Stephen D. Atkinson

Molecular and Morphological Analysis of Myxobolus Spp. of Salmonid Fishes with the Description of a New Myxobolus Species

Abstract While investigating the parasite fauna of wild coho salmon. Oncorhynchus kisutch (Walbaum, 1792), histological examination provided evidence of a new species of Myxobolus (Myxozoa: Myxosporea) infecting nerves of skeletal muscle. Spores were morphologically similar to those of the intramuscular Myxobolus insidiosus Wyatt and Pratt, 1963, both having pyriform spores with clavate polar capsules. However, the former developed exclusively in the nerves of skeletal muscle rather than in myocytes. We examined both species of Myxobolus derived from coho salmon; Chinook salmon, Oncorhynchus tshawytscha (Walbaum, 1792); cutthroat trout, Oncorhynchus clarkii (Richardson, 1836); and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from freshwater in Oregon. Spore morphology, small subunit ribosomal RNA gene (rDNA) sequences, and site of infection were compared. Myxobolus arcticus Pugachev and Khokhlov, 1979 has pyriform spores, infects the central nervous system of many salmonids, and is found in the Pacific Northwest. It was therefore included in the analyses to rule out conspecificity with the new species. Together, these data show that the Myxobolus sp. from peripheral nerves in the skeletal musculature of coho salmon, rainbow trout, and cutthroat trout is a new species, described herein as Myxobolus fryeri n. sp.

INVOLVEMENT OF MANAYUNKIA SPECIOSA (ANNELIDA: POLYCHAETA: SABELLIDAE) IN THE LIFE CYCLE OF PARVICAPSULA MINIBICORNIS, A MYXOZOAN PARASITE OF PACIFIC SALMON

A coelomic myxozoan infection was detected in freshwater polychaetes, Manayunkia speciosa from the Klamath River, Oregon/California, a site enzootic for the myxozoan parasites Ceratomyxa shasta and Parvicapsula minibicornis. The tetractinomyxon type actinospores had a near-spherical spore body 7.9 × 7.1 μm, with 3 spherical, protruding polar capsules, no valve cell processes, and a binucleate sporoplasm. Parvicapsula minibicornis-specific primers Parvi1f and Parvi2r amplified DNA from infected polychaetes in a polymerase chain reaction (PCR) assay. The small subunit 18S rRNA gene of the spores was sequenced (GenBank DQ231038) and was a 99.7% match with the sequence for P. minibicornis myxospore stage in GenBank (AF201375). Chinook salmon (Oncorhynchus tshawytscha) exposed to a dose of 1,000 actinospores per fish tested PCR positive for P. minibicornis at 14 wk postinfection and presporogonic stages were detected in the kidney tubules by histology at 20 wk. This life cycle is 1 of only about 30 known from more than 1,350 myxozoan species, and only the second known from a freshwater polychaete.

Density of the Waterborne Parasite Ceratomyxa shasta and Its Biological Effects on Salmon

ABSTRACT The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter−1 for Chinook but only 5 spores liter−1 for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.

Myxozoan parasitism in waterfowl

Myxozoans are spore-forming, metazoan parasites common in cold-blooded aquatic vertebrates, especially fishes, with alternate life cycle stages developing in invertebrates. We report nine cases of infection in free-flying native and captive exotic ducks (Anseriformes: Anatidae) from locations across the United States and describe the first myxozoan in birds, Myxidium anatidum n. sp. We found developmental stages and mature spores in the bile ducts of a Pekin duck (domesticated Anas platyrhynchos). Spores are lens-shaped in sutural view, slightly sigmoidal in valvular view, with two polar capsules, and each valve cell has 14-16 longitudinal surface ridges. Spore dimensions are 23.1 microm x 10.8 microm x 11.2 microm. Phylogenetic analysis of the ssrRNA gene revealed closest affinity with Myxidium species described from chelonids (tortoises). Our novel finding broadens the definition of the Myxozoa to include birds as hosts and has implications for understanding myxozoan evolution, and mechanisms of geographical and host range extension. The number of infection records indicates this is not an incidental occurrence, and the detection of such widely dispersed cases suggests more myxozoans in birds will be encountered with increased surveillance of these hosts for pathogens.

Disparate infection patterns of Ceratomyxa shasta (Myxozoa) in rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus tshawytscha) correlate with internal transcribed spacer-1 sequence variation in the parasite☆

Ceratomyxa shasta is a virulent myxosporean parasite of salmon and trout in the Pacific Northwest of North America. The parasite is endemic in the Klamath River, Oregon/California, where a series of dams prevent movement of fish hosts between the upper and lower parts of the basin. Ceratomyxa shasta exhibits a range of infection patterns in different fish species above and below the dams. We hypothesised that the variations in infection and disease are indicators that different strains of the parasite exist, each with distinct host associations. Accordingly, we sought to identify strain-specific genetic markers in the ssrRNA and internal transcribed spacer region 1 (ITS-1). We examined 46 C. shasta isolates from water samples and two fish hosts, from June 2007 field exposures at upper and lower Klamath River sites with similarly high parasite densities. We found 100% of non-native rainbow trout became infected and died at both locations. In contrast, mortality in native Chinook salmon was <10% in the upper basin, compared with up to 40% in the lower basin. Parasite ssrRNA sequences were identical from all fish. However, ITS-1 sequences contained multiple polymorphic loci and a trinucleotide repeat (ATC)(0-3) from which we defined four genotypes: 0, I, II and III. Non-native rainbow trout at both sites were infected with genotype II and with a low level of genotype III. Chinook salmon in the upper basin had genotypes II and III, whereas in the lower basin genotype I predominated. Genotype I was not detected in water from the upper basin, a finding consistent with the lack of anadromous Chinook salmon there. Genotype O was only detected in water from the upper basin. Resolution of C. shasta into sympatric, host-specific genotypes has implications for taxonomy, monitoring and management of this significant parasite.

Sphaerospora sensu stricto: Taxonomy, diversity and evolution of a unique lineage of myxosporeans (Myxozoa)

Myxosporeans (Myxozoa) are eukaryotic parasites, primarily of fish, whose classification is in a state of flux as taxonomists attempt to synthesize the traditional morphology-based system with emerging DNA sequence-based phylogenies. The genus Sphaerospora Thélohan, 1892, which includes pathogenic species that cause significant impacts on fisheries and aquaculture, is one of the most polyphyletic taxa and exemplifies the current challenges facing myxozoan taxonomists. The type species, S. elegans, clusters within the Sphaerospora sensu stricto clade, members of which share similar tissue tropism and long insertions in their variable rRNA gene regions. However, other morphologically similar sphaerosporids lie in different branches of myxozoan phylogenetic trees. Herein, we significantly extend taxonomic sampling of sphaerosporids with SSU+LSU rDNA and EF-2 sequence data for 12 taxa including three representatives of the morphologically similar genus Polysporoplasma Sitjà-Bobadilla et Álvarez-Pellitero, 1995. These taxa were sampled from different vertebrate host groups, biogeographic realms and environments. Our phylogenetic analyses and statistical tests of single and concatenated datasets revealed Sphaerospora s. s. as a strongly supported monophyletic lineage, that clustered sister to the whole myxosporean clade (freshwater+marine lineages). Generally, Sphaerospora s. s. rDNA sequences (up to 3.7 kb) are the longest of all myxozoans and indeed metazoans. The sphaerosporid clade has two lineages, which have specific morphological, biological and sequence traits. Lineage A taxa (marine Sphaerospora spp.) have a single binucleate sporoplasm and shorter AT-rich rDNA inserts. Lineage B taxa (freshwater/brackish Sphaerospora spp.+marine/brackish Polysporoplasma spp.) have 2-12 uninucleate sporoplasms and longer GC-rich rDNA inserts. Lineage B has four subclades that correlate with host group and habitat; all Polysporoplasma species, including the type species, cluster together in one of these subclades. We thus suppress the genus Polysporoplasma and the family Polysporoplasmidae and emend the generic diagnosis of the genus Sphaerospora. The combination of morphological, biological and DNA sequence data applied in this study helped to elucidate an important part of the taxonomic puzzle within the phylum Myxozoa.

Spatial, temporal and host factors structure the Ceratomyxa shasta (Myxozoa) population in the Klamath River basin

The myxozoan parasite Ceratomyxa shasta is a virulent pathogen of salmonid fish in the Klamath River, Oregon/California, USA. We previously defined four principal genotypes of the parasite (O, I, II, III) based on a trinucleotide repeat (ATC)(0-3) in Internal Transcribed Spacer region 1 sequences. Genotypes occur in sympatry and show marked host preference: I in Chinook salmon (Oncorhynchus tschawytscha) and II in non-native rainbow trout (O. mykiss). In the present study, we sequenced the parasite from river water samples collected in May, June and September at three localities below, above and between the Klamaths five dams. We also sampled adult and juvenile coho salmon (O. kisutch), steelhead trout (O. mykiss, anadromous form) and native redband rainbow trout (O. mykiss, freshwater form) and additional Chinook salmon and non-native rainbow trout. We found that the C. shasta population was highly structured spatially, temporally and with respect to fish host species. Genotype O was present in water throughout the basin but detected almost exclusively in steelhead and native rainbow trout. Genotype I was in water only below the dams and detected only in Chinook salmon. Genotype II was detected in coho salmon below the dams, and in non-native rainbow trout exposed both above and below the dams. The same genotypes were detected in adult and juvenile fish of the same species. These findings have major implications for the design of effective surveillance and control programs for this economically and ecologically important fish parasite.

Some remarks on the occurrence, host-specificity and validity of Myxobolus rotundus Nemeczek, 1911 (Myxozoa: Myxosporea)

Myxobolus rotundus Nemeczek, 1911 is a common and specific parasite of the common bream Abramis brama (L.). Small, round or ellipsoidal shaped plasmodia of this species develop in the gill and exhibit strong histotropism to the secondary gill lamellae with plasmodial development in their capillary network. M. rotundus is frequently found in mixed infection with M. bramae Reuss, 1906, a parasite of the afferent arteries of gill filaments. The round spores of M. rotundus resemble several other Myxobolus spp., but can be distinguished from these by their small subunit ribosomal RNA gene sequence (GenBank accession no. EU710583), which also differs from the sequence for ‘M. rotundus’ from the skin of Chinese goldfish Carassius auratus auratus (L.), which we suggest has been misidentified. The SSU rRNA gene sequence of M. rotundus myxospores from bream corresponded to Triactinomyxon type 4 actinospores (AY495707) isolated from Tubifex tubifex (Müller) by Hallett et al. (2005), and we infer from this that these are alternate life stages.

Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage.

In order to clarify the phylogenetic relationships among the main marine myxosporean clades including newly established Ceratonova clade and scrutinizing their evolutionary origins, we performed large-scale phylogenetic analysis of all myxosporean species from the marine myxosporean lineage based on three gene analyses and statistical topology tests. Furthermore, we obtained new molecular data for Ceratonova shasta, C. gasterostea, eight Ceratomyxa species and one Myxodavisia species. We described five new species: Ceratomyxa ayami n. sp., C. leatherjacketi n. sp., C. synaphobranchi n. sp., C. verudaensis n. sp. and Myxodavisia bulani n. sp.; two of these formed a new, basal Ceratomyxa subclade. We identified that the Ceratomyxa clade is basal to all other marine myxosporean lineages, and Kudoa with Enteromyxum are the most recently branching clades. Topologies were least stable at the nodes connecting the marine urinary clade, the marine gall bladder clade and the Ceratonova clade. Bayesian inference analysis of SSU rDNA and the statistical tree topology tests suggested that Ceratonova is closely related to the Enteromyxum and Kudoa clades, which represent a large group of histozoic species. A close relationship between Ceratomyxa and Ceratonova was not supported, despite their similar myxospore morphologies. Overall, the site of sporulation in the vertebrate host is a more accurate predictor of phylogenetic relationships than the morphology of the myxospore.

Complete life cycle of Myxobolus rotundus (Myxosporea: Myxobolidae), a gill myxozoan of common bream Abramis brama.

The life cycle of Myxobolus rotundus Nemeczek, 1911, a myxosporean parasite of the gills of common bream Abramis brama L., was studied under laboratory conditions. Mature Myxobolus spp. spores from plasmodia in the gills of wild bream were used to infect naïve oligochaete worms in a flow-through system of aquaria. Triactinomyxon-type actinospores were released from the oligochaetes 1 yr later and allowed to continually flow into a tank containing uninfected bream fry. The gills of the fry were checked for development of plasmodia in squash preparations 3 d postexposure, and then at weekly intervals for 8 wk. Tissue samples were fixed at each time point. Developing plasmodia were first observed 17 d post-exposure (Day 17). Mature spores were collected from plasmodia on Day 56 and were added to plastic dishes containing parasite-free Tubifex tubifex oligochaetes. Second-generation actinospores were released from these worms 8 mo post-exposure, and were morphologically identical to first-generation spores. Myxospores obtained from the bream fry were morphologically identical to those identified in wild bream as M. rotundus. Small subunit ribosomal RNA gene sequences obtained from first- and second-generation actinospores and the bream fry myxospores were 100% similar to M. rotundus spores from the original wild fish.