Catherine M. Collins

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea)from five fish families (Teleostei)

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.

Redescription of Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 (Monogenea: Gyrodactylidae); a species identified by ribosomal RNA sequence

Molecular and morphological features of Gyrodactylus specimens from Oncorhynchus mykiss, Salmo trutta and Salmo salar were examined. Sequences from variable region V4 of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacers, produced by the FRS Marine Laboratory, revealed that these were not the same as other well-characterised Gyrodactylus that are common on European salmonids and were in fact a distinct species. Initial morphological examination of the opisthaptor indicated that this species very closely resembles G. salaris Malmberg, 1957. More detailed analysis revealed differences in the shape of the marginal hook sickle of these two species and thus Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 was erected. Analysis of the ribosomal RNA gene or spacer sequences remains the most reliable method of identifying this species. This is believed to be the first record of a Gyrodactylus species identified first from molecular data and confirmed by morphological examination; previous molecular analyses had provided alternative methods for identifying species that had already been described using morphological characters.

SEQUENCE AND SECONDARY STRUCTURE VARIATION IN THE GYRODACTYLUS (PLATYHELMINTHES: MONOGENEA) RIBOSOMAL RNA GENE ARRAY

Nucleotide sequences were determined for the rRNA internal transcribed spacers 1 and 2 (ITS1 and 2) and the 5′ terminus of the large subunit rRNA in selected Gyrodactylus species. Examination of primary sequence variation and secondary structure models in ITS2 and variable region V4 of the small subunit rRNA revealed that structure was largely conserved despite significant variation in sequence. ITS1 sequences were highly variable, and models of structure were unreliable but, despite this, show some resemblance to structures predicted in Digenea. ITS2 models demonstrated binding of the 3′ end of 5.8S rRNA to the 5′ end of the large subunit rRNA and enabled the termini of these genes to be defined with greater confidence than previously. The structure model shown here may prove useful in future phylogenetic analyses.

Isolation and expression profile of a gene encoding for the Signal Transducer and Activator of Transcription STAT2 in Atlantic salmon (Salmo salar)

Signal Transducer and Activator of Transcription (STAT)-2 is a molecule involved in the type I interferon (IFN) signalling pathway. The full length cDNA sequence of Atlantic salmon (Salmo salar) ssSTAT2 was determined and phylogenetic analysis of the amino acid sequence grouped this novel salmon gene to the STAT2 clade. This represents the first fish STAT2 report. The gene encodes for a 802 aa polypeptide that has 38% identity to the human or murine STAT2. The expression was monitored by qPCR in the kidney of animals over the time of infection with the Infectious Salmon Anaemia Virus (ISAV) and in TO cells infected with Infectious Pancreatic Necrosis Virus (IPNV) or with the Salmon Alphavirus (SAV). SAV and ISAV induced an approximate 10-fold increase in the level of expression of ssSTAT2 gene whilst IPNV only induced a 1.5-fold increase.

Gyrodactylus derjavinoides sp. nov. (Monogenea, Platyhelminthes) on Salmo trutta trutta L. and G. derjavini Mikailov, 1975 on S. t. caspius Kessler, two different species of Gyrodactylus — combined morphological and molecular investigations

The paper deals with two morphologically similar but molecularly clearly different species of Gyrodactylus: G. derjavinoides sp. nov. on Salmo trutta trutta L. in Western Europe and G. derjavini Mikailov. 1975 collected on Salmo trutta caspius Kessler in Iran. The new species is described and its opisthaptoral hard parts compared to those of G. derjavini. Our molecular analysis of G. derjavinoides and G. derjavini confirmed that the morphological differences between them are species differences and not intraspecific variations. Phylogenetic analysis using the ITS rDNA region placed both species within the subgenus G. (Limnonephrotus) and within the G. wageneri-group, quite in accordance with morphological results. The two species, however, did not cluster as sister taxa. The correspondence between molecular-based clades within G. (Limnonephrotus) and the morphological shapes of marginal hooks within these clades are discussed. The importance of combined molecular and morphological analyses when describing or redescribing Gyrodactylus species is stressed.

Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe.

Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.

Pseudalataspora vanderlingeni n. sp. (Myxosporea: Bivalvulida) from gall bladders of the Cape hakes Merluccius capensis Castelnau, and M. paradoxus Franca (Teleostei: Merlucciidae)

A new species of myxosporean parasite is described from the gall bladders of the hakes Merluccius capensis Castelnau and M. paradoxus Franca (Pisces: Teleostei) caught off the west and south coasts of South Africa. The new species, Pseudalataspora vanderlingeni, is described morphologically and molecularly and compared with the 15 other species of Pseudalataspora previously described from marine fish. Although a molecular description is available on GenBank for only one of these 15 species, the morphological description supports the status of P. vanderlingeni as a new species. Earlier reports, without detailed descriptions, of Leptotheca sp. and Ceratomyxa sp. from the same hosts caught off Namibia were very likely to have been of P. vanderlingeni. These earlier studies reported high prevalences of infection, similar to those of >60% described in the present study. The effects of fixation and freezing on the dimensions of spores of Pseudalataspora spp. are described, and the status of the genus Pseudalataspora within the family Ceratomyxidae is discussed.