Parasites as biological tags of divergence of black-striped pipefish, Syngnathus abaster (Actinopterygii: Syngnathiformes: Syngnathidae), populations in their natural and acquired range

Parasites as biological tags of divergence of black-striped pipefish, Syngnathus abaster (Actinopterygii: Syngnathiformes: Syngnathidae), populations in their natural and acquired range


Introduction
Species range extension is a natural process stretched over time and it ultimately contributes to the formation of biodiversity (Alexandrov et al. 2007;Polačik et al. 2008).However, over recent decades, human activity has caused natural range borders to change significantly, spreading many species, including fishes (Bij de Vaate et al. 2002;García-Berthou et al. 2005;Hirsch et al. 2016).As a result, non-native (or alien) species are now having adverse effects (e.g., Ponto-Caspian gobiids in the Rhine River basin and the North-American Great Lakes, rainbow trout in European mountain locations, etc.) in many recipient ecosystems and, as such, they represent one of the biggest challenges for global biodiversity today (Leppäkoski et al. 2002;Hirsch et al. 2016).
Ukraine is located at a major crossing of transport corridors (land, freshwater, and marine), representing important routes for the spread of alien aquatic species (Alexandrov et al. 2007;Semenchenko et al. 2016).At least two crucial aquatic biological invasion routes pass through Ukraine's aquatic ecosystems, the so-called Southern and Central Corridors (Panov et al. 2009), and many Ponto-Caspian and Mediterranean species have now increased their ranges along these corridors (Tutman et al. 2012;Semenchenko et al. 2016;Marenkov 2018;Dobrzycka-Krahel et al. 2023).One such group is the "neolimnetics", which have a marine/brackish water origin but have spread into freshwater habitats in Ukraine and other European countries.These include species such as the Ponto-Caspian gobiids, the Ukrainian stickleback, Pungitius platygaster (Kessler, 1859); the black-striped pipefish, Syngnathus abaster Risso, 1827; and the big-scale sand smelt, Atherina boyeri Risso, 1810 (see Kvach and Kutsokon 2017).
The black-striped pipefish is an Atlanto-Mediterranean fish species with a natural range from the southern Gulf of Biscay in the north to Gibraltar in the south, the Mediterranean and Black Seas, and the estuarine zones of their rivers (Dawson 1986).It has a maximum body length of 21.5 cm (more commonly 16.0 cm) and a maximum weight of 3.0 g (age unknown).It lives along the coastal zone, mainly in shallow waters, and is tolerant of waters with different salinities (e.g., it is found along the coast of Crimea and in the Dnipro River near Kyiv), though it is far more common and numerous in estuaries and desalinated coasts than in open sea areas.The species matures at 2-3 years of age with a body length of ca.7.0 cm and spawns from April to September, laying up to 200 eggs (usually ca.100).The male accepts eggs from several females and carries them around in a brood sac.The species feeds on plankton and benthic organisms, tiny and medium-sized crustaceans, insect larvae, fish roe, and occasionally algae.
While the species has a brackish water origin, its high tolerance to freshwater has allowed it to spread up all large rivers in the Black Sea basin, i.e., the Danube, Dniester, Southern Buh, and Dnipro (Slastenenko 1956).It has been recorded in the Ros River (Dnipro basin) since the begin-ning of the 20 th century and is now present in all reservoirs and the rivers Prypyat and Desna, though it is presently only found in small tributaries in the lower part of the basin (Beling 1923;Zimbalevskaya et al. 1989;Movchan 2012;Movchan and Roman 2014).In the Danube, the species has been recorded as far as Serbia and Bosnia and Herzegovina (Sekulić et al. 1999;Tutman et al. 2012).
Parasites are commonly used as biological tags of their host populations (Moser 1991;Mackenzie 2002).Fish parasite communities can be used to discriminate fish stocks and populations and, as such, a process known as "biological tagging" (Catalano et al. 2014;Poulin and Kamiya 2015;Kvach et al. 2017;Kutsokon et al. 2022) and the same biological tag can be used as a marker to clarify the possible origin of invasive populations (Hohenadler et al. 2018a(Hohenadler et al. , 2018b;;Ondračková et al. 2012b;2019;2021).A total of 18 parasite species have been recorded for populations of the Black Sea basin blackstriped pipefish (Gaevskaya et al. 1975).However, data for parasites in its acquired range are primarily related to the Danubian population in Bulgaria, where nine species were recorded (Ondračková et al. 2012a), and the Dnipro population in Ukraine, where just two ciliate species were recorded (Trichodina partidisci Lom, 1962 andTrichodina acuta Lom, 1961;see Yurishinets 2010).Consequently, very little is known about parasites acquired in the species' non-native freshwater habitats.
Therefore, the presently reported study aimed to describe the current range of black-striped pipefish in Ukrainian waters and obtain biological tag data (parasites) for the populations.

Materials and methods
The fish were sampled using the 10 × 1 m dipnet and a 1 × 0.5 m diameter hand net (0.5 cm mesh).In total, 107 fish were sampled from five different localities during the warm seasons of 2020-2021 (Table 1; Fig. 1).These included one marine locality (Gulf of Odesa), two deltaic zones (Danube and Dniester deltas), and two localities in the middle Dnipro basin (Lake Vyazky and the Stuhna River).In the Danube Delta, fish were sampled from three different sites (Fig. 1), considered the same locali- ty for further analysis.The salinity of the Gulf of Odesa is 10‰-17‰ depending on the season (Zaitsev 1992).
Once caught, the fish were transported alive to the laboratory in aerated cans filled with water from the sampling site, where they were placed in aerated aquaria containing water from the place of capture for no more than two days (Kvach et al. 2016) before being examined for parasites.Findings of black-striped pipefish from other sites were registered (without removing the fish) and used to evaluate the range of the species (Fig. 1).In addition, a literature search was undertaken (Movchan 2012;Movchan and Roman 2014;Demchenko 2017;Kutsokon and Kvach 2021;Kutsokon and Roman 2021;Kutsokon et al. 2021aKutsokon et al. , 2021b) ) to obtain the latest information on the species' present-day range.
Prior to dissection, the standard length (SL, mm) and sex was determined for each fish (Table 1).After humanely sacrificing the fish, smears of gill and fins mucosa were taken, dried, and stained according to Klein (1958) to identify ciliates.Likewise, blood smears were dried and stained in hematoxylin (Giemsa 1904) to identify other unicellular organisms.Fresh smears from muscle and the gall and uterine bladders were examined for myxozoans and any living spores mounted onto gelatin gel as semi-permanent preparations for further identification.Monogeneans were placed onto glass slides and mounted in glycerin-ammonium-picrate for morphological study (Malmberg 1957).Cestodes, digeneans, and nematodes were fixed with hot 4% formalin (Cribb and Bray 2010), and glochidia and crustaceans in cold 4% formalin.Acanthocephalans and nematodes were mounted in glycerin as temporary slides for further species identification, while cestodes and digeneans were stained in iron acetocarmine and mounted onto Canada balsam slides (Georgiev et al. 1986).All parasites were identified to the lowest possible taxa, with parasite taxonomy presented following the World Register of Marine Species (WoRMS 2022).Parasitological terminology and principal indices, such as prevalence, intensity, mean intensity, and abundance, were used in accordance with Bush et al. (1997): where n is the number of infected fish and N is the number of fish examined.Intensity (of infection) (I) is the number of individuals of a particular parasite species in a single infected host (individual fish) usually presented as the intensity range Mean intensity (of infection) (I M ) where N TP is the total number of individuals of a particular parasite species found in all fish examined and N inf is the number of infected fish.Abundance (A) where N T is the total number of individuals of a particular parasite species found in all fish examined and N is the number of fish examined.
For microparasites (unicellular and myxozoans), only the prevalence was calculated, with the intensity of infection evaluated by the presence of microparasites in the microscope's field of view (Mierzejewska et al. 2012)   The Czekanowski-Sørensen index (CSI) was calculated (Sørensen 1948) to analyze differences in parasite fauna at different localities, with differences considered high in cases where index parameters were < 50%.
Statistical differences in length were evaluated using t-tests and F-tests for comparing two samples in Statistica for Windows 10 (StatSoft).Standard deviation values (SD) were calculated for mean parameters in each case.Visualization of fish size data was carried out in PAST v.4.03 (Paleontological Statistics Software system), using the search statistics methods (box and whisker), comparative analysis, and cluster analysis (Hammer et al. 2001).Discriminant analysis was then performed to evaluate differences in the respective parasite communities.

Results
We observed no significant difference in the length of black-striped pipefish from marine (Gulf of Odesa), estuarine (Danube and Dniester deltas), or freshwater (Lake Vyazky and Stuhna River) localities (Fig. 2).
Based on published literature and databases, we consider the modern range of black-striped pipefish in Ukraine to include both brackish and freshwaters of the Black Sea basin (Fig. 1).In rivers where the populations have been established since the middle of the 20 th century, the species is still to be found mainly in the lower reaches, though it has spread as far as the Belarus border in the Dnipro Reservoir cascade (Fig. 1), and into many of the lower first-order tributaries of the Dnipro River, regardless of size (i.e., medium rivers such as the Ros and Desna, and small rivers such as the Leglych and Mokra Moskovka).Despite much long-term research in the area, the black-striped pipefish has not been found in upper-flow tributaries to date.While the highest recorded finding was in the middle flow of the Ros River in 1923 (Beling 1923), all later records of the species have been from the lower reaches of the river (Kutsokon 2010).The species is also found in the lower reaches of the Dniester, Danube (along the entire stretch within the borders of Ukraine), Tyligul, Southern Buh, and rivers of the Crimean and Northern Azov coasts (Demchenko 2017;Kutsokon and Kvach 2021;Kutsokon and Roman 2021;Kutsokon et al. 2021b).
The most numerous species were ciliates of Trichodina spp., with the prevalence varying from 5.9% to 100% and intensity of infection of up to several thousand cells (Table 2).Among the ciliates, we identified at least three species, most represented by Trichodina partidisci, found in both freshwater (except the isolated Lake Vyazky) and marine/brackish sites.This is a smallsized species (23.7-31.5 µm; 27.6 ± 2.9), with a denticle ring diameter of 10.4-16.1 µm (13.5 ± 2.0) and a denticle number of 18-24 (22).It can be recognized by the broadly rounded distal surface of its denticle blade and the (usually) few, irregular unstained granules in the central part of its adhesive disc (Fig. 3).Trichodina rectuncinata, a species common on marine fish, was only found in the Gulf of Odesa.Again, this is a small-sized species (23.0-30.5 µm, 28.8 ± 1.7) with a denticle ring diameter of 12.3-15.2µm (13.6 ± 1.2) and a denticle number of 22-25 (24), recognized by a triangular blade with a cavity in the center (Fig. 3).Trichodinella epizootica, a small mobilid typical of freshwaters, was registered in just one fish from the Danube Delta.
Ukrainian pipefish populations differed with the abundance values for Nicolla skrjabini and Diplostomum spp.3), with a comparative analysis of parasite communities using the Czekanowski-Sørensen index (CSI) distinguished marine populations from freshwater/brackish populations (Fig. 4).CSI, Mahalanobis distance, and the Fischer criterium all showed significant differences between the Gulf of Odesa and the Danube and Dniester deltas (Table 4), with CSI parameters showing the largest differences between all localities.

Discussion
This study provides comprehensive new data on the parasites of black-striped pipefish in Ukraine, along with supporting data on its acquired range in Ukrainian freshwaters.Our new data confirm that the species is now found in the coastal zones of the Black Sea and the Sea of Azov and the deltaic zones of rivers and reservoirs of   the Dnipro basin (Movchan 2011;Snigirov et al. 2020).
However, the only new findings we recorded were inside the species' established range in the Lower Dnipro basin, suggesting that its Ukrainian range has probably now stabilized since it started spreading in the middle of the 20 th century.
Several parasite species were registered on this host for the first time, i.e., Trichodinella epizootica, Trypanosoma sp., Bothriocephalus scorpii, Progrillotia dasyatidis, Ophiotaenia europaea, Cryptocotyle jejuna, Metorchis xanthosomus, Tylodelphys clavata, Holostephanus luehei, Contracaecum rudolphii, Mothocya epimerica; and Unionidae gen.sp.(Table 2).In addition, we confirmed the presence of several species previously recorded on the black-striped pipefish, including several ciliate species.While these are common on a wide spectrum of freshwater and brackish water hosts (Kostenko 1981;Grupcheva et al. 1989;Yurishinets 2010), no specific ciliates were previously known for black-striped pipefish.The most common species found was Trichodina partidisci, a parasite of mugilid fish in the Black Sea (Lom 1962) that has a wide spectrum of hosts (Grupcheva et al. 1989;Lom and Dyková 1992).In Ukrainian freshwaters, it is known from pipefish in the middle Dnieper basin (Yurishinets 2010).A second species, Trichodina rectuncinata, a widespread marine fish parasite, has previously been   recorded in different areas of the Atlantic and Pacific oceans (Xu et al. 2001;Islas-Ortega et al. 2020;Öztürk and Güven 2022).
While it has previously been registered in fish from the Danube Delta (Kvach et al. 2020), this is the first time it has been registered in the Dniester River.
In the Gulf of Odesa and the Danube Delta, sporadic cases of parasitism by Contracaecum rudolphii nematode larvae were noted, the adult worms being common parasites of pelicans, herons, mergansers, and cormorants (Sreedevi et al. 2017).Previous authors (e.g., Gaevskaya et al. 1975) have also noted the presence of another species of this genus, Contracaecum microcephalum (Rudolphi, 1809), along with larvae of Agamonema sp.nematode in the parasitefauna of marine pipefish.
The isopod Mothocya epimerica, a parasite of the branchial and oral cavities of sand-smelts (Atherina spp.), was only noted at a marine location with few indications of invasion (Bruce 1986;Leonardos and Trilles 2004).The parasitic copepods Ergasilus lizae Krøyer, 1863 and Ergasilus ponticus Markevich, 1940 have also previously been reported from marine populations of the blackstriped pipefish (Gaevskaya et al. 1975).
The black-striped pipefish is now widespread in Ukrainian bodies of water, particularly in coastal brackish and freshwaters of the Black Sea and the Sea of Azov basins.In Ukraine, it is found along all shores of the Black and Azov Seas and in the estuaries, near-estuary and estuarine zones of their rivers, from where it has entered reservoirs and rivers connected to the sea (below the Danube, Dniester, and Southern Buh).It is also found in all reservoirs along the Dnipro and the Siversky Donets River (Movchan 2011).Previous studies have confirmed differences in biotope preferences between marine pipefish, which prefer plant thickets, and those in freshwaters, which prefer muddy biotopes (Ondračková et al. 2012a; middle Danube).Ondračková et al. (2012a) noted the absence of any parasites specific to this fish species, suggesting that the formation of the species' parasite component communities depends entirely on the environmental factors affecting each population.In such cases, the local features of the parasite communities will depend on the following: • The presence of "marine" species of unicellular parasites (ciliophores) in marine localities (10‰-17‰ salinity) only as a refraction of relative stenohalinity (Trichodina rectuncinata), or findings of "marine" ciliate species in freshwater locations, as an example of successful osmoconformation (Trichodina partidisci).• The presence of multicellular parasites in localities with abiotic/biotic conditions that allow completion of complex life cycles, i.e., trematodes (freshwater/ marine mollusks as obligate first hosts) or cestodes (freshwater/marine invertebrates as intermediate hosts or marine/freshwater vertebrates as definitive hosts).
Overall, the parasite fauna of neolimnetic black-striped pipefish exhibits two main parasite community formation strategies in their acquired ecosystems: • Parasite release (very poor communities in freshwater).• Acquisition of local parasite species, which have overcome the filters of encounter and adaptation (Combes 1995).
Our findings confirm that analyzing changes in the structure of neolimnetic fish parasitic communities that over-come geographical and ecological barriers is a convenient model for establishing the patterns and features of hydrobiont distribution beyond the boundaries of natural habitats.
as: • Sporadic (S), from 1 to < 10 individuals in the material examined; • Not numerous (NN), < 10 individuals in < 10% of field of view; • Numerous (N), up to 20 individuals in > 50% of field of view; • Very numerous (VN), > 20 individuals in > 50% of field of view; • Mass (M), dozens of individuals in each field of view.

Figure 1 .
Figure 1.Map of Ukraine indicating the sampling localities with current sampling sites (2020-2021) marked with triangles.

Figure 4 .
Figure 4. Dendrogram of similarity expressed by Czekanowski-Sørensen index for parasites component communities of Syngnathus abaster at different localities in Ukraine.

Table 1 .
Number (n)and standard length (SL) of Syngnathus abaster from different localities in Ukraine.

Table 2 .
Parasite communities of Syngnathus abaster from various localities in Ukraine (as determined in the presently reported study).

Table 3 .
Discriminant function analysis of parasite communities of Syngnathus abaster from different localities in Ukraine.

Table 4 .
Matrix of differences between parasite fauna/communities of pipefish from different localities.