Comparison of small subunit ribosomal RNA gene and internal transcribed spacer sequences among isolates of the intranuclear microsporidian Nucleospora salmonis

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.     

Wild arctic char Salvelinus alpinus and trout Salmo trutta: hosts and reservoir of the salmonid pathogen Spironucleus salmonicida (Diplomonadida; Hexamitidae)

Spironucleus salmonicida is a diplomonad flagellate known to cause systemic infections in farmed salmonids. In northern Norway, outbreaks of spironucleosis in farmed Atlantic salmon Salmo salar have been a recurring problem. Common to all these outbreaks was the origin of smolts: all came from the same farm. In the present study, wild Arctic char Salvelinus alpinus and brown trout Salmo trutta were sampled from the lakes used as a water source for the smolt supplier. In addition, smolt and three-spined sticklebacks Gasterosteus aculeatus were sampled from the smolt farm. Bile and intestinal contents from the sampled fish were examined by light microscopy and PCR. Spironucleus salmonicida was identified in both wild Arctic char and brown trout from the lakes used as water sources by the smolt farm, suggesting that the farmed fish were exposed to this pathogen before transfer to the sea. Spironucleus barkhanus and Spironucleus salmonis were also identified in the sampled fish. The present study also demonstrated that infections with multiple Spironucleus species are present in wild salmonids. No indications of disease related to diplomonad infections were observed in the wild fish, suggesting that wild salmonids are reservoir hosts of Spironucleus salmonicida.     

Evidence from SSU rRNA phylogeny that Octomitus is a sister lineage to Giardia

Octomitus intestinalis is a diplomonad flagellate inhabiting the digestive tract of rodents and amphibians. Octomitus is of evolutionary interest because, based on ultrastructural characteristics, it is thought to be closely related to the morphologically derived genus Giardia, and together they have been proposed to make up the Giardiinae. In molecular trees of diplomonads, Giardia is the deepest branching lineage, so identifying a sister group to Giardia that is less derived would be informative. Octomitus is a logical candidate for this position, but unfortunately there are no molecular data from it, and it is not available in culture. To determine the position of Octomitus, and specifically test whether it is more closely related to Giardia than other diplomonads, we have isolated it directly from the caecum of wild mice and characterized its small subunit ribosomal RNA (SSU rRNA) gene. Phylogenetic analysis showed Octomitus to be the sister to Giardia with strong support, together occupying one side of the deepest split in the diplomonad tree.     

生物的分界与前源真核生物界的提出

根据本实验室对贾第虫（Ｇｉａｒｄｉａ）细胞核的观察、研究,并参阅有关文献及电镜照片报道,发现双滴虫类的细胞核有两个极原始的特征：１）还没有进化出核仁;２）核被膜尚不完整。它们在核分裂方面也是极原始的。据此,作者建议把Ｃａｖａｌｉｅｒ—Ｓｍｉｔｈ（１９８９）提出的源真核生物（Ａｒｃｈｅｚｏａ）超界中的源真核生物界划分为两个界,即以双滴虫门为代表的前源真核生物（Ｐｒｏａｒｃｈｅｚｏａ）界和包含目前所知的其他源真核生物的后源真核生物（Ｍｅｔａｒｃｈｅｚｏａ）界。     

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.     

Intestinal pH profile in rainbow trout Oncorhynchus mykiss and microhabitat preference of the flagellate Spironucleus salmonis (Diplomonadida)

In farmed rainbow trout Oncorhynchus mykiss, the flagellate Spironucleus salmonis (Diplomonadida) is often found in the pyloric region of the intestine. While previous in vitro studies report a pH of 7.5 to 8.0 as optimal for presumed S. salmonis, no previous in vivo studies have investigated the relationship between pH and microhabitat preference. Therefore, in 698 rainbow trout (75% were 5 to 6 mo old juveniles, 10 to 20 cm total length), we recorded occurrence and density of S. salmonis, and pH, in the pyloric, anterior, middle, and posterior intestine. There were no significant differences in total length or weight between infected and uninfected fish. S. salmonis preferred the pyloric region, with occurrence and density decreasing significantly from pyloric to posterior regions. In infected fish, pH in pyloric (6.8 to 7.9, mean 7.3) and posterior regions (6.5 to 8.0, mean 7.1) was significantly lower than in anterior (6.5 to 8.5, mean 7.7) and middle (6.8 to 8.2, mean 7.7) regions; in uninfected fish, the pH profile was similar. At the individual level, 90 % of infected fish and 79% of uninfected fish showed this pH profile. In the pyloric region, pH was not significantly different among uninfected fish, and fish with light, moderate, or heavy infections. Our in vivo study suggests the optimal pH for S. salmonis is between 7.1 and 7.5, possibly close to 7.3 (the mean in pyloric region of infected fish). We conclude that while the presence of S. salmonis reflected tolerable pH, density of infection was not correlated with pH, and thus a causal relationship between microhabitat preference and pH is unlikely.     

Distribution of the flagellate Hexamita salmonis Moore, 1922 and the microsporidian Loma salmonae Putz, Hoffman and Dunbar, 1965 in brown trout, Salmo trutta L., and rainbow trout, Salmo gairdneri Richardson, in the River Itchen (U.K.) and three of its fish farms

The occurrence of Hexamita salmonis Moore, 1922 and Loma salmonae Putz, Hoffman and Dunbar, 1965 was investigated at 10 sites on the R. Itchen (five for brown trout only, three for rainbow trout only, and two for both brown trout and rainbow trout) and at three of its nine fish farms (two for rainbow trout, one for brown trout). Hexamita salmonis was recorded in brown trout from three river sites and the farm, and in rainbow trout from both farms and four river sites. Prevalence of Hexamita salmonis in farmed rainbow trout was higher than in farmed brown trout and was consistent with the former species being more susceptible to infection. H. salmonis was at significantly higher prevalence in rainbow trout from farm no. 5 than farm no. 2 for three size classes of fish. In wild brown trout and feral rainbow trout, the highest prevalences of H. salmonis were recorded at sites in the vicinity of farm no. 2. This distribution was consistent with an area of naturally high infection levels, and with infected fish unintentionally released from farm no. 2 serving as a source of infection, the infection subsequently becoming established in the river fish. Loma salmonae was recorded in wild brown trout and in rainbow trout from both farms. This appears to be the first recording of this parasite from British salmonids and also the first recording of the parasite from brown trout. The distribution of the parasite (particularly the prevalence being higher at farm no. 2 than farm no. 5) was consistent with it being introduced into the R. Itchen via rainbow trout from farm no. 2 (and probably no. 3) much of whose stock derived from imported Californian 'Shasta' rainbow trout.     

Distribution and phylogenetic relationships of freshwater Thaumatomonads with a description of the new species Thaumatomonas coloniensis n. sp

The order Thaumatomonadida includes biflagellated heterotrophic flagellates that form filopodia and typically possess siliceous surface scales. We found thaumatomonads to contribute on average about 5%-10% to flagellate abundance in different benthic habitats. A new species of thaumatomonads, Thaumatomonas coloniensis n. sp., is described on the basis of morphological and molecular biological features. This new species was isolated both from groundwater at Appeldorn near Rees (Germany) and from the Rhine River at Cologne (Germany). We have sequenced the small subunit rRNA (ssu rRNA) gene and a fragment of the large subunit rRNA (lsu rRNA) gene (D3-D5 region) from the isolates of the new species, including the first sequence of a representative of the thaumatomonad genus Gyromitus. In agreement with previous studies, the differences in ribosomal genes of different thaumatomonad species are very small. For understanding the phylogenetic relationships of Thaumatomonadida and to explore their sister group relationships, we have created three sequence data sets (ssu rRNA, partial lsu rRNA, concatenated alignment of both) with the same composition of isolates (from Thaumatomonadida, Euglyphida, Cercomonadidae, and Heteromitidae). According to a Kishino-Hasegawa test, Thaumatomonadida evolved within the Cercozoa as a sister taxon to the Heteromitidae. A possibly close relationship to the Euglyphida, recently grouped together with the Thaumatomonadida in the class Imbricatea/Silicofilosea based on the rRNA data sets was not supported by our analyses.     

Spironucleus vortens N. Sp. from the Freshwater Angelfish Pterophyllum scalare: Morphology and Culture

ABSTRACT. A new diplomonad flagellate, Spironucleus vortens n. sp., is described from the intestinal lumen of the freshwater angelfish, ( Pterophyllum scalare ), bred in Florida. Live organisms are pyriform, and measure 12.5–20.5 μm long by 5.0–11.2 üm wide. Scanning electron microscopy shows that the trophozoite bears two compound lateral longitudinal ridges, each originating posterior to three emerging anterior flagella, and continuing posteriorly to the emergence of the posterior flagellum. Each ridge comprises a broad central part, surrounded by a peripheral ridge. At the opening of the flagellar pocket, the broader right peripheral ridge crosses to the other side of the body, and then back again. The posterior end of the body bears two papillae. Transmission electron microscopy shows that the compound lateral ridges are supported by microtubules, and bear microfibrillar structures in discrete longitudinal plaques. The serendipitous growth of S. vortens in a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose). The flagellate is now routinely maintained in an axenic TYI-S-33 medium (trypticase, yeast extract, iron serum), and is stabilized in the cryopreserved state. Spironucleus vortens is an aerotolerant anaerobe that can be cultured at 25° C, 28° C and 30° C.     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain.

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.     

Phylogenetic affinity of a Giardia lamblia cysteine desulfurase conforms to canonical pattern of mitochondrial ancestry

Among a few potential archezoan groups, only the Metamonada (diplomonads, retortamonads, and oxymonads) still retain the status of amitochondriate protists that diverged before the acquisition or retention of mitochondria. Indeed, finding that diplomonad genomes harbor a gene encoding a mitochondrial type chaperonin 60, the most compelling evidence for their secondarily amitochondriate nature, may be interpreted as an acquisition of this important general chaperone during some transient alpha-proteobacterial endosymbiosis. Recently published data on the cysteine desulfurase IscS demonstrated an alpha-proteobacterial origin of mitochondrial enzymes including a diplomonad Giardia lamblia homolog. An extended phylogenetic analysis of IscS is reported here that revealed a full canonical pattern of mitochondrial ancestry for the giardial enzyme. The above canonical pattern, a sister group relationship of mitochondria and rickettsiae exclusive of free-living alpha-proteobacteria, was robustly confirmed by a comprehensive analysis of Cob and Cox1 subunits of the respiratory chain encoded by resident mitochondrial genes. Given that Fe-S cluster assembly involving IscS represents an essential mitochondrial function, these data strongly suggest that diplomonads once harbored bona fide mitochondria.     

Local patchiness of Gyrodactylus colemanensis and G. salmonis parasitizing salmonids in the South River watershed, Nova Scotia, Canada

Prevalence and intensity of Gyrodactylus colemanensis and G. salmonis (Monogenea) parasitizing juvenile/adult brook trout Salvelinus fontinalis, rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta, and Atlantic salmon Salmo salar at 3 localities over an 8 km stretch in the South River, Nova Scotia, Canada, were calculated 4 times over a 9 mo period (October 2009, December 2009, March 2010, June 2010). G. colemanensis was on all 4 salmonids (endemic and non-endemic), while G. salmonis parasitized mostly S. fontinalis (endemic) and occasionally S. trutta (non-endemic). At an upstream locality, beyond a waterfall barrier, in a small tributary of the main river, G. colemanensis was more common than G. salmonis. In the main river, 7 km downstream, prevalence of G. colemanensis on S. fontinalis was comparable, or higher, than that of G. salmonis, while intensity of G. salmonis was higher than that of G. colemanensis. Downstream a further 1 km, in a tributary of the main river, both prevalence and intensity of G. salmonis on brook trout were higher than those of G. colemanensis. Stocks at a local trout hatchery had only G. colemanensis. The present study reports on a method by which exit water from such farms can be monitored for gyrodactylid parasites through a simple settling procedure. We estimated that up to 230,000 dislodged, live G. colemanensis exit the hatchery daily in discharge water entering the river. It is suggested that such systems are ideal for studying the impact of such parasite export on the nature of local parasite populations.     

Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids

Adult and mobile preadult sea lice Lepophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sealice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis-derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.     

A wide diversity of previously undetected free‐living relatives of diplomonads isolated from marine/saline habitats

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free‐living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas‐like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small‐subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad‐retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation – it is likely that undersampling at the level of major lineages is still widespread for protists.     

Lateral transfer of the gene for a widely used marker, alpha-tubulin, indicated by a multi-protein study of the phylogenetic position of Andalucia (Excavata)

alpha-Tubulin is one of the most widely used markers for estimating deep-level phylogenetic relationships amongst eukaryotes. We sequenced 6-7 nuclear protein-coding genes, including alpha-tubulin, from the two described species of the enigmatic jakobid(-like) excavate protist Andalucia. Concatenated protein phylogenies place Andalucia in a clade with other jakobids, Euglenozoa and Heterolobosea. Individual gene trees, except that of alpha-tubulin, do not conflict strongly with this position. In alpha-tubulin trees, Andalucia instead falls in a strongly supported clade with diplomonads, parabasalids and opisthokonts (including animals and fungi), and branches with diplomonads. This clade is robust to changes in taxon sampling, and is unlikely to represent long-branch attraction, compositional heterogeneity artefact, or segmental gene conversion. Phylogenies estimated without alpha-tubulin strongly support the original position for Andalucia, and also reinforce recent studies in placing diplomonads and parabasalids with Preaxostyla, not opisthokonts. alpha-Tubulin seems to have experienced two or more eukaryote-to-eukaryote lateral gene transfer (LGT) events, one perhaps from an ancestral opisthokont to an ancestor of diplomonads and parabasalids, or vice versa, and one probably from the diplomonad lineage to Andalucia. Like EF-1alpha/EFL, alpha-tubulin has a complex history that needs to be taken into account when using this marker for deep-level phylogenetic inference.