Molecular methods of distinguishing Gyrodactylus species parasitising salmonid fish
Gyrodactylus salaris Malmberg, 1957, a monogenean parasite of salmonid fish, has caused the death of up to 95% of salmon parr in 37 Norwegian rivers. In order to prevent further spread of this parasite, a reliable method of identifying G. salaris and distinguishing it from other closely related species is required. This study, the first investigation of Gyrodactylus genetics, has demonstrated that DNA technology can provide methods of gyrodactylid species identification suitable for routine use. DNA was extracted from G. salaris and two other species common on salmonid fish; G. derjavini and G. truttae. The small subunit ribosomal RNA (srRNA) gene was amplified from this DNA by polymerase chain reaction (PCR). The complete nucleotide sequence of the srRNA gene from G. salaris was determined. This was used to predict a secondary structure for gyrodactylid srRNA and to construct molecular phylogenies of platyhelminths including Gyrodactylus. Fragments of the srRNA gene from each species were compared by Denaturing Gradient Gel Electrophoresis. Mobility differences in G. truttae fragments were found and one fragment showed variation between and within species. The V4 region of srRNA was amplified from single specimens of gyrodactylids using a combined lysis and PCR reaction and sequenced. Examination of these sequences enabled prediction of Restriction Fragment Length Polymorphisms (RFLPs) between species and the design of oligonucleotide probes specific for each species. Digestion of the srRNA gene V4 region with two restriction enzymes produced restriction fragment polymorphisms that can be used to discriminate between G. salaris, G. derjavini and G. truttae. The digoxigenin labelled oligonucleotides GsV4, GdV4 and GdV4 are specific for G. salaris, G. derjavini and G. truttae respectively and can detect PCR amplified DNA from single specimens in dot blots. Both RFLP and probe methods of identifying Gyrodactylus species are suitable for use in diagnostic laboratories.