CULTIVATION‐INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE‐CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.     

Host specificity and incidence of Trypanosoma in some African rainforest birds: a molecular approach

Studies of host-parasite interactions in birds have contributed greatly to our understanding of the evolution and ecology of disease. Here we employ molecular techniques to determine the incidence and study the host-specificity of parasitic trypanosomes in the African avifauna. We developed a polymerase chain reaction (PCR)-based diagnostic test that amplified the small subunit ribosomal RNA gene (SSU rRNA) of Trypanosoma from avian blood samples. This nested PCR assay complements and corroborates information obtained by the traditional method of blood smear analysis. The test was used to describe the incidence of trypanosomes in 479 host individuals representing 71 rainforest bird species from Cameroon, the Ivory Coast and Equatorial Guinea. Forty-two (59%) of these potential host species harboured trypanosomes and 189 individuals (35%) were infected. To examine host and geographical specificity, we examined the morphology and sequenced a portion of the SSU rRNA gene from representative trypanosomes drawn from different hosts and collecting locations. In traditional blood smear analyses we identified two trypanosome morphospecies, T. avium and T. everetti. Our molecular and morphological results were congruent in that these two morphospecies had highly divergent SSU rRNA sequences, but the molecular assay also identified cryptic variation in T. avium, in which we found seven closely allied haplotypes. The pattern of sequence diversity within T. avium provides evidence for widespread trypanosome mixing across avian host taxa and across geographical locations. For example, T. avium lineages with identical haplotypes infected birds from different families, whereas single host species were infected by T. avium lineages with different haplotypes. Furthermore, some conspecific hosts from geographically distant sampling locations were infected with the same trypanosome lineage, but other individuals from those locations harboured different trypanosome lineages. This apparent lack of host or geographical specificity may have important consequences for the evolutionary and ecological interactions between parasitic trypanosomes and their avian hosts.     

High diversity of the 'Spumella-like' flagellates: an investigation based on the SSU rRNA gene sequences of isolates from habitats located in six different geographic regions

We isolated 28 strains of 'Spumella-like' flagellates from different freshwater and soil habitats in Austria, People's Republic of China, Nepal, New Zealand, Uganda, Kenya, Tanzania and Hawaii by use of a modified filtration-acclimatization method. 'Spumella-like' flagellates were found in all of the samples and were often among the dominant bacterivorous flagellates in the respective environments. The small subunit ribosomal RNA (SSU rRNA) gene sequence of the isolates was determined and aligned with previously published sequences of members belonging to the Chrysophyceae sensu stricto. Phylogenetic analysis of the 28 new sequences confirmed their position within the Chrysophyceae sensu stricto and positioned them within different clades. Most of the sequences grouped within clade C and formed several subclusters separated from each other by green taxa including flagellates belonging to Ochromonas, Dinobryon, Poterioochromonas and others. All soil isolates clustered together (subcluster C1) with the soil strain Spumella elongata and the undescribed soil strain 'Spumella danica'. Aquatic isolates were affiliated with at least two branches (C2 and C3). Sequence similarity to the closest related member of the Chrysophyceae ranged between 92% and 99.6%, sequence divergence among the 'Spumella-like' flagellates was as high as 10%. We conclude that (i) the 'Spumella-like' flagellates are a diverse group both in terms of sequence dissimilarity between isolates and in terms of the number of genotypes, (ii) Spumella and Ochromonas are polyphyletic, and (iii) based on the SSU rRNA gene no biogeographical restriction of certain branches could be observed even though different ecotypes may be represented by the same genotype.     

Blood parasites of chickens in Uganda and Cameroon with molecular descriptions of Leucocytozoon schoutedeni and Trypanosoma gallinarum

Using microscopy and PCR, we determined the prevalence of blood parasites in village chickens in Uganda and Cameroon. Of 148 individuals tested, 18.3% were infected with Leucocytozoon schoutedeni (Haemosporida, Leucocytozoidae) and 4.1% were infected with Trypanosoma gallinarum (Kinetoplastida, Trypanosomatidae). No other blood parasites were detected. Subsequent phylogenetic analysis of the cytochrome b gene of L. schoutedeni identified 2 distinct lineages that were found at all 3 sampling locations in Uganda. The sequence divergence between these 2 lineages is 1.5%. One of these lineages was also found in chickens in Cameroon, nearly 2,000 km distant. There are no morphological differences between blood stages of the parasites represented by the 2 different lineages, suggesting that cytochrome b gene sequence divergence can be as high as 1.5% within a single well-defined morphospecies of Leucocytozoon. We sequenced a portion of the small subunit ribosomal RNA gene (SSU rRNA) of T. gallinarum, and redescribe T. gallinarum for the first time since its discovery in 1911. These are the first assignments of DNA sequence data to these morphospecies of Leucocytozoon and Trypanosoma and may represent an example of intraspecific sequence divergence.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Multiple cosmopolitan ecotypes within a microbial eukaryote morphospecies

Microbial eukaryotes that are morphologically indistinguishable (i.e. 'morphospecies') tend to be genetically diverse. While most protist morphospecies have cosmopolitan distribution, it has been suggested that ribotypes (unique rRNA gene sequences) or rRNA sequence clusters do have biogeography and such clusters may correlate with particular (non-morphological) adaptations. We have studied this in the ciliated protozoan morphospecies Cyclidium glaucoma. Fifty-four isolates collected worldwide represented 31 distinct ribotypes. There was no evidence of biogeographic distribution patterns. For example, identical ribotypes occurred in samples from Argentina, Peru, Morocco, Russia and Ukraine; in samples from Denmark and Australia; and in samples from Great Salt Lake and hyperhaline ponds in Spain. The morphospecies Cyclidium glaucoma is euryhaline and occurs in freshwater, brackish water, seawater, and hyperhaline waters. Evidence suggests that one ribotype cluster occurs only in marine or brackish habitats, and another one has so far been found only in hyperhaline habitats. Two clades seem to occur only in freshwater, but one clade includes ribotypes that were found in freshwater as well as in brackish water.     

Cryptic diversity within the choanoflagellate morphospecies complex Codosiga botrytis - Phylogeny and morphology of ancient and modern isolates

Choanoflagellates are closely related to metazoans and fungi according to recent phylogenetic studies; therefore the systematics of these organisms is of particular interest. The choanoflagellate morphospecies Codosiga botrytis is the first described choanoflagellate, and is one of the most frequently reported choanoflagellate species. In this study we present phylogenetic and morphological data on eight different strains of Codosiga botrytis. Among these there are five ancient strains; these cultures have been established from up to 43,000 years old cysts from Siberian permafrost. We found that based on the variable V4 region of the small subunit (SSU) of the rDNA, all the investigated freshwater isolates of Codosiga botrytis, together with Sphaeroeca volvox, form a cluster at the base of all other choanoflagellate species. Moreover, the morphospecies described classically as Codosiga botrytis contains at least four different genotypes separated by considerably high genetic distance. All these 'cryptic species' have identical general morphology and cell structure. Strains have a similar life cycle with several different life forms and large morphological plasticity. For the first time we were able to establish cultures from cryo-conserved cysts of choanoflagellates. The ancient strains did not differ significantly in partial SSU rDNA from the modern ones. Besides, no biogeographically pattern could be established. This fact and the low genetic distances of some strains from remote locations support the distribution of dormant stages via air.     

High diversity and isolated distribution of aquatic heterotrophic protists in salars of the Atacama Desert at different salinities

The species richness of eukaryotes in the hypersaline environment is generally thought to be low. However, recent studies showed a high degree of phylogenetic novelty at these extreme conditions with variable chemical parameters. These findings call for a more thorough look into the species richness of hypersaline environments. In this study, various hypersaline lakes (salars, 1–348 PSU) as well as further aquatic ecosystems of northern Chile were investigated regarding diversity of heterotrophic protists by metabarcoding studies of surface water samples. Investigations of genotypes of 18S rRNA genes showed a unique community composition in nearly each salar and even among different microhabitats within one salar. The genotype distribution showed no clear connection to the composition of main ions at the sampling sites, but protist communities from similar salinity ranges (either hypersaline, hyposaline or mesosaline) clustered together regarding their OTU composition. Salars appeared to be fairly isolated systems with only little exchange of protist communities where evolutionary lineages could separately evolve.     

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.     

Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.     

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene. Accepted: 25 October 2000     

Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5'-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5'-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5'-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.     

Morphological and genetic evidence that the cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck encompasses at least two species

Dense blooms of the cyanobacterium Lyngbya wollei are increasingly responsible for declining water quality and habitat degradation in numerous springs, rivers, and reservoirs. This research represents the first molecular phylogenetic analysis of L. wollei in comparison with the traditional morphological characterization of this species. Specimens were collected from several springs in Florida and a reservoir in North Carolina. Segments of the small-subunit (SSU) rRNA and nifH genes were PCR amplified, cloned, and sequenced. The phylogenetic analysis of the SSU rRNA gene revealed sequences that fell into three distinct subclusters, each with >97% sequence similarity. These were designated operational taxonomic unit 1 (OTU1), OTU2, and OTU3. Similarly, the nifH sequences fell into three distinct subclusters named S1, S2, and S3. When either bulk samples or individual filaments were analyzed, we recovered OTU1 with S1, OTU2 with S2, and OTU3 with S3. The coherence between the three SSU rRNA gene and nifH subclusters was consistent with genetically distinct strains or species. Cells associated with subclusters OTU3 and S3 were significantly wider and longer than those associated with other subclusters. The combined molecular and morphological data indicate that the species commonly identified as L. wollei in the literature represents two or possibly more species. Springs containing OTU3 and S3 demonstrated lower ion concentrations than other collection sites. Geographical locations of Lyngbya subclusters did not correlate with residual dissolved inorganic nitrogen or phosphorus concentrations. This study emphasizes the need to complement traditional identification with molecular characterization to more definitively detect and characterize harmful cyanobacterial species or strains.     

Solenicola setigera is the first characterized member of the abundant and cosmopolitan uncultured marine stramenopile group MAST‐3

Culture‐independent molecular methods based on the amplification, cloning and sequencing of small‐subunit (SSU) rRNA genes are a powerful tool to study the diversity of prokaryotic and eukaryotic microorganisms for which morphological features are not conspicuous. In recent years, molecular data from environmental surveys have revealed several clades of protists lacking cultured and/or described members. Among them are various clades of marine stramenopiles (heterokonts), which are thought to play an essential ecological role as grazers, being abundant and distributed in oceans worldwide. In this work, we show that Solenicola setigera, a distinctive widespread colonial marine protist, is a member of the environmental clade MArine STramenopile 3 (MAST‐3). Solenicola is generally considered as a parasite or an epiphyte of the diatom Leptocylindrus mediterraneus. So far, the ultrastructural, morphological and ecological data available were insufficient to elucidate its phylogenetic position, even at the division or class level. We determined SSU rRNA gene sequences of S. setigera specimens sampled from different locations and seasons in the type locality, the Gulf of Lions, France. They were closely related, though not identical, which, together with morphological differences under electron microscopy, suggest the occurrence of several species. Solenicola sequences were well nested within the MAST‐3 clade in phylogenetic trees. Since Solenicola is the first identified member of this abundant marine clade, we propose the name Solenicolida for the MAST‐3 phylogenetic group.     

A Novel Alveolate in Bivalves with Chemosynthetic Bacteria Inhabiting Deep‐Sea Methane Seeps

It has recently been unveiled that a wide variety of microbial eukaryotes (protists) occur in chemosynthetic ecosystems, such as hydrothermal vents and methane seeps. However, there is little knowledge regarding protists associated with endemic animals inhabiting these environments. In the present study, utilizing PCR techniques, we detected fragments of the small subunit ribosomal RNA gene (SSU rRNA gene) from a particular protist from gill tissues of a significant fraction of the vesicomyid clams Calyptogena soyoae and C. okutanii complex and of the mussel Bathymodiolus platifrons and B. japonicus, all of which harbor chemosynthetic endosymbiont bacteria and dominate methane seeps in Sagami Bay, Japan. Based on the phylogeny of SSU rRNA gene, the organism in question was shown to belong to Alveolata. It is noteworthy that this protist did not affiliate with any known alveolate group, although being deeply branched within the lineage of Syndiniales, for which the monophyly was constantly recovered, but not robustly supported. In addition, the protist detected using PCR followed by sequencing was localized within gill epithelial cells of B. platifrons with whole‐mount fluorescence in situ hybridization. This protist may be an endoparasite or an endocommensal of Calyptogena spp. and Bathymodiolus spp., and possibly have physiological and ecological impacts on these bivalves.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.