Molecular studies on Sphaerospora truttae and other freshwater myxozoans

Abstract

This study investigates the life cycle of Sphaerospora truttae, a myxozoan parasite of the Atlantic salmon, using molecular methods based on the 185 rONA. DNA sequencing showed that the 185 rONA of S. truttae differs substantially from the sequence obtained from its proposed alternate actinosporean life cycle stage, Echinactinomyxon type 5. With more than 90% sequence identity Echinactinomyxon type 5 is closely related to Myxobolus portucalensis whereas S. truttae with an extraordinary long 185 sequence (2541 bp), with inserts in the variable regions of the gene, does not relate closely to any myxozoans. On the basis of the obtained sequence for S. truttae, a single round nested peR assay was developed which allows low-level detection and specific identification of S. truttae in all life cycle stages. Furthermore, two of the primers from the peR assay were successfully used on tissue sections in an optimised in situ hybridisation (ISH) protocol. ISH experimentally identified the gills as the predominant entry locus of S. trottse into the fish host and it detected the spatiotemporal migration of the parasite via the vascular system into the target organ, the kidney. The ISH protocol and the peR assay were also used to screen oligochaetes and other co-occurring invertebrates for S. truttae infection but an alternate host for S. truttse could not be identified. However, 12 actinosporean stages were found and they were characterized on the basis of their 185 rONA, together with 9 further myxosporean species from wild fish in the same riverine habitat. Three actinosporeans were found to be genetically identical with three myxosporeans (Myxidium truttae, Chloromyxum truttse and Chloromyxum sp.) and thus represent alternate life cycle stages of these species. Phlyogenetic analysis of the myxozoans identified a very basal position of S. truttae and S. elegans, as a sister group to the marine species. All other species were nested in the freshwater clades and clustered according to host tissue localization, but independent from host species or myxozoan spore taxonomy.