Carey O. Cunningham

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.

Molecular phylogenetic analysis of the genus Gyrodactylus (Platyhelminthes: Monogenea) inferred from rDNA ITS region: subgenera versus species groups.

Analyses of small subunit ribosomal RNA gene sequences of representatives of major taxa of Monopisthocotylea were performed to identify the sister group of Gyrodactylus. Nuclear ribosomal DNA sequences from the complete internal transcribed spacer (ITS) region were used to infer phylogeny of 37 Gyrodactylus species and Gyrodactyloides bychowskii, Macrogyrodactylus polypteri and Gyrdicotylus gallieni, using maximum likelihood, parsimony and Bayesian inference. The genus Gyrodactylus appeared to be a monophyletic group in all analyses, based on the present data set. Within the genus, there were 3 major groups recognized by high bootstrap values and posterior probabilities. None of the 6 subgenera appeared to be monophyletic, and the most basal subgenus G. (Gyrodactylus) was paraphyletic. Characteristics of the excretory system of Gyrodactylus do not seem to be conservative enough to reveal subgenera within Gyrodactylus and we suggest abandoning existing subgenera as indicators of phylogeny. The grouping of species based on the morphology of the ventral bar and marginal hooks seems to have sufficient power to infer relationships between the Gyrodactylus species.

Characterization of a Gyrodactylus salaris variant: infection biology, morphology and molecular genetics

A laboratory population of a Danish Gyrodactylus salaris variant founded by 1 single specimen was established and infection studies performed. Rainbow trout as well as Atlantic salmon of 3 different stocks were infected both in cohabitation systems and as single-parasite infections on isolated hosts. Both infection systems revealed that this particular morphotype exhibits low virulence towards Atlantic salmon. Thus, in isolated hosts, the parasites could either not establish or only reproduce to a limited degree on salmon. Rainbow trout, in contrast, proved to be rather susceptible to infection with this G. salaris variant and abundances on this host species always attained significantly higher values in cohabitation systems compared to salmon. Detailed morphological examination confirmed the very high resemblance to G. salaris (sensu stricto), as the range of variation in sclerite characters of the morphotype is almost fully covered by the total range of variation reported for reference G. salaris. Morphological similarities to the closely related congeneric species G. bohemicus were noted. Molecular studies, however, showed that the morphotype most likely represents a G. salaris variant, as it differed only slightly from G. salaris sensu Malmberg, 1957, which is also known to inhabit Danish watercourses. The genomic target region investigated does not allow us to rule out the possibility that it represents a variant form of G. thymalli. Sequences of the ribosomal RNA internal transcribed spacer (ITS) revealed that single individuals contained 2 different ITS sequences, one identical to reference sequence of G. salaris while the other differed at 3 positions. This can be interpreted as an example of a hybrid or, more likely, as intra-individual variation of ITS within single individuals. As one of the nucleotide changes in the variant ITS affects an Hae III restriction site, the current G. salaris variant can be distinguished from G. salaris sensu Malmberg by RFLP diagnosis.

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.

The use of host specificity, pathogenicity, and molecular markers to differentiate between Gyrodactylus salaris Malmberg, 1957 and G. thymalli Zitnan, 1960 (Monogenea: Gyrodactylidae)

The validity of Gyrodactylus thymalli has been questioned, based on its morphological and genetic resemblance to G. salaris. This taxonomic problem has practical implications regarding correct diagnosis of G. salaris, which has proved to be highly pathogenic to stocks of wild Norwegian Atlantic salmon. The host specificity and pathogenicity of G. salaris and G. thymalli were experimentally tested on salmon and grayling. Both parasite species were able to infect, live and reproduce on both fish species. G. salaris was highly pathogenic for the experimental stock of salmon, while grayling mounted an effective response against this parasite. Both fish species responded to an infection with G. thymalli. The results did not support the hypothesis of conspecificity between G. thymalli and G. salaris. The ribosomal RNA gene intergenic spacer of both species was sequenced. Variation in sequence was lower than expected for different species. Variation in the sequences of tandemly repeated elements was found and may prove useful in distinguishing G. salaris and G. thymalli.

Genetic characterization of six species of diplozoids (Monogenea; Diplozoidae)

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.

Spatial and temporal variations in parasite prevalence and infracommunity structure in herring ( Clupea harengus L.) caught to the west of the British Isles and in the North and Baltic Seas: implications for fisheries science

Herring Clupea harengus L. viscera were examined for endoparasitic infections as part of a multidisciplinary stock identification project (WESTHER, EU Contract no. Q5RS-2002-01 056) which applied a range of stock discrimination techniques to the same individual fishes to obtain comparable results for multivariate analysis. Spawning and non-spawning adults, and juvenile herring were caught, over 3 years, by commercial and research vessels from numerous locations to the west of the UK and Ireland, along with control samples of spawning fish from the eastern Baltic Sea, and juveniles from sites in the eastern and western North Sea, and the north of Norway. The metacercariae of two renicolid digeneans (Cercaria pythionike and Cercaria doricha), one larval nematode (Anisakis simplex s.s.) and one larval cestode (Lacistorhynchus tenuis) were selected as tag species. Results were compared with those from herring collected between 1973 and 1982, which suggested remarkable stability in the parasite fauna of herring in the study area. These species were used to compare the parasite infracommunities of spawning herring. A significant variation in infracommunity structure was observed between different spawning grounds. These results suggest that the parasite fauna of herring are spatially variable but remain temporally stable in both the short and long term. Significant differences in prevalence and abundance of infections and comparisons of parasite infracommunity enabled the separation of putative herring stocks west of the British Isles. Distinctive patterns of parasite infection in two different spawning groups off the north coast of Scotland suggest that this area is occupied by two spawning populations, one recruiting from the west of Scotland, the other from outside this area, and most likely from the eastern North Sea. The distribution patterns of L. tenuis, C. doricha and C. pythionike suggest the potential for fish that spawn in three distinct International Council for the Exploration of the Seas (ICES) management units to be present in mixed aggregations found over the Malin Shelf, with significant implications for management in this area.

IDENTIFICATION OF EUROPEAN DIPLOZOIDS (MONOGENEA, DIPLOZOINAE) BY RESTRICTION DIGESTION OF THE RIBOSOMAL RNA INTERNAL TRANSCRIBED SPACER

The second internal transcribed spacer (ITS2) of the ribosomal RNA genes of Diplozoon paradoxum and Paradiplozoon nagibinae were amplified and sequenced. The polymerase chain reaction product of D. paradoxum was bigger (840 bp) than that of P. nagibinae (820 bp). There was no intraspecific variability recorded in sequences from either species. Sequence comparisons and ITS2 restriction fragment length polymorphism (RFLP) pattern of 8 European diplozoid species aimed to resolve their identification and amend the previous studies. RFLP was used to distinguish the 2 species from each other and from P. bliccae, P. homoion, P. megan, P. pavlovskii, P. sapae, and Eudiplozoon nipponicum, using restriction enzymes AluI, HaeIII, HinfI, RsaI, and SphI. The criteria for morphological identification of 8 European diplozoids are also included, with the main morphological characters of clamps, trapeze spur, and anterior joining sclerites of 8 diplozoid species being illustrated. Combination of the shape and comparison of length of the trapeze spur and anterior joining sclerites could lead to accurate identification of diplozoid species.

Random amplified polymorphic DNA (RAPD) analysis of three Norwegian Gyrodactylus salaris populations (Monogenea:Gyrodactylidae).

Gyrodactylus salaris Malmberg, 1957 from Atlantic salmon (Salmo salar L.) parr in 3 Norwegian rivers have been compared genetically by the use of random amplified polymorphic DNA (RAPD) analysis. Two primers revealed polymorphism between different populations of G. salaris. This variation has probably arisen since the introduction of G. salaris to Norway in the 1970s and complements results showing morphological variation between G. salaris from different geographical areas.