Identification of Gyrodactylus ectoparasites in Polish salmonid farms by PCR-RFLP of the nuclear ITS segment of ribosomal DNA (Monogenea, Gyrodactylidae)

Abstract

The Gyrodactylus fauna of 274 fish taken from ten salmonid farms in Poland was sampled in 2006. Four fish species were investigated: rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta (morphs fario, lacustris, and trutta), grayling Thymallus thymallus and huchen Hucho hucho. No parasites were observed on huchen. No indications of gyrodactylosis were observed, but an unexpected parasite species diversity was found. A molecular species identification by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of ITS1 + 5.8S + ITS2 was utilized, with addition of morphometric methods. The most frequent parasite was a new record in Poland, G. teuchis. It was present in two molecular forms on brown trout and rainbow trout, which also carried G. derjavinoides and G. truttae. Three molecular forms of G. salaris/G. thymalli were found, the standard type ITS only on grayling. A heterozygous (or heterogenic) G. salaris type described earlier in Denmark was found in seven farms on rainbow trout, and a complementary homozygous clone which differs from the standard by three nucleotides, in two farms. This homozygous form has not been recorded earlier. The PCR-RFLP results were confirmed by sequencing ITS segment from representative specimens of each type and comparing them with all available salmonid-specific Gyrodactylus sequences in GenBank. The Polish fauna with seven different Gyrodactylus clones separated by PCR-RFLP was the most diverse reported in fish farms in any country so far.

The first report of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes, Monogenea) on Italian cultured stocks of rainbow trout (Oncorhynchus mykiss Walbaum)

“…skin scrapes), the likelihood of G. truttae occurring on stock held at the other farms cannot be ruled out. Gyrodactylus teuchis has previously been reported in France, sporadically in Denmark and the UK and appears to be common on rainbow trout in Polish fish farms (Lautraite et al, 1999;Cunningham et al, 2001;Rokicka et al, 2007). Lautraite et al (1999) found G. teuchis to be widely distributed on both wild and farmed salmonids from Brittany to the Western Pyré né es, and the current survey now extends its distribution into Italy.…”

“…This survey together with recent studies on gyrodactylids in rainbow trout farms in Europe (e.g. Rokicka et al, 2007;Dzika et al, 2009) points to the importance of this industry for spreading of G. salaris in Europe. It seems more than likely that the examination of rainbow trout farms in other countries will extend the range further.…”

“…Throughout Europe, G. salaris has also been reported from Norway (Johnsen and Jensen, 1991;Johnsen et al, 1999), from rivers on the Swedish west coast Alenä s, 1998), Denmark (e.g. Buchmann and Bresciani, 1997;Buchmann et al, 2000), Finland (Rimaila-Pä rnä nen and Wiklund, 1987;Kerä nen et al, 1992;Koski Malmberg, 1995;Koski, 1996;Rintamä ki-Kinnunen and Valtonen, 1996), Russia (Ergens, 1983;Ieshko et al, 1996Ieshko et al, , 1997Meinilä et al, 2004), Germany (Lux, 1990;Dzika et al, 2009), Spain (Malmberg, 1993), France (Johnston et al, 1996) and, most recently, from Poland (Rokicka et al, 2007). While the presence of G. salaris has been confirmed by molecular methods for many of these, the reports from certain countries, i.e.…”

Gyrodactylus malalai sp. nov. (Monogenea, Gyrodactylidae) from Nile tilapia, Oreochromis niloticus (L.) and Redbelly tilapia, Tilapia zillii (Gervais) (Teleostei, Cichlidae) in the Lake Turkana, Kenya

“…The remaining parts of the parasite´s bodies were stored in absolute ethanol SPECTRANAL (Allied-Signal, Riedel-de Haën). The specimens collected in Sudan were placed directly in absolute ethanol and the slides for morphological analysis were subsequently prepared in the home laboratory according to the methods previously used by Rokicka et al (2007).…”

“…The DNA was eluted in 50 μl. The Internal Transcribed Spacer (ITS) region of the ribosomal DNA was amplified with ITS1F (5'-GTTTCCGTAGGTGAACCT-3') (Rokicka et al 2007) and ITS2R (5'-TCCTCCGCTTAGTGATA-3') (Matějusová et al 2001), and a partial fragment of 18S was amplified using primers S1 (5'-ATTCCGATAACGAACGAGACT-3'), which anneal in the internal region of the 18S gene (Sinnappah et al 2001) and ITSR3A (5'-GAGCCGAGTGATCCACC-3') (Matějusová et al 2001), in a Mastercycler eP gradient (Eppendorf).…”