The application of new biosystematic techniques in the discrimination of the genus Gryodactylus (monogenea) on samonoid fish

Abstract

Prior to 1989 the total number of Gyrodactylus species recorded for all British freshwater fish numbered 20. The fauna present on the British Salmonidae was poorly documented and frequently not identified to species level. The European free market, created in 1992, resulted in legislative changes allowing the movement of live fish stocks, albeit under strict disease monitoring conditions, into the UK. One stipulation maintains that the fish stock be free of the ectoparasitic monogenean Gyrodactylus salaris Malmberg, 1957, a parasite made notifiable in the UK in 1987 (Diseases of Fish Act, 1937) owing to its pathogenicity and damage to Norwegian salmon populations in 38 rivers. Although this parasite has been reported since 1957 throughout mainland Europe, its occurrence in the UK was unknown.

This project set out to make a national survey of British salmon ids and investigated 250 sample sites, examined four salmonid hosts, Atlantic salmon Salmo safar, brown trout Salmo trutta, rainbow trout Oncorhynchus mykiss and Arctic charr Salvelinus alpinus. Seventy of the sites were found to be positive for Gyrodactylus. Distinctions were made between wild and farmed fish and prevalence, abundance and intensity data collected for comparison. Species determination within the genus Gyrodactylus is based upon subtle differences in hook morphology and has long posed a taxonomic problem. The discrimination of collected specimens was based on two platforms. The initial approach used classical morphometrics from the light microscope, the results being processed using multivariate analyses to separate species. The second approach analysed morphometric data collected from scanning electron micrographs. This was made possible by the development of a sclerite release technique utilising a source of ultrasound to liberate hooks from surrounding tissue and a subsequent flotation stage which permitted flat preparations. Sonication of fresh and frozen material retained the structures that would be lost by enzymatic digestion. The description of new morphometric parameters using digital image analysis allowed the subtle differences in hamuli and marginal hook shape to be discriminated when analysed using principal components analysis (PCA).

Four species were identified following multivariate and morphological analyses of opisthaptoral sclerites. G. truttae Glaser, 1975 was found to occur on S. trutta and G. derjav;ni Mikailov, 1975 was found to occur on S. trllfta, S. salar and O. mykiss. Two hitheno undescribed forms, one on S. salar and one on S. alpinus which may be a new species are desclibed. In addition, two forms of G. derjavilli from S. salar and S. alpinlls and one form of G. truttae from S. tnltta are described. Sub-populations of Gyrodactylus sp. were found to be determined by the pattern of distribution of the host; S. salar. The two sub-populations were divided into a southern celtic population (Morph 1) and a nonhern boreal population (Morph 2). Water temperature, was found to be an important environmental parameter influencing sclerite size. The principal component analyses identified key characters which could discriminate G. salaris from the native British species using novel parameters based upon both single elements and the full complement of sclerites. Of these new parameters, the hamulus angle and the size of the marginal hook sickle aperture were the most discriminating. Electronmicrographs of hamuli were traced using a digitising tablet and prepared for image processing. The hamulus angle was measured on original hook images and on enhanced (skeletonised) images using an image analyser. Skeletonisation investigated the reliability of the hook angle as a taxonomic criterion by the removal of possible age-related sclerotisation of the hamulus. Statistical analysis of the data revealed that there are significant differences in hook angle between some species. The isolation of sclerites by sonication enabled their elemental composition to be investigated. The hamuli and marginal hooks were found to have a high sulphur content, indicative of a keratin-like substance. The ventral bar composed of sulphur and calcium is weakly keratinised. The hamulus and the ventral bar were also found to contain vanadium. the significance of which is unknown. The detailed morphology and composition of the individual sclerites is discussed in relation to the functional mechanics of the entire haptoral complex.

The protein profiles of G. salaris. G. truttae and G. derjvani were investigated using SDS-PAGE gel electrophoresis, with four proteins common to the three species. Two antibodies raised against G. salaris were found using Western blots.

The chaetotaxy of argentophilic structures on three species of the genus Gyrodactyllis was investigated to ascertain the usefulness of this technique in distinguishing species of this genus. Chaetotaxy maps were prepared for G. salaris from Scandinavia and compared to native species of Gyrodactylus parasitizing salmonids in Britain. A formula for the arrangement of the sensilla analogues and the evolutionary position of the genus Gyrodactylus is commented upon.