonging to the genera Micropterusand Lepomis.

Figure 4.Histological section of the atrium of the heart of a pumpkinseed infected with metacercariae ofPosthodiplostomum centrarchi.M:

metacercaria. HE staining. Scale bar5100m^m

Figure 5.Histological section of the liver of a pumpkinseed containing two metacercariae of Posthodiplostomum centrarchi and several specimens ofSchulmanela petruschewskiinematodes (arrows). M: metacercaria. HE staining. Scale bar5100m^m

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The occurrence of P. centrarchi in Hungary showed a unique pattern. After ﬁnding the parasite in a small water reservoir, several efforts were made to recover it from other areas in Hungary where pumpkinseed are abundant. Lake Balaton is the largest natural water in Hungary and the adjacent countries, where the most abundant population of pumpkinseeds is present. Despite dissecting pumpkinseeds of different size from distinct parts of Lake Balaton, no infected specimens were detected. Surprisingly, a relatively heavy infection was recorded in the Sio channel, only a few metres

away from Lake Balaton. In contrast, the native P. cuticola was detectable in cyprinidﬁshes all over Lake Balaton as well as in the river Danube and its tributaries.

The spread of trematodes by their possible final host is understudied due to the difficulties of following water-birds and their parasites across countries. Up to now, Stoyanov et al. (2017a) has reported an adult Posthodiplostomum centrarchifrom the small intestine of a grey heron (Ardea cinerea L.) at a bird recovery centre in Catalonia (Spain), originating from the Lagoon Bassa de les Olles, Ebro Delta, Spain. Our research group tried to get additional data on the host range of this trematode but wild water-birds were not available for research owing to the strict protection of wildlife. However, the infection experiments were successful, although the adult stages in chicks do not necessarily prove the wide host range of this trematode because these freshly hatched birds can be infected by several unspeciﬁc parasites.

Nevertheless, the adult life stages obtained in infection experiments might serve as a basis for further research.

Despite heavy infections with P. centrarchi metacercariae, clinical changes were not found in the dissected pumpkinseed specimens. Remarkable histological changes with a general host reaction were not diagnosed either in the case when metacercariae were located inside internal organs or in the case when dozens of metacercariae covered the Figure 6.Micrographs of adult of Posthodiplostomum centrarchi

collected post mortem from the guts of experimentally infected chicks. Scale bar5100m^m

Figure 7.Maximum likelihood tree of the samples ofPosthodiplostomumspp. from the present study (A: ITS region, B: COI) in relation to other diplostomid sequences deposited in GenBank. Bootstrap values are given at the nodes. Samples from the present study are in bold. The

scale bar indicates the expected number of substitutions per site 799

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serous membranes in the abdominal cavity. Infection of the liver of pumpkinseed with Schulmanela petruschewskii nematodes also seems to be common in the former USSR as Moravec (1994)reported the results ofShulman (1948)who had recorded heavy infections similar to our cases.Stoyanov et al. (2017b) also found this parasite frequently in pumpkinseed in Bulgaria. Nevertheless, changes in the liver due to P. centrarchiinfection, especially in specimens also infected with Schulmanela nematodes, indicate the potential patho- genicity of P. centrarchi.

ACKNOWLEDGEMENTS

This work was supported by the European Regional and Development Fund and the Government of Hungary project GINOP-2.3.2-15-2016-00004: ‘Establishing the sustainable angling-aimed management of Lake Balaton’.

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