The round goby (
In European waters, beyond the area of its natural occurrence, the species was first found in 1990 in Gdańsk Bay (Poland), a confluence of the Baltic Sea and Martwa Wisła river (26). In the subsequent years, its presence was also noticed in nearby reservoirs (25), and probably since 1996, it has been present in the western Baltic Sea and the Oder River estuary (8). In the German region of the Baltic Sea, the round goby was first found in 1998 in fish catches in the vicinity of the Zicker Peninsula (Rügen Island) (31), while in 2002, it was found further to the west, near the Darß Peninsula (Darßer Ort) (5, 31). In the following years, the species colonised the Rhine Delta and the Lek River near the village of Schoonhoven (2004), the Elbe River (2008), the Scheldt and Albert Canals in Belgium (2010), and the Weser River (2012) (3, 13, 27, 28). At the beginning of the 21st century, the species was also seen in the northern and eastern parts of the Baltic Sea (21).
It is believed that the second route of colonisation of European waters by the round goby was the catchment area of the Danube River, as evidenced by the successive occurrence of this fish at sites in the middle and upper courses of the river (22, 24). For example, in the Austrian section of the Danube near Vienna, the species was found in 2000 (30), while in the German section of the river between Passau and Straubing, it was noted in 2004 (23).
Such a rapid spread of the round goby is a potential threat, not only due to disturbance of the ecosystems it colonises, but also from the point of view of epidemiology, as it can be a source of pathogens introduced into the new environment. This aspect is particularly relevant as the species can penetrate into fish ponds during filling and become an intermediate host in the chain of transmission of parasitic diseases. With regard to viral diseases, there is a possibility of the round goby transmitting viral diseases to species of great economic importance, as it has been found in the stomachs of such species as cod, perch, pikeperch, and brill (29).
The study materials were 35 individuals of round goby (
DNA was isolated using a DNA Mini Kit (Syngen Biotech, Wrocław, Poland). Qualitative and quantitative assessment of the extracted DNA was conducted by measurement of absorbance using the NanoDrop 2000 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and electrophoretic separation on a 1.5% agarose gel (Prona, Tychy, Poland).
All samples were analysed molecularly, which allowed the presence of sequence fragments of KHV (Cyprinid herpesvirus 3, CyHV-3), and frequently co-occurring CEV to be confirmed. Detection was conducted using PCR and nested PCR, using four pairs of specific primers for KHV and two pairs of primers for CEV.
PCR products obtained by using the KHV-F/KHV-R primer pair were used as a template for the second nested PCR employing the KHV-1Fn/KHV-1Rn primer pair (2, 9). The second set comprised the KHV-TK-F and KHV-TK-R primers used in the first round, and the KHV-TK-Fn/KHV-TK-Rn used in the nested PCR step (1, 16). The PCR products of the CEVforB/CEVrevJ primer pair were used as a template for the second nested PCR with CEVforBint/CEVrevJint. The PCR assays were performed according to previously published methods. The primers and methods used in this study are presented in Table 1.
Primer pairs used in the study to detect the KHV and CEV genomes
Primer | Primer sequence (5′–3′) | Product size | References |
---|---|---|---|
KHV-F | GACGACGCCGGAGACCTTGTG | 486 bp | Gilad |
KHV-R | CACAAGTTCAGTCTGTTCCTCAAC | 486 bp | Gilad |
KHV-1Fn | CTCGCCGAGCAGAGGAAGCGC | 414 bp | Bergmann |
KHV-1Rn | TCATGCTCTCCGAGGCCAGCGG | 414 bp | Bergmann |
KHV-TK-F | GGGTTACCTGTACGAG | 409 bp | Bercovier |
KHV-TK-R | CACCCAGTAGATTATGC | 409 bp | Bercovier |
KHV-TK-Fn | CGTCGTGAGGAATACGACG | 348 bp | Unpublished: Way in Kempter |
KHV-TK-Rn | ACCGTACAGCTCGTACTGG | 348 bp | Unpublished: Way in Kempter |
CEVforB | ATGGAGTATCCAAAGTACTTAG | 528 bp | Matras |
CEVrevJ | CTCTTCACTATTGTGACTT TG | 528 bp | Matras |
CEVforBint | GTTATCAATGAAATTTGTGTATTG | 478 bp | Matras |
CEVrevJint | TAGCAAAGTACTACCTCATCC | 478 bp | Matras |
The products of each PCR were assessed by separation on a 1.5% agarose gel followed by bidirectional Sanger sequencing (Genomed, Warsaw, Poland). The results of sequencing were aligned and analysed using BLAST-N and GENEIOUS PRIME software (
The qualitative and quantitative analysis of the DNA obtained from the samples demonstrated that the method employing spin columns yielded a high degree of purity of the obtained DNA samples (A260/A280 = 1.8–2.0). As a result of electrophoretic separation of the nested PCR products obtained using the KHV-CT-Fn/KHV-CT-Rn primer pair, the presence of KHV in six samples from round gobies was determined (Fig. 1). The obtained results confirmed the effectiveness of this method for detection of parts of the KHV genome.
Positive results were obtained from samples of one individual caught in the Spandowerhagen facility in the Greifswalder Bodden (Fig. 1, sample 52RG) and in five individuals from the Szczecin Lagoon on the Polish side (Fig. 1, samples 2Z, 23P, 14ZAL, 1ZAL, 4ZAL). For the remaining 64 samples, negative results of detection of the KHV genome fragment were obtained, or the number of copies of the virus was below the threshold of detection of the method.
The results of the assay aiming to detect CEV were negative for all 70 investigated round gobies. Bidirectional Sanger sequencing and alignment of sequences in the GENEIOUS and BLAST-N software confirmed that the obtained 310 bp fragments showed 100% similarity to the reference KHV sequences published in the National Center for Biotechnology Information (NCBI) GenBank database (Fig. 2).
Viral infections are one of the causes of many mass mortalities among the indigenous ichthyofauna. The disease caused by KHV is a herpesviral infection (12) capable of inducing contagious and acute viraemia in common carp (
In conclusion, the results of our study suggest that as one of the most invasive species in European waters, the round goby not only jeopardises the biodiversity of the indigenous ichthyofauna but also poses an epidemiological threat in regards to KHV and possibly CEV. Its presence as a potential vector or carrier of KHV increases the risk and provides an additional opportunity for the transmission of the disease to new environments that this fish species colonises in the course of its expansion.