Phylogeny of spironucleus (eopharyngia: diplomonadida: hexamitinae).

Diplomonad flagellates from the genera Spironucleus and Hexamita (Hexamitinae) inhabit the digestive tract of a variety of animal hosts, some as important pathogens, and others as commensals. Species descriptions of many diplomonads have been based on light microscopy only, causing many taxa to be misidentified. Presently, electron microscopy is considered the appropriate tool for description of diplomonads. The existence of morphologically very similar genotypes/species has in addition prompted the need for molecular tools to resolve the true identity of many diplomonad taxa. To further explore the relationship within the Hexamitinae we have sequenced most of the SSU rRNA gene from Spironucleus torosa isolated from Atlantic cod, Spironucleus meleagridis isolated from turkey, Spironucleus vortens isolated from ide and Hexamita nelsoni isolated from oyster. Phylogenetic analyses recovered three non-monophyletic Spironucleus clades that may have originated separately in the sea, in freshwater or on land. Spironucleus torosa was identified as a sister taxon to Spironucleus barkhanus and Spironucleus salmonicida. Spironucleus vortens from ide appeared to be genetically very different from Spironucleus vortens isolated from angelfish.

Ultrastructure and molecular diagnosis of Spironucleus salmonis (Diplomonadida) from rainbow trout Oncorhynchus mykiss in Germany.

Diplomonad flagellates infect a wide range of fish hosts in aquaculture and in the wild in North America, Asia and Europe. Intestinal diplomonad infection in juvenile farmed trout can be associated with morbidity and mortality, and in Germany, diplomonads in trout are commonly reported, and yet are poorly characterised. We therefore undertook a comprehensive study of diplomonads from German rainbow trout Oncorhynchus mykiss, using scanning and transmission electron microscopy, and sequencing of the small subunit (ssu) rRNA gene. The diplomonad was identified as Spironucleus salmonis, formerly reported from Germany as Hexamita salmonis. Our new surface morphology studies showed that the cell surface was unadorned and a caudal projection was present. Transmission electron microscopy facilitated new observations of functional morphology, including vacuoles discharging from the body surface, and multi-lobed apices of the nuclei. We suggest the lobes form, via hydrostatic pressure on the nucleoplasm, in response to the beat of the anterior-medial flagella. The lobes serve to intertwine the nuclei, providing stability in the region of the cell exposed to internal mechanical stress. The ssu rRNA gene sequence clearly distinguished S. salmonis from S. barkhanus, S. salmonicida, and S. vortens from fish, and can be used for identification purposes. A 1405 bp sequence of the ssu rRNA gene from S. salmonis was obtained and included in a phylogenetic analysis of a selection of closely related diplomonads, showing that S. salmonis was recovered as sister taxon to S. vortens.

Severe mortality in wild Atlantic salmon Salmo salar due to proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae (myxozoa).

Extensive mortality in Atlantic salmon fry was reported in the River Aelva from 2002 to 2004. Dead fish were collected in late summer 2006, and live fish were sampled by electrofishing in September the same year. At autopsy and in histological sections, the fish kidneys were found to be pale and considerably enlarged. Proliferative lesions with characteristic PKX cells were seen in a majority of the fish. DNA from kidney samples of diseased fish was subjected to PCR and sequencing, and the amplified sequences matched those of Tetracapsuloides bryosalmonae. We concluded that this myxozoan transmitted from bryozoans was the main cause of the observed mortality in salmon fry in 2006. Results from quantitative electrofishing in 2005 and 2006, combined with the observed fry mortality from 2002 to 2004, show that the smolt production in the river is severely reduced and that T. bryosalmonae is the most likely explanation for this decline. The present study is the first to report a considerable negative population effect in wild Atlantic salmon due to proliferative kidney disease (PKD). It also represents the northernmost PKD outbreak in wild fish. The river is regulated for hydroelectric power purposes, causing reduced water flow and elevated summer temperatures, and the present PKD outbreak may serve as an example of increased disease vulnerability of northern fish populations in a warmer climate.

Phylogenetic analysis of the SSU rRNA gene from the piscine diplomonad Spironucleus torosus (Diplomonadida: Hexamitinae).

Previous studies have recorded Spironucleus torosus Poynton et Morrison, 1990 from several species of gadoid fishes, including the only freshwater gadoid, the burbot Lota lota (L.). Two morphologically different isolates of S. torosus have been described (elongate and pyriform). Both have been found in saltwater, while only the elongate has been found in freshwater. To address the conspecificity of the two morphs of S. torosus, and to identify the source of S. torosus in burbot in Norway, we have sequenced the small subunit ribosomal RNA (SSU rRNA) gene from 43 isolates of S. torosus from six species of gadoid fishes sampled at 15 localities in Norway, Sweden and the Baltic Sea. Phylogenetic analyses of the SSU rRNA gene sequence data recovered two major clades, one containing mainly isolates from burbot, while the other contained isolates from marine gadoid fishes only. The genetic distance (based on 25 nucleotide substitutions in 789 base pairs) separating the two assemblages was not large enough to consider the two groups separate species. Spironucleus torosus isolated from burbot displayed limited genetic variation in the small subunit ribosomal RNA (SSU rRNA) gene along the post-Pleistocene migration route of its host. The present study is the first report of S. torosus in tusk Brosme brosme (Ascanius), whiting Merlangius merlangus (L.), and fourbeard rockling Enchelyopus cimbrius (L.).

Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar.

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.

Parasites of wild sea bass Dicentrarchus labrax from Norway.

Thirteen wild sea bass from the Oslo fjord in south-eastern Norway were examined for parasites. Nineteen species were found, comprising 5 protozoans, 1 monogenean, 8 digeneans, 1 cestode, 2 nematodes and 2 crustaceans. Based on the similarity to the parasitic fauna of Mediterranean sea bass, it is predicted that sea bass farmers in Northern Europe will experience the same parasite problems as sea bass farmers in warmer regions.

SSU rRNA gene sequence reveals two genotypes of Spironucleus barkhanus (Diplomonadida) from farmed and wild Arctic charr Salvelinus alpinus.

Spironucleus barkhanus isolated from the blood of Arctic charr Salvelinus alpinus from a marine fish farm were genetically compared with S. barkhanus isolated from the gall bladder of wild Arctic charr. The wild Arctic charr were caught in the lake used as the water source for the hatchery from which the farmed fish originated. Sequencing of the small subunit ribosomal RNA gene (SSU rDNA) from these 2 populations showed that the isolates obtained from farmed and wild Arctic charr were only 92.7 % similar. Based on the sequence differences between these isolates, it is concluded that the parasites isolated from the farmed fish have not been transmitted from wild Arctic charr in the hatchery's fresh water source. It is therefore most likely that the farmed fish were infected by S. barkhanus after they were transferred to seawater. S. barkhanus isolated from diseased farmed Arctic charr were 99.7% similar to the isolates obtained from diseased farmed Chinook (Canada) and Atlantic salmon (Norway). The high degree of sequence similarity between S. barkhanus from farmed Arctic charr, Chinook and Atlantic salmon indicates that systemic spironucleosis may be caused by specific strains/variants of this parasite. The genetic differences between the isolates of farmed and wild fish are of such magnitude that their conspecificity should be questioned.

Intracellular infection with Spironucleus barkhanus (Diplomonadida: Hexamitidae) in farmed Arctic char Salvelinus alpinus.

A case of intracellular systemic infection with the diplomonad flagellate Spironucleus barkhanus in farmed Arctic char Salvelinus alpinus is described. The parasites were widely disseminated throughout the vasculature and in most organs. Aggregates of the parasites were seen within well-defined structures regarded as host cells in capillaries and sinusoids of the liver, spleen and head kidney. Intracellular infection with Spironucleus spp. has never previously been reported. The prevalence of infection and mortality in the affected farm was low. In contrast to systemic spironucleosis in farmed Atlantic salmon, and despite huge numbers of flagellates in the vasculature, the tissues of the organs were remarkably unaffected. The relatively few gross and histopathological lesions may indicate that Arctic char are more tolerant to this parasite than Atlantic salmon.

Infection by Parvicapsula sp. (Myxozoa) is associated with mortality in sea-caged Atlantic salmon Salmo salar in northern Norway.

In March 2002, 3 seawater farms in northern Norway experienced high mortality among Atlantic salmon postsmolts. A myxosporean parasite assigned to the genus Parvicapsula was detected in the pseudobranchs of diseased fish, and extensive destruction of this organ was observed. The parasite was also found in the gills, liver and kidney of some fish. Based on host species, spore morphology, and the unusual site preference of the parasite, it is likely that it represents a hitherto undescribed species. The diseased fish had been transferred to seawater in September 2001, and it is believed that the infection took place shortly after exposure to seawater. The source of infection is unknown.

Isolation of a novel fish thymidylate kinase gene, upregulated in Atlantic salmon (Salmo salar L.) following infection with the monogenean parasite Gyrodactylus salaris.

Analysis of differential gene expression in salmon (Salmo salar) blood following infection with the monogenean parasite Gyrodactylus salaris, resulted in the isolation of a thymidylate kinase gene not previously described from fish and which showed similarity to an LPS-inducible thymidylate kinase gene isolated from mouse macrophages. This salmon TYKi-like gene may play a role in an innate generalised response to pathogen infection as it was upregulated in salmon following infection with the parasite, and also in response to injection with the immunostimulants LPS and Poly I:C, used to emulate bacterial and viral infections, respectively. The possible role of this gene in the biosynthesis of mitochondrial DNA in activated macrophages, in response to G. salaris infection is discussed.

The marine pathogenic genotype of Spironucleus barkhanus from farmed salmonids redescribed as Spironucleus salmonicida n. sp.

There are two genotypes of the diplomonad Spironucleus barkhanus. Based on sequence data from the small subunit ribosomal RNA gene the conspecificity of these two genotypes has been questioned. Therefore, we have sampled Spironucleus from 27 fish, representing 14 populations, five species, and four genera. Partial nucleotide sequences from the three genes; small subunit ribosomal DNA, glutamate dehydrogenase 1 and alpha-tubulin were compared. The pathogenic isolates of S. barkhanus, which causes systemic spironucleosis in Atlantic salmon, Chinook salmon, and Arctic charr, all farmed in sea water, were genetically very different from the commensal isolate found in wild freshwater populations of Arctic charr and grayling. The genetic distances between the genotypes were of the same magnitude as those separating species of Giardia. Based on these genetic and ecological data, the pathogenic genotype from farmed salmonids is described as a new species, Spironucleus salmonicida n. sp. Scanning and transmission electron microscopy showed no specific morphological or ultrastructural features distinguishing S. salmonicida n. sp. from S. barkhanus. The present study clearly demonstrates the value of applying genetics in identification of Spironucleus species. Phylogenetic analyses that included the isolates of S. salmonicida n. sp. did not change the phylogenetic relationship within the genus Spironucleus.

Spironucleus vortens (Diplomonadida) in the Ide, Leuciscus idus (L.) (Cyprinidae): a warm water hexamitid flagellate found in northern Europe.

The hexamitid flagellate Spironucleus vortens, previously reported from Pterophyllum scalare from Florida, was found in the intestine of Leuciscus idus in Norway. The flagellate was cultivated and studied by scanning and transmission electron microscopy. Identification was based on a suite of ultrastructural features unique for S. vortens: compound lateral ridges, a swirled posterior end, and a distinctive microtubular cytoskeleton. Microfibrillar structures with a periodicity of 0.13 microm in the right peripheral part of the compound lateral ridges were shown to be responsible for the distinctive rope-like appearance of the peripheral ridge seen in scanning electron micrographs, and not previously reported for S. vortens. The present results show a wide geographic distribution and a wide temperature tolerance for S. vortens. The flagellate was successfully cultivated at 5 degrees C and 15 degrees C, having previously been cultivated between 2-34 degrees C. Spironucleus vortens is believed to be endemic in Norwegian waters, but an introduction hypothesis is also discussed. The similarity is striking between S. vortens and S. elegans, previously described from amphibians and fish in Europe, and the possibility of conspecificity is believed to be high.