Spin-off from routine parasite diagnostics of Atlantic salmon; first report of Gyrodactylus alexanderi in Norway

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1 14, Bull. Eur. Ass. Fish Pathol., 32(1) 2012 Spin-off from routine parasite diagnostics of Atlantic salmon; first report of Gyrodactylus alexanderi in Norway H. Hansen*, A. Jørgensen and T. A. Mo Norwegian Veterinary Institute, P.O. Box 750 Sentrum, NO-0106 Oslo, Norway Abstract During a routine investigation of farmed Atlantic salmon, Salmo salar, the monogenean Gyrodactylus arcuatus was diagnosed. As this is a common species on sticklebacks, Gasterosteus aculeatus, we examined sticklebacks from the inlet water of the fish farm to establish a possible source of the infection. These investigations confirmed the presence of G. arcuatus on the sticklebacks but on the same host we also recovered another species, G. alexanderi. This is the first report of G. alexanderi in Norway. Introduction Due to the economic and ecological damage caused by Gyrodactylus salaris Malmberg, 1957 in Norwegian wild populations of Atlantic salmon, Salmo salar L. (Johnsen et al., 1999), farmed Atlantic salmon in freshwater are routinely examined for Gyrodactylus infections. If confirmed, Gyrodactylus specimens are sent to the national reference laboratory at the Norwegian Veterinary Institute for species diagnosis. In 2005 a routine examination of farmed Atlantic salmon from a farm located on the island of Frøya, South Trøndelag county, revealed the presence of Gyrodactylus specimen on the fins. The initial sample was stored in formalin and this is not suitable for molecular diagnostic methods. However, upon morphological examination the specimens were tentatively identified as G. arcuatus Bychowsky, 1933, a species commonly reported from the three-spined stickleback, Gasterosteus aculeatus L. To confirm the diagnosis by molecular methods and to establish a possible source of the infection on the salmon, new samples of both sticklebacks and salmon were obtained. Materials and methods In October 2005, fins from twenty Atlantic salmon from a salmon farm and twenty whole sticklebacks from the intake water to the farm in the Skagvatn Lake, Sør-Trøndelag County ( N, E), were fixed in 96% EtOH and sent to the Norwegian Veterinary Institute for parasitological examination and species diagnosis. The salmon fins and the sticklebacks were examined under a stereo microscope at times magnification for the presence of Gyrodactylus parasites. Parasites were sampled with pair of tweezers and stored in 96% EtOH. The opisthaptor was severed from the parasite bodies with a scalpel blade. * Corresponding author s haakon.hansen@vetinst.no

2 Bull. Eur. Ass. Fish Pathol., 32(1) 2012, 15 The so tissue was then digested and the hard parts prepared for morphological analyses according to standard procedures (Harris et al., 1999). Morphological preparations were examined and photographed under a dissection microscope (Leica DM5000) at times magnification. Parasite bodies were subjected to molecular analyses. DNA extraction, PCR of the ribosomal internal transcribed spacer (ITS) region and DNA sequencing was performed according to standard protocols (Matejusová et al., 2001; Ziętara and Lumme, 2003). Sequences were proofread in VectorNTI (Invitrogen) and species identity was established by subjecting the sequences to a GenBank BlastN search (Zhang et al., 2000). In May 2009, additional samples of sticklebacks were obtained from the same site and examined as described above. Results Parasitological examination of the salmon fins revealed approximately thirty Gyrodactylus parasites in total (maximum 10 on one fin). A number of parasites were found on the skin, the fins, the buccal cavity and the gill arches of the sticklebacks. Morphological analyses identified these specimens on the salmon and those in the buccal cavity and gill arches of the sticklebacks as G. arcuatus. The specimens on the skin and fins of the sticklebacks were identified as G. alexanderi Mizelle and Kritsky, 1967 (see Figure 1). Approximately 1000 bp of the ribosomal DNA cluster, including the complete internal transcribed spacers (ITS) 1 and 2, and the 5.8S gene, were amplified and sequenced from six specimens from the different hosts and sites sampled in 2005 and from 5 specimens from the skin of sticklebacks sampled in A BLASTN search of these sequences in GenBank confirmed the morphological diagnoses. The sequences from parasites infecting the skin and fins of sticklebacks in this study (GenBank acc. No. JN695633) corresponded to the sequence of G. alexanderi in GenBank (GenBank acc. no. FJ435201), except the la er contains one ambiguous nucleotide in position 115 of ITS1. The sequences from parasites on the fins of Atlantic salmon, and those from the buccal cavity and gill arches of sticklebacks (GenBank acc. no. JN703797), were identical to G. arcuatus (GenBank acc. no AF328865). Discussion We here report G. alexanderi from Norway for the first time. This species was first described from California (Mizelle and Kritsky, 1967) and is now confirmed present in British Columbia (Cone and Wiles, 1985) in the Western Hemisphere and in Kamchatka (Sokolov, 2002), in Iceland (Tine Huyse, pers. comm.), in Scotland and England (Harris, 2008) and now also in Norway in the Eastern Hemisphere. Gyrodactylus alexanderi seems to have a disjunctive geographic distribution and is less common than several of the other species infecting sticklebacks. It is speculated that G. alexanderi invaded the Atlantic basin along with its natural host years ago (Harris, 2008) and has survived in glacial refugia during the last glacial maximum. In addition, G. alexanderi, as opposed to G. arcuatus, seems to favour freshwater habitats and might not be able to remain on its euryhaline host when moving between water of different salinities. The fact that we did not find G. alexanderi on Atlantic salmon in the current study is interesting. Although this

3 16, Bull. Eur. Ass. Fish Pathol., 32(1) 2012 Figure 1. Light micrographs of the haptoral hard parts of Gyrodactylus alexanderi Mizelle and Kritsky, A: hamuli, B: marginal hooks, and C: ventral bar. All scale bars are 10μm.

4 Bull. Eur. Ass. Fish Pathol., 32(1) 2012, 17 absence could be a result of only examining the fins from twenty salmon, it could also indicate that G. alexanderi is more host specific than G. arcuatus, the la er which is found on a number of different fish species (Harris et al., 2004). More extensive sampling and host specificity experiments will give a be er understanding of the biogeography of G. alexanderi. Only a few investigations of the gyrodactylid fauna of sticklebacks have been carried out in Norway (see Sterud, 1999) and G. arcuatus is the most commonly encountered species. This species is also sporadically found on Atlantic salmon when examined during routine investigations and in the national surveillance programs for G. salaris. Of the other species, only G. branchicus is reported from Norway previously (Sterud, 1999). The finding of G. arcuatus on salmon is quite common, but the relatively high number seen on the salmon fins (indicating an even higher total infection on the fish), and the relatively low number on the sticklebacks in the intake water, might mean that G. arcuatus has reproduced on salmon. Reproduction on salmon has also been indicated by other authors (Ziętara et al., 2008). Acknowledgements We thank Havbrukstjenesten A/S for sampling sticklebacks and salmon in 2005 and T. T. Poppe for the additional samples taken in References Cone D and Wiles M (1985). The systematics and taxonomy of Gyrodactylus species (Monogenea) parasitizing gasterosteid fishes in North America. Canadian Journal of Zoology 63, Harris PD (2008). The evolution of fish ectoparasite communities - the role of the ice ages. Wiadomości Parazytologiczne 54, Harris PD, Cable J, Tinsley RC and Lazarus CM (1999). Combined ribosomal DNA and morphological analysis of individual gyrodactylid monogeneans. Journal of Parasitology 85, Harris PD, Shinn AP, Cable J and Bakke TA (2004). Nominal species of the genus Gyrodactylus von Nordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species. Systematic Parasitology 59, Johnsen BO, Møkkelgjerd PI and Jensen AJ (1999). The parasite Gyrodactylus salaris on salmon parr in Norwegian rivers, status report at the beginning of year 2000, NINA Oppdragsmelding, pp (In Norwegian with English summary). Matejusová I, Gelnar M, McBeath AJA, Collins CM and Cunningham CO (2001). Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei). International Journal for Parasitology 31, Mizelle JD and Kritsky DC (1967). Studies on monogenetic trematodes XXXVI. Gyrodactylid parasites of importance to California fishes. California Fish and Game 53, Sokolov SG (2002). The first record of Gyrodactylus alexanderi (Plathelminthes, Monogenea, Gyrodactylidae) in Eurasian fauna. Vestnik Zoologii 36, Sterud E (1999). Parasitter hos norske ferskvannsfisk. Norsk Zoologisk Forening, Oslo (in Norwegian). Zhang Z, S wartz S, Wagner L and Miller W (2000). A greedy algorithm for aligning DNA sequences. Journal of Computational Biology 7, Ziętara MS, Kuusela J, Veselov A and Lumme J (2008). Molecular faunistics of accidental

5 18, Bull. Eur. Ass. Fish Pathol., 32(1) 2012 infections of Gyrodactylus Nordmann, 1832 (Monogenea) parasitic on salmon Salmo salar L. and brown trout Salmo tru a L. in NW Russia. Systematic Parasitology 69, Ziętara MS and Lumme J (2003). The crossroads of molecular, typological and biological species concepts: two new species of Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidae). Systematic Parasitology 55,

Natural History Museum, Department of Zoology, University of Oslo, P.O. Box 1172, NO-0318 Oslo, Norway 2

Natural History Museum, Department of Zoology, University of Oslo, P.O. Box 1172, NO-0318 Oslo, Norway 2 Host-based identification is not supported by morphometrics in natural populations of Gyrodactylus salaris and G. thymalli (Platyhelminthes, Monogenea) 2041 K. OLSTAD 1 *, A. P. SHINN 2, L. BACHMANN 1