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

Two species of the genus Posthodiplostomum (Digenea: Diplostomatidae) ( Posthodiplostomum brevicaudatum Nordmann, 1832 and Posthodiplostomum cuticola Nordmann, 1832) are known as parasites of Hungarian native ﬁ shes. Metacercariae of P. cuticola are widespread in Europe and cause black spot disease. Several species of Posthodiplostomum were described also from North America but none of them has been isolated in Hungary up to now. Posthodiplostomum centrarchi Hoffman, 1958 has been detected recently in pumpkinseeds ( Lepomis gibbosus L., 1758) in several European countries. Posthodiplostomum centrarchi was isolated for the ﬁ rst 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 Si (cid:1) o channel) were sampled in order to determine its presence and distribution. Only the native species P. cuticola was detected in Lake Balaton on cyprinids but a relatively high infection rate was observed in the Si (cid:1) o 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 of P. centrarchi and P. cuticola clustered into two distinct branches, which was in agreement with the morphological results.

New record of metacercariae of the North American Posthodiplostomum Centrarchi (digenea, diplostomidae) in pumpkinseed (Lepomis Gibbosus) in Hungary INTRODUCTION Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) species are distributed worldwide (L opez-Hern andez et al., 2018;Niewiadomska 2002;Ritossa et al. 2013) and well known as fish 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 Posthodiplostomum infection that occurs on the body surface, fins, scales, or in the musculature and body cavity of fishes (Hor ak et al. 2014;Ondra ckov a et al. 2004;Tobler and Schlupp 2008;Zrn ci c et al. 2009). These encysted stages often induce developmental disorders of the fish skeleton that may be lethal in the case of fingerlings (Luck y 1970). The most common signs are weight loss due to appetite decrease, lesions of the liver and kidney, and digestive dysfunction (Iqbal et al. 2014;Lane and Morris 2000;L opez-Hern andez et al. 2018;Rolbiecki 2004;Sch€ aperclaus 1990).
More than 25 species of Posthodiplostomum have been described in the world (L opez-Hern andez 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;Moln ar 1969;Moravec 2001;Moshu 2012Moshu , 2014Roman-Chiriac 1960). In Hungary, the native species P. cuticola and P. brevicaudatum were documented by Jacz o (1941) and Moln ar (1968), while Sz ekely and Moln ar (1996) reported only P. cuticola. Recently, the presence of P. centrarchi has been reported published from several European countries: Bulgaria, Slovakia, the Czech Republic, Portugal, Ukraine and Germany (Kvach et al. 2017(Kvach et al. , 2018aOndra ckov a et al., 2019;Stoyanov et al. 2017a).
The nomenclature and the fish intermediate host specificity of P. centrarchi are still under debate. Hoffman (1958Hoffman ( , 1999 considered P. centrarchi a subspecies of P. minimum and named it P. minimum centrarchi because he proved that it could infect only centrarchid fishes while P. minimum minimum was able to infect only cyprinids. The strict host specificity of the P. minimum subspecies was supported by Locke et al. (2010a,b), Lane et al. (2015), Stoyanov et al. (2017a) and Boone et al. (2018). Accordingly, Stoyanov et al. (2017a) elevated P. minimum centrarchi to species rank as P. centrarchi and this novel name has become general since then (Kvach et al. 2018a;Ondra ckov a et al. 2019).
Metacercariae of an unknown Posthodiplostomum species, non-indigenous in Hungary, were first 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 centrarchid fishes. 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 of P. 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 Si o 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 conditions. Both metacercariae and adult stages were studied by morphological and molecular 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 by P. centrarchi metacercariae 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 Si o channel (Table 1). In the littoral zone of Lake Balaton, the pumpkinseed is the most abundant non-native fish species (Czegl edi 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 Si o 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 mm 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 interference contrast optics, then photographed with an Olympus DP 20 digital camera.

Experimental infection
Two experimental infections were carried out to obtain adult stages of Posthodiplostomum spp. 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 domesticus L., 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 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 commercial supplier (Hegyh at BR Kft., Szentgotth ard-R abaf€ uzes, Hungary). Formal ethical approval was given by the Government Office of Pest County (permit PEI/001/1004-4/ 2015).

Molecular methods
Samples preserved in 80% ethanol were centrifuged at 8,000 g 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 mL 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 S andor et al. (2017). The amplification of COI was performed using the primers (Plat-diploCOX1F and Plat-diploCOX1R) and protocol described by Moszczynska 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 (5 0 -TGTCGAT-GAAGAGCGCAGC-3 0 ) and 5.8S2 (5 0 -TAAGCCGACCCTCGG-ACAGG-3 0 ) (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 (MN080274À92, MN179280À90, 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 in Table 3. Bolbophorus damnificus 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 HKY þ G model for the ITS region and GTR þ G 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 corresponded to P. centrarchi (Figs 1 and 2) as characterised and depicted by Hoffman (1999) and Kvach 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 Si o 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 Si o channel showed infection with 20-150 metacercariae. These two Posthodiplostomum species could be identified and distinguished easily under compound and dissecting microscope based their morphology and predilection site. All specimens of P. centrarchi were found in the abdominal cavity contrary to metacercariae of P. cuticola, which were located in the skin, muscles and fins. In histological sections, the P. centrarchi cysts 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 Si o 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 with P. centrarchi metacercariae 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 with P. cuticola metacercariae, only 2 adult flukes were found in the bursa of Fabricius of a single chick after incubation for 8 days.

DISCUSSION
In this study, the first finding of Posthodiplostomum centrarchi metacercariae 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 Si o 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 of P. centrarchi.
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 centrarchi from 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 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 of Shulman (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. centrarchi infection, especially in specimens also infected with Schulmanela nematodes, indicate the potential pathogenicity of P. centrarchi.