First Record of Psorospermium sp. (Class: Mesomycetozoea) in Northern Clearwater crayfish Faxonius propinquus Girard 1852 (Decapoda: Cambaridae) from Michigan, U.S.A.

Psorospermium cf. haeckeli Hilgendorf 1883 is a unicellular, eukaryotic protozoan within the class Mesomycetozoea, phylogenetically situated near the animal-fungal divergence(Cavalier-Smith 1998; Ragan et al. 1998). Although only one species has been identified, there have been four morphotypes described in 17 species of crayfish from the Holarctic, Neotropical, and Australasian regions (Herbert 1987; Henttonen et al. 1992, 1994; Rug Vogt 1994). However, molecular analyses of the internal transcribed spacer DNA suggest morphotypes may represent distinct species (Bangyeekhun et al. 2001).

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Two New Species of Marine Saprotrophic Sphaeroformids in the Mesomycetozoea Isolated From the Sub-Arctic Bering Sea.

The genus Sphaeroforma previously encompassed organisms isolated exclusively from animal symbionts in marine systems. The first saprotrophic sphaeroformids (Mesomycetozoea) isolated from non-animal hosts are described here. Sphaeroforma sirkka and S. napiecek are also the first species in the genus possessing endogenous DNA-containing motile propagules and central vacuoles, traits that have previously guided morphological differentiation of sphaeroformids from the genus Creolimax. Phylogenetic analysis of DNA sequences from the 18S rRNA and the ITS1-5.8S--ITS2 loci firmly place S. sirkka and S. napiecek within Sphaeroforma, extending the number of known species to six within this genus. The discovery of these species increases the geographical range, cellular variation and life history complexity of the sphaeroformids.

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

Diversity and distribution of unicellular opisthokonts along the European coast analysed using high-throughput sequencing.

The opisthokonts are one of the major super groups of eukaryotes. It comprises two major clades: (i) the Metazoa and their unicellular relatives and (ii) the Fungi and their unicellular relatives. There is, however, little knowledge of the role of opisthokont microbes in many natural environments, especially among non-metazoan and non-fungal opisthokonts. Here, we begin to address this gap by analysing high-throughput 18S rDNA and 18S rRNA sequencing data from different European coastal sites, sampled at different size fractions and depths. In particular, we analyse the diversity and abundance of choanoflagellates, filastereans, ichthyosporeans, nucleariids, corallochytreans and their related lineages. Our results show the great diversity of choanoflagellates in coastal waters as well as a relevant representation of the ichthyosporeans and the uncultured marine opisthokonts (MAOP). Furthermore, we describe a new lineage of marine fonticulids (MAFO) that appears to be abundant in sediments. Taken together, our work points to a greater potential ecological role for unicellular opisthokonts than previously appreciated in marine environments, both in water column and sediments, and also provides evidence of novel opisthokont phylogenetic lineages. This study highlights the importance of high-throughput sequencing approaches to unravel the diversity and distribution of both known and novel eukaryotic lineages.

Impact of Ichthyophonus infection on spawning success of Yukon River Chinook salmon Oncorhynchus tshawytscha.

We examined the impacts of Ichthyophonus infection on spawning success of Yukon River Chinook salmon Oncorhynchus tshawytscha at spawning grounds of the Chena and Salcha Rivers, Alaska, USA. During the period 2005 to 2006, 1281 salmon carcasses (628 male, 652 female) were collected throughout the spawning season and from the entire spawning reaches of the Chena and Salcha Rivers. For each fish, infection status was determined by culture method and visual inspection of lesions of heart tissue as uninfected (culture negative), infected without lesions (culture positive with no visible lesions), and infected with lesions (culture positive with visible lesions), and spawning status was determined by visually inspecting the percentage of gametes remaining as full-spawned (<10%), partial-spawned (10-50%), and unspawned (>50%). Among the 3 groups, the proportion of full-spawned (i.e. spawning success) females was lower for those infected without lesions (69%) than those uninfected (87%) and infected with lesions (86%), but this did not apply to males (uninfected 42%, infected without lesions 38%, infected with lesions 41%). At the population level, the combined (infected and uninfected) proportion of female spawning success was 86%, compared to 87% when all females were assumed uninfected. These data suggest that while Ichthyophonus infection slightly reduces spawning success of infected females, its impact on the spawning population as a whole appears minimal.

Capturing the population structure of microparasites: using ITS-sequence data and a pooled DNA approach.

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is a common marker not only for the molecular identification of different taxa and strains, but also for the analysis of population structure of wild microparasite communities. Importantly, the multicopy nature of this region allows the amplification of low-quantity samples of the target DNA, a common problem in studies on unicellular, unculturable microparasites. We analysed ITS sequences from the protozoan parasite Caullerya mesnili (class Ichthyosporea) infecting waterflea (Daphnia) hosts, across several host population samples. We showed that analysing representative ITS-types [as identified by statistical parsimony network analysis (SPN)] is a suitable method to address relevant polymorphism. The spatial patterns were consistent regardless of whether parasite DNA was extracted from individual hosts or pooled host samples. Remarkably, the efficiency in detecting different sequence types was even higher after sample pooling. As shown by simulations, an easily manageable number of sequences from pooled DNA samples are sufficient to resolve the spatial population structure in this system. In summary, the ITS region analysed from pooled DNA samples can provide valuable insights into the spatial and temporal dynamics of microparasites. Moreover, the application of SPN analysis is a good alternative to the well-established neighbour-joining method (NJ) for the identification of representative ITS-types. SPN can even outperform NJ by joining most of the singleton sequences to representative sequence clusters.

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

Proposed changes to the nomenclature of Ichthyophonus sp. life stages and structures.

Much of the terminology describing Ichthyophonus sp. life stages and structures can be traced to the mistaken classification of this organism as a fungus. This misidentification led early investigators to use mycological terms for the structures they observed; while some terminology is not so easily explained, it appears to have been co-opted from the fields of botany and bacteriology. The purpose of this exercise is to attempt to standardize the terminology associated with Ichthyophonus and to bring it into agreement with terminology currently used to define similar life stages of other protists. The proposed changes are (1) spore/macrospore/mother spore to "schizont," (2) microspore/endospore to "merozoite," and (3) pseudohyphae to "hyphae" or "germ tube."

Comparative morphology and genealogical delimitation of cryptic species of sympatric isolates of Sphaeroforma (Ichthyosporea, Opisthokonta).

Of the ancient clades of unicellular relatives of the multicellular animals, ichthyosporea are among the easiest to collect, cultivate, and analyze at the population level. Once identified, species can be correlated with their animal hosts and geographical ranges. However, the spherical stages common to many ichthyosporea provide little basis for morphological species identification. This study of the genus Sphaeroforma is the first to apply patterns of genetic discontinuity to delimit species among any of the unicellular 'holozoa.' Sequences of three loci from 148 sympatric isolates, along with type cultures, provided concordant support for new species "Sphaeroforma nootkatensis" and "Sphaeroforma gastrica," and for formally describing 'Pseudoperkinsus tapetis,' as "Sphaeroforma tapetis". We document light and electron microscopic characters that distinguish the genus but not its species. "S. tapetis" sometimes had brief amoeboid or plasmodial motile stages and endospore release through pores. Unlike closely related Creolimax, Sphaeroforma lacked a central vacuole but had multiple peripheral nucleoli. Like distantly related eccrinales, Sphaeroforma cell walls had pores and a calyx. Analyses of allele frequencies in "S. tapetis" indicated geographical differentiation but no host specificity. Accurate molecular identification of species will increase the feasibility and reliability of further studies of Sphaeroforma in its natural habit.

Inability to demonstrate fish-to-fish transmission of Ichthyophonus from laboratory infected Pacific herring Clupea pallasii to naïve conspecifics.

The parasite Ichthyophonus is enzootic in many marine fish populations of the northern Atlantic and Pacific Oceans. Forage fishes are a likely source of infection for higher trophic level predators; however, the processes that maintain Ichthyophonus in forage fish populations (primarily clupeids) are not well understood. Lack of an identified intermediate host has led to the convenient hypothesis that the parasite can be maintained within populations of schooling fishes by waterborne fish-to-fish transmission. To test this hypothesis we established Ichthyophonus infections in Age-1 and young-of-the-year (YOY) Pacific herring Clupea pallasii (Valenciennes) via intraperitoneal (IP) injection and cohabitated these donors with naïve conspecifics (sentinels) in the laboratory. IP injections established infection in 75 to 84% of donor herring, and this exposure led to clinical disease and mortality in the YOY cohort. However, after cohabitation for 113 d no infections were detected in naïve sentinels. These data do not preclude the possibility of fish-to-fish transmission, but they do suggest that other transmission processes are necessary to maintain Ichthyophonus in wild Pacific herring populations.

Fossilized nuclei and germination structures identify Ediacaran "animal embryos" as encysting protists.

Globular fossils showing palintomic cell cleavage in the Ediacaran Doushantuo Formation, China, are widely regarded as embryos of early metazoans, although metazoan synapomorphies, tissue differentiation, and associated juveniles or adults are lacking. We demonstrate using synchrotron-based x-ray tomographic microscopy that the fossils have features incompatible with multicellular metazoan embryos. The developmental pattern is comparable with nonmetazoan holozoans, including germination stages that preclude postcleavage embryology characteristic of metazoans. We conclude that these fossils are neither animals nor embryos. They belong outside crown-group Metazoa, within total-group Holozoa (the sister clade to Fungi that includes Metazoa, Choanoflagellata, and Mesomycetozoea) or perhaps on even more distant branches in the eukaryote tree. They represent an evolutionary grade in which palintomic cleavage served the function of producing propagules for dispersion.

Facing unknowns: living cultures (Pirum gemmata gen. nov., sp. nov., and Abeoforma whisleri, gen. nov., sp. nov.) from invertebrate digestive tracts represent an undescribed clade within the unicellular Opisthokont lineage ichthyosporea (Mesomycetozoea).

During a culture-based survey of opisthokonts living in marine invertebrate digestive tracts, we isolated two new eukaryotes that differed from described taxa by more than 10% in their small subunit ribosomal DNA sequences. Phylogenetic analysis showed that the two isolates represented a divergent clade of ichthyosporeans known previously only from environmental clone sequences. We used light and electron microscopy to describe the isolates as new genera and species Pirum gemmata and Abeoforma whisleri. A. whislerihad a complex life cycle that remains incompletely known but involved walled spherical cells, plasmodia and amoebae. Asexual reproduction occurred via dispersal amoebae, endospores, binary fission and budding. In contrast P. gemmatahad a less complex life cycle with no amoeboid or plasmodial stages. Both species had membrane-bound tubular extensions of the cytoplasm embedded in the inner layers of their cell walls. By comparing P. gemmata and A. whislerito other ichthyosporea we speculate on the characters that may have been present in the ancestral ichthyosporean. P. gemmata and A. whisleri illustrate the unique and diverse forms that can be found by capturing taxa belonging to divergent and uncultured lineages.

Dermocystid infection and associated skin lesions in free-living palmate newts (Lissotriton helveticus) from Southern France.

Since the early 1900s, mesomycetozoan parasites have been reported in both European anuran and caudate species. These reports have primarily been descriptive, which has made assessing the impact of these parasites on host populations difficult. Anecdotal reports of dermocystidium-like parasites are becoming widespread across Europe, possibly indicating that these mesomycetozoan parasites are increasing in distribution and/or abundance. This highlights the need for further investigations into the occurrence, pathogenesis and effects on host health of these parasitic infections for free-living amphibian populations, particularly those which are already stressed or threatened by other factors. Here we report the results of pathological, microbiological and molecular investigations used to characterize unidentified skin lesions in palmate newts (Lissotriton helveticus) from Larzac, France. We confirm that the lesions are the result of infection with a novel dermocystidium-like parasite, which is related to Amphybiocystidium ranae. We also show that the same parasite is distributed across several newt breeding sites. The lesions that result from infection with this parasite range from single or few vesicular or nodular cutaneous lesions to multiple coalescing skin ulcers with extensive hemorrhages. The latter have not been previously described in amphibians due to mesomycetozoan parasitic infection. Dermocystid DNA was detected only in newts that showed lesions, providing comparative evidence of the parasite's pathogenicity. We discuss the potential significance of the presence of this pathogen in the context of the population health of palmate newts.

A Daphnia parasite (Caullerya mesnili) constitutes a new member of the Ichthyosporea, a group of protists near the animal-fungi divergence.

Caullerya mesnili is a protozoan endoparasite in the gut epithelium of Daphnia, which causes regular epidemics in lakes throughout Europe. Its classification has remained unchanged for over a century, leaving it placed with the Haplosporidia, despite speculation that this position is incorrect. The difficulty in classifying C. mesnili stems from its few known morphological and ecological characteristics, as well as a lack of genetic markers. Here we sequenced the nuclear small subunit (SSU) and internal transcribed spacer rDNA regions of C. mesnili samples from 10 locations. Based on sequence similarities, we suggest the re-classification of C. mesnili to the Ichthyosporea, a class of protists near the animal-fungi divergence. We report average intragenomic variation of 0.75% and 2.27% in the SSU and internal transcribed spacer regions, respectively. From electron micrographs and light microscopy of histological sections we determined that C. mesnili spores grow within the intestinal epithelium where they establish themselves intercellularly. In addition, we confirmed previous accounts regarding the high virulence of this parasite. Caullerya mesnili reduces host lifespan, the number of clutches, and the total number of offspring. This high selection pressure placed on hosts supports the importance of C. mesnili as a model parasite for the study of host-parasite biology in permanent lakes.

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout.

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of approximately 740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

Microscopic eukaryotes living in a dying lake (Lake Koronia, Greece).

The morphological and phylogenetic diversity of the microscopic eukaryotes of the Lake Koronia water column was investigated during a mass kill of birds and fish in August-September 2004. The dominant morphospecies corresponded to the known toxin-producing species Prymnesium parvum, followed by Amoebidium sp., a taxon belonging to the group of parasitic Mesomycetozoea, and the common chlorophyte Pediastrum boryanum. Prymnesium exhibited heteromorphic life-cycle stages (flagellate and nonmotile coccoid cells). Phylogenetic analysis with 18S rRNA gene suggested that these heteromorphic stages belonged to the Platychrysis-Prymnesium monophyletic group. The most abundant phylotype was almost identical to P. boryanum. The fungal phylotypes were related to the Chytridiomycota, and the ciliate-like ones were closely related to Enchelys polynucleata and Pattersoniella vitiphila. Two phylotypes representing novel members belonging to the Jakobida and the Apicomplexa were also found. The microscopic eukaryotes of Lake Koronia include several organisms that are related to parasitic life modes.

Multiple isolations of a culturable, motile Ichthyosporean (Mesomycetozoa, Opisthokonta), Creolimax fragrantissima n. gen., n. sp., from marine invertebrate digestive tracts.

A fragrant, spherical, osmotrophic eukaryote was isolated 27 times from the digestive tracts of marine invertebrates collected from the Northeast Pacific. The isolates were cultured from 7 animal collections over a 2-year period, most from the peanut worm, Phascolosoma agassizii. A small subunit ribosomal DNA phylogeny placed the spherical organism within the ichthyosporea, closest to Sphaeroforma arctica and Pseudoperkinsus tapetis. Supporting the close relationship of isolates, the sequences of ribosomal gene internal transcribed spacers determined for 26 isolates were identical, as were the elongation factor 1-alpha-like gene fragments from 7 isolates. Dispersal via amoeboid cells distinguished this species from its closest relatives and led to the erection of a new genus and species, "Creolimax fragrantissima." Vegetative cells reproduced asexually in vitro after they reached 30-60 microm in diameter by producing amoebae or endospores, which escaped through openings in the parent cell wall. Ultrathin sections of vegetative cells prepared by high-pressure-freeze substitution provided some of the first images of ichthyosporean spindle pole bodies and document, for the first time, tubular extensions of the plasma membrane into an electron-translucent inner layer of the cell wall. Ichthyosporeans are parasites and commensals of animals and culturable species are few. Because "C. fragrantissima" can be isolated regularly and repeatedly from nature and then grown easily through cycles of asexual reproduction, it has the potential to serve as a model organism for further research into marine ichthyosporeans.