New record of monogenean parasites on non-indigenous fishes in the Ukrainian Danube Delta

The Danube Delta is a recognised hot-spot for non-indigenous aquatic species, with 11 new species recorded over the last 20 years. The North-American pumpkinseed (Lepomis gibbosus L., 1758), has been common in the Ukrainian Danube Delta since 1918, while the Chinese sleeper (Perccottus glenii Dybowski, 1877) has only recently been introduced. In April 2017, 21 fish species were recorded during a study of the fish fauna around Vylkove (Ukraine, Danube Delta). The Chinese sleeper was the dominant sampled fish from the studied locality, comprising 41.4% of the fish community, while pumpkinseed comprised only 1.1%. Three non-indigenous monogenean parasites were registered: Gyrodactylus perccotti Ergens and Yukhimenko, 1973 infecting the Chinese sleeper; and Gyrodactylus avalonia Hanek and Threlfall, 1969 and Onchocleidus similis Müller, 1936 infecting pumpkinseed. The American monogenean G. avalonia is reported for the first time in Europe though further molecular/genetic characterisation is needed for species validation. Likewise, G. perccotti, a specific parasite of the Chinese sleeper, is recorded in the Danube Delta for the first time. Finally, our data confirm the presence of the monogenean O. similis, a specific parasite of centrarchid fishes, in the Ukrainian part of the Danube.


Introduction
Non-indigenous host-species can introduce alien parasites into its new range or play a role in distributing local parasites into new habitats (Taraschewski 2006).Many non-native parasites are co-introduced with their original host species.Such parasites often remain a part of the host's parasite fauna and are spread only as the host increases its range (Lymbery et al. 2014).Some co-introduced parasites can also switch to new native host species, however, greatly increasing the speed at which it spreads through the new environment (Galli et al. 2005).In this case the parasite forms a complicated bicomponent parasitic system, where parasite success in the new environment depends on the predisposition of the new hosts to broader ecological and physiological parameters (Machkevskyi et al. 2015).
The Chinese sleeper is an Asian fish species that was first introduced into the Danube basin via the upper River Tisza drainage in Ukraine in 1995 (Sivokhop 1998) and is now invasive in many European waters (Kottelat and Freyhof 2007).In Europe, the fish hosts two co-introduced helminth species: the monogenean Gyrodactylus perccotti Ergens and Yukhimenko, 1973 in the River Vistula, Poland (Ondračková et al. 2012); and the cestode Nippotaenia perccotti (Akhmerov, 1941) in the River Tisza, Slovakia (Košuthová et al. 2004).Moreover, G. perccotti has been reported from the River Dnieper in Ukraine and the River Tisza in Hungary (Antal et al. 2015;Zaichenko 2015) and N. perccotti from the Rivers Dniester and Tisza (Kvach et al. 2013(Kvach et al. , 2016b;;Antal et al. 2015), and in the River Vistula in Poland (Mierzejewska et al. 2010).
Pumpkinseed, which is one of the most widespread non-native fish species in Europe, has been found in around 30 different countries (Copp and Fox 2007).In Ukraine, the species has a range stretching from the Kalmius river basin in the east (Diripasko et al. 2008) to the Middle Dnieper basin near Kiev in the north (Tsyba 2011;Afanasyev et al. 2017).Specific monogenean species are known to parasitise pumpkinseed in different parts of Europe, such as France (River Durance; Havlátová et al. 2015), the Ukraine (Rubtsova 2015), Italy (subalpine lakes; Galli et al. 2003Galli et al. , 2005)), Norway (ponds; Sterud and Jørgensen 2006) and in southwestern England (Hockley et al. 2011).In addition to the co-introduced monogeneans, pumpkinseed act as host to the American digenean, Posthodiplostomum minimum centrarchi Hoffmann, 1958, which has also been recorded in the Danube basin (Kvach et al. 2017;Stoyanov et al. 2017).All other pumpkinseed parasites registered in Europe are of local origin.
The aim of the present study was to describe the presence of non-indigenous parasites co-introduced along with their non-indigenous fish hosts, the Chinese sleeper and the pumpkinseed, in the Ukrainian part of the Danube Delta.

Material and methods
On 25.04.2017,we sampled the fish community in a canal connected with the River Danube at the town of Vylkove, Danube Delta, south-western Ukraine (45º24′N; 29º35′E).The canal has muddy bottom overgrown with submerged aquatic vegetation.Fish were sampled using a 10-m beach-seine at a depth of around 1 m.The seine was towed about 50 m along the shore with about 50% capture (in total of 250 m 2 was covered by sampling).All fish were identified to species, counted and species dominance calculated as the percentage of all species caught.There are no published studies of fish samples in this region, so we present the data from the sample in Table 1.Two invasive fish species, the Chinese sleeper and the pumpkinseed, were caught and individuals larger than 20 mm were taken for further parasite analysis in the laboratory; all other species being released alive where they were caught.The fish were kept in aerated water and transported to the laboratory of the Institute of Marine Biology, National Academy of Sciences of Ukraine in Odessa.
Eleven Chinese sleeper (standard length [SL]; mean ± S.D. = 69 ± 8.3; range 50-83 mm) and two pumpkinseed (SL 42 and 44 mm) were examined for parasites within 48 h of capture (Kvach et al. 2016a).Monogeneans were mounted in glycerine-ammoniumpicrate solution (GAP) in accordance with Malmberg (1970).The slides were then studied under an Olympus BX50 light microscope equipped with phase contrast, differential interference contrast and Olympus MicroImage ™ Digital Image Analysis software (Olympus Optical Co.).The voucher material has been deposited in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (acronym IPCAS).

Results
A total of 21 fish species were recorded in the fish sample (Table 1), with the invasive Chinese sleeper dominant at the sampling locality (41.4% of the fish sample), followed by native bleak Alburnus alburnus (L., 1758) (7.2%), loach Cobitis elongatoides Băcescu and Mayer, 1969 (6.1%) and three-spined stickleback Gasterosteus aculeatus L., 1758 (6.1%).Pumpkinseed represented just 1.1% of the fish sample.One of the 11 Chinese sleepers caught was found to be infected with a specimen of an Asian monogenean, G. perccotti (Gyrodactylidae).Both pumpkinseed were infected with North-American monogeneans, one with two specimens of Gyrodactylus avalonia Hanek and Threlfall, 1969 (Gyrodactylidae) and the second with two specimens of Onchocleidus similis Müller, 1936 (Ancyrocephalidae).

Discussion
In this paper, we report the presence of an American gyrodactylid monogenean G. avalonia in Europe for the first time (though species identification is still awaiting molecular/genetic characterisation).In addition, our data confirm the presence of the ancyrocephalid monogenean O. similis, a specific parasite of centrarchid fishes, in the Ukrainian stretch of the River Danube; and the presence of G. perccotti, a specific parasite of the Chinese sleeper, in the Danube Delta for the first time.
Gyrodactylus avalonia was first described on the fins of a three-spined stickleback from the brackish waters of Witless Bay Estuary in Newfoundland, Canada (Hanek and Threlfall 1969).Later, the species was documented on the fins of pumpkinseed in the freshwater Bay of Quinte in Lake Ontario, Canada (Hanek and Fernando 1971).This parasite is described as an extreme generalist among gyrodactylids, infecting eight host species from five different orders (Bakke et al. 2002).While G. avalonia is suspected of being a junior synonym of G. arcuatus Bychowsky 1933, it remains valid as a species until molecular/genetic characterisation is conducted (J.Lumme and S.D. King, pers. comm., in Rubio-Godoy et al. 2012).
According to the "parasite spillover" concept, an introduced host may serve as a source of new parasitic agents for native hosts (Kelly et al. 2009).As G. avalonia displays broad halotolerance and halohabitat distribution (Cone and Wiles 1985), shows low host specificity (Bakke et al. 2002) and its main host, G. aculeatus, is relatively abundant in the Danube Delta, the consequent spread of this species in Europe is highly likely.Moreover, as G. avalonia has been shown to pass on pathogenic bacteria to its fish host, further dispersal potentially represents a threat to local fish fauna (Cusack and Cone 1985).
Gyrodactylus perccotti was recorded in Europe for the first time in the Vistula Basin (Ondračková et al. 2012).The parasite was later documented in the Middle Dnieper near Kiev, Ukraine (Zaichenko 2015) and in the River Tisza Basin and the Lake Balaton drainage in Hungary (Antal et al. 2015) (Figure 2).The results of this study also confirm the presence of G. perccotti in the Ukrainian stretch of the Danube.Given the non-indigenous origin of this parasite and its host specificity (Sokolov et al. 2012), its presence on the Chinese sleeper could point to the species' fish host invasion history (Kvach et al. 2016b;Reshetnikov et al. 2017).Gyrodactylus perccotti was not recorded during a study investigating 70 individuals of the Chinese sleeper in the River Prut drainage (Moldova) in 2012 (Sokolov and Moshu 2013).Therefore, it is more likely that the Chinese sleeper was introduced (or expanded) into the Danube Delta from the Tisza basin via the main Danube channel.
The first study on pumpkinseed parasites in the River Danube was undertaken by Roman (1953Roman ( , 1955)), who recorded two monogenean species (Onchocleidus dispar Müller, 1936 and O. similis) in the lower Danube near Corabia and Drobeta-Turnu Severin, Romania.Both of these species were later recorded in the Ukrainian stretch of the Danube (Pashkevichute 1971).The occurrence of O. similis on the pumpkinseed in our study corresponds with the results of Kulakovskaya and Koval (1973), who also reported this species at Vylkove in the Danube Delta over 50 years ago.Since its first record in Europe, O. similis has been documented from several other sites, including Slovakia (summarised in Moravec 2001), the middle and lower Danube (Molnár 1968;Ondračková et al. 2011), subalpine lakes in Italy (Galli et al. 2003(Galli et al. , 2005) ) and the River Durance in south-eastern France (Havlátová et al. 2015) (Figure 4).As with other parasites collected from the lower Danube basin (e.g.Roman 1953;Ondračková et al. 2011), O. similis from the Danube Delta exhibited smaller haptoral hard parts than those observed in the parasite's native area (Hoffman 1998), probably reflecting host size.
The Danube Delta region has a strong influence on waters of the Black Sea, with sea water reaching the town of Vylkove (Liashenko andZorina-Sakharova 2014, 2015) and branch waters and tributaries along the Ukrainian coast reaching an average salinity of around 0.29-0.81‰(max.4.72‰).This provides appropriate conditions for the survival of the parasite G. avalonia, whose natural range includes harbours along the coastal waters of Newfoundland and whose type host is the brackish-water three-spined stickleback (Hanek and Threlfall 1969).Two species of Onchocleidus, O. similis and O. dispar, occur along the middle and lower stretch of the Danube (Ondračková et al. 2011), aside from the delta where only O. similis has been recorded (Kulakovskaya and Koval 1973;current data).Spreading of pumpkinseed in the Black sea region occurs during the spring floods, when riverine flood waters deliver fish to the open sea and thence on to the deltas of other rivers (Afanasyev et al. 2017).This same method was the vector for pumpkinseed expansion into the rivers Dniester and Dnieper, where pumpkinseed populations are genetically identical to those in the Danube (Slynko et al. 2014).At least one parasite species, O. dispar, has been co-introduced into the River Dnieper with its host (Rubtsova 2015).We can therefore expect further range expansion of O. similis into other river drainages in the Black Sea basin along with its host, the pumpkinseed.