Ecological study on helminths of three species of Gobiidae from the Danube River, Bulgaria

The Danube River crosses ten countries and flows into the Black Sea through a vast delta. The ichthyofauna of the river includes over 100 species, and 68 have been reported for the ichthyofauna of the Bulgarian section of the Danube (Zarev et al., 2013). For the section of the Danube River between Bulgaria and Romania, six species of gobies have been reported, including racer goby, Babka gymnotrachelus (Kessler, 1857; syn. Neogobius gymnotrachelus); monkey goby, Neogobius fluviatilis (Pallas, 1814); round goby, Neogobius melanostomus (Pallas, 1814; Vassilev et al., 2008). Four species of gobies, including B. gymnotrachelus, N. fluviatilis, and N. melanostomus, are spread upstream of the Danube River, outside their natural habitats (Ondračková et al., 2009). The Danube River plays an important role in expanding the range of various fish species (Bódis et al., 2012), which can lead to the spread of their parasites (Juhásová et al., 2019). Some non-native fish species become food for many native predatory fish species (Bódis et al., 2012). The infection of the fish is carried out through feeding (Docan et al., 2019). The parasites’ life cycles involve several intermediate hosts (Kuzmanova et al., 2019), including fish. Fish are rich in nutrients (Ljubojevic et al., 2015) and are often present in the human diet (Bănăduc et al., 2016). A person can become infected by consuming raw fish or fish without sufficient heat treatment (Scholz, 1999; Ljubojevic et al., 2015). Among the endoparasitic species causing zoonoses are E. excisus (Ljubojevic et al., 2015; Juhásová et al., 2019) and Contracaecum sp. (Demir & Karakişi, 2014; Shamsi, 2019). Different authors have studied the parasite fauna of gobies from the Danube River in other countries (Ondračková et al., 2005; Molnár, 2006; Kosuthova et al., 2009; Ondračková et al., 2009; Mühlegger et al., 2010; Francová et al., 2011; Ondračková et al., 2012; and others). The parasite fauna of N. fluviatilis, N. melanostomus, and others from the Black Sea was studied by Kvach (2005). Helminthological studies on gobies, including racer goby, monkey goby, and round goby, have also been carried out in the Danube River and the river basin in Bulgaria (Francová et al., 2011; Ondračková et al., 2010; Ondračková et al., 2012; Kirin et al., 2013; and others).

The present study aims to provide new data on the helminths and helminth communities of the three species of gobies (B. gymnotrachelus, N. fluviatilis, N. melanostomus) from the upper section of the Danube River (near the villages Kudelin, Novo selo, Koshava, and Kutovo) in Northwestern Bulgaria. The study enriches the data on the ecological indices of endohelminths of the three species of gobies. More research is needed on the parasite fauna of fish, especially those frequently consumed by humans and infested with pathogenic endohelminth species.

In 2019 – 2021, during the spring, summer, and autumn, an ecologohelminthological study was conducted on a total of 72 specimens of gobies (Gobiidae) of the species: racer goby, Babka gymnotrachelus (Kessler, 1857); monkey goby, Neogobius fluviatilis (Pallas, 1814); round goby, Neogobius melanostomus (Pallas, 1814) near the villages Kudelin, Novo selo, Koshava and Kutovo, denoted as sampling sites (44°12′07.9″N 22°41′27.0″E; 44°09′52.4″N 22°47′09.8″E; 44°03′32.3″N 23°02′02.8″E and 44°00′58.4″N 22°58′30.3″E), the Danube River, Northwestern Bulgaria (Fig. 1). B. gymnotrachelus, N. fluviatilis are freshwater, brackish, and benthopelagic species. N. melanostomus is a marine, freshwater, brackish and benthic species. They are found in the Danube River, the lower current and mouths of its tributaries, in the mouths of rivers flowing into the Black Sea and others. The three species feed on crustaceans, insects, and others. B. gymnotrachelus inhabits muddy, sandy, or stony bottoms; N. fluviatilis prefers muddy and sandy bottoms; N. melanostomus is found on stony and sandy bottoms (Karapetkova & Zhivkov, 2006; Vassilev et al., 2012). The three studied species of gobies are in the category “LC = Least Concern” on the IUCN Red List. The monkey goby is also included in Annex No. 3 of the Bern Convention (Freyhof & Brooks, 2011; https://www.iucnredlist.org).

Fig. 1.

Collecting fish samples was carried out under the permit for fishing for scientific research purposes from the Executive Agency for Fisheries and Aquaculture, Ministry of Agriculture. Nine specimens of B. gymnotrachelus (Kudelin and Novo selo), 48 specimens of N. fluviatilis (Kudelin, Koshava, Novo selo, and Kutovo) and 15 specimens of N. melanostomus (Kudelin, Koshava, and Novo selo) were subjected to ecologohelminthological examination.

The scientific names of the fish species are used according to Vassilev et al. (2012); Karapetkova and Zhivkov (2006). Immediately after their capture, all fish were weighed and measured; the metric data – total length, L, cm; maximum height, H, cm; body weight, W, g, were recorded (Table 1).

Fish were euthanized by being in a container with a drop of oil of cloves. A visual inspection of the fish's body's surface was performed, followed by dissection and fixation of the internal organs in ethyl alcohol for further processing. All caught specimens of gobies were examined for the presence of parasites according to standard methods (Zashev & Margaritov, 1966; Bauer (Ed), 1987; Moravec, 2013). The isolated parasites were fixed and preserved in 70 % ethyl alcohol until further processing. From the isolated helminths from class Trematoda, permanent microscope slides (by Georgiev et al., 1986), and from the representatives of classes Acanthocephala and Nematoda – temporary microscope slides (by Zashev & Margaritov, 1966; Petrochenko, 1956; Moravec, 2013) were prepared. Basic ecological indices: total number of species; the total number of specimens; mean intensity (MI); mean abundance (MA); prevalence (P%), and range (R; min – max; by Bush et al., 1997) were presented.

This research on fish has complied with all the relevant national regulations and institutional policies for the care and use of animals.

During the ecologohelminthological research of the three species of gobies, 6 endohelminth species, of which 3 pathogenic species were found. E. excisus is a pathogenic species to fish (Novakov et al., 2015) and humans (Juhásová et al., 2019). P. laevis is a pathogenic fish species (Novakov et al., 2015). Contracaecum sp. is pathogenic to fish and humans (Zashev & Margaritov, 1966; Demir & Karakişi, 2014). In this study, P. laevis was found in the intestines of the examined fish; E. excisus is localized under the serous membrane on the surface of the mesentery and internal organs, in the body cavity, and the muscle under the skin; Contracaecum sp., larvae were found capsulated on the serous membrane of the organs in the abdominal cavity. P. laevis affects its hosts, damaging their intestines and slowing their growth. E. excisus damages the organs of fish (Novakov et al., 2015), and in humans, it causes intestinal perforation (Juhásová et al., 2019). Contracaecum sp., larvae can reduce fish weight and liver lipid content and cause their death. In humans, they cause the disease anisakidosis (Zashev & Margaritov, 1966; Demir & Karakişi, 2014). Eustrongylides spp. cause gastritis and intestinal perforation in humans infected with this species (Ljubojevic et al., 2015).

The ecological indices of helminths of the three fish species from the present study were compared with those from previous helminthological studies of the same fish species from the Bulgarian section of the Danube River. The helminths of B. gymnotrachelus from the Danube River in the area of Vidin town and the Koshava and Gomotartsi villages were studied by Ondračková et al. (2012). The authors reported 4 endohelminth species. Common helminth species with those established in the present study of racer goby were: N. skrjabini and E. excisus. The mean abundance of N. skrjabini from Kudelin and Novo selo was higher, while the prevalence was lower than those reported by Ondračková et al. (2012). The mean abundance and prevalence of E. excisus from Kudelin and the mean abundance of E. excisus from Novo selo were higher than those reported for Vidin, Koshava and Gomotartsi (Table 6). B. gymnotrachelus is a new host of Ac. lucii from the Danube River and the river basin in other countries and Bulgaria. Ac. lucii is reported for the first time for the helminth fauna of B. gymnotrachelus in Bulgaria. Kudelin and Novo selo are new habitats for N. skrjabini, Ac. lucii and E. excisus of B. gymnotrachelus.

More recent studies on the helminths of N. fluviatilis from the Bulgarian section of the Danube River have been carried out by Ondračková et al. (2012; in the area of the Koshava and Gomotartsi villages) and by Kirin et al. (2013; in the area of the Vetren village). Three helminth species were reported by Ondračková et al. (2012). Comparing the ecological indices (MA and P%) of N. skrjabini from Kudelin, Novo selo, and Kutovo with those indicated by Ondračková et al. (2012), it was found that generally, the ecological indices in the present study were lower, except the prevalence of N. skrjabini from Novo selo and Kutovo. The mean abundance and prevalence of E. excisus from Kudelin were higher, while those from Novo selo were lower than those indicated by Ondračková et al. (2012). The acanthocephalan Pomphorhynchus tereticollis (Rudolphi, 1809) Meyer, 1932, which was not found in the present study in N. fluviatilis, was reported by Kirin et al. (2013; Table 6). N. fluviatilis is a new host of Ac. lucii from the Danube River and the river basin. Ac. lucii and others were reported in N. fluviatilis from the Black Sea (Kvach, 2005). Ac. lucii is reported for the first time for the helminth fauna of monkey goby from the Danube River and the river basin. Kudelin, Novo selo, and Kutovo are new habitats for the established helminths of N. fluviatilis.

Studies on the helminths of N. melanostomus from the Bulgarian section of the Danube River were carried out by Ondračková et al. (2010), Ondračková et al. (2012) in the area of Vidin town, and Francová et al. (2011) in the vicinities of the towns of Vidin and Ruse. Two, four and five helminth species were reported in round goby by Ondračková et al. (2010), Ondračková et al. (2012) and Francová et al. (2011), respectively. The mean abundance and prevalence of N. skrjabini and P. laevis in round goby from the present study were compared with those reported in earlier studies from the Bulgarian section of the Danube River. The MA and P% values of N. skrjabini from Kudelin and Koshava were higher than those reported by Francová et al. (2011) and by Ondračková et al. (2012). The mean abundance and prevalence of P. laevis in round goby from Koshava were lower than those indicated by Ondračková et al. (2010), Francová et al. (2011) and Ondračková et al. (2012) (Table 6). N. melanostomus is a new host of Ac. lucii, Ac. anguillae and Contracaecum sp. in Bulgaria. Ac. lucii, Contracaecum rudolphii Hartwich, 1964 L3, Contracaecum microcephalum (Rudolphi, 1809) Baylis, 1920 L3 and others were reported in N. melanostomus from the Black Sea (Kvach, 2005). The helminths Ac. lucii, Ac. anguillae and Contracaecum sp. are reported for the first time for the helminth fauna of N. melanostomus in Bulgaria. Kudelin, Koshava and Novo selo are new habitats for the established helminths of round goby.

Due to the place of localization of the helminths and the inclusion of the three species of gobies in the human diet, it is recommended to perform a visual inspection of the abdominal cavity and the muscles of the three species of gobies; to remove the internal organs, especially of the smaller specimens of fish and to cook to a sufficiently high internal temperature. Due to the presence of pathogenic helminth species, not only for fish but also for humans, it is desirable to conduct systematic studies on the parasite fauna of the three species of gobies, not only from the freshwater ecosystem of the Danube River but also from other aquatic ecosystems. It is important to carry out helminthological studies on predatory fish species that feed on the gobies and are consumed by humans. It is also desirable to monitor the ecological indices of the helminths.