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.

Sphaeromyxa artedielli sp. n. (Myxozoa: Sphaeromyxidae), a parasite of sculpins (Cottidae) in northern Norway.

Sphaeromyxa artedielli sp. n. is described from the gall bladder of the Atlantic hookear sculpin Artediellus atlanticus Jordan et Evermann (Cottidae; type host) from northern Norway. The parasite was also found to infect Triglops murrayi Günther (Cottidae). Spores are produced in disporic pansporoblasts in large flat plasmodia. Spores are straight and fusiform with truncated ends, and measure 16.5-18.7 microm x 4.9-6.2 microm. Valves are thick, striated and suture line is straight. Two equal ovoid polar capsules measure 4.2-6.8 microm x 2.9-4.4 microm and contain irregularly folded polar filaments. Distinctive features include spore shape and size, spore length/width relationship, striated valves, equal polar capsules and a short intercapsular distance. Sphaeromyxa bonaerensis Timi et Sardella, 1998, Sphaeromyxa cannolii Sears, Anderson et Greiner, 2011, and Sphaeromyxa sevastopoli Naidenova, 1970 produce straight spores with truncated ends that are of similar length as those of the new species. Sphaeromyxa cannolii differs in showing smooth spores with unequal polar capsules. The new species differs from S. bonaerensis and S. sevastopoli in significantly wider spores and polar capsules. Sphaeromyxa balbianii Thélohan, 1892, a species originally described with significantly smaller spores than S. artedielli sp. n., has previously been recorded from T. murrayi. We show that S. artedielli sp. n. differs from S. balbianii from the type host Gaidropsarus vulgaris (Cloquet) by its SSU rDNA sequence, and suggest that Atlantic records of Sphaeromyxa spp. from T. murrayi represent S. artedielli sp. n. The closest relative to S. artedielli sp. n. according to the SSU rDNA sequences, S. longa Dunkerly, 1921, differs clearly by spore size and shape. In the SSU rDNA-based phylogenetic analyses, S. artedielli sp. n. groups with other Sphaeromyxa spp. with straight spores and truncated ends in a clade that represents a sister-group to Sphaeromyxa spp. with arcuate spores and rounded ends. Our results indicate that an SSU rDNA pseudogene is present in S. balbianii.

Characterization of gut-associated cathepsin D hemoglobinase from tick Ixodes ricinus (IrCD1).

To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.

Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade.

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.

Molecular evidence for the existence of cryptic species assemblages of several myxosporeans (Myxozoa).

Myxosporeans Chloromyxum cristatum, Chloromyxum fluviatile and Zschokkella nova (Myxozoa) are common gall bladder parasites of the cyprinid fishes frequently persisting as co-infections. Despite the fact that they are believed to be innocuous endocommensals, C. cristatum clearly displays the potential of a serious pathogen since it may pervade fish liver parenchyma and cause its necrosis. Employing the comparison of genetic distances among the myxosporean rDNA sequences and performing phylogenetic analyses, we demonstrate that cryptic species assemblages exist in C. fluviatile and Z. nova. Sequence comparison revealed that Chloromyxum legeri, previously assigned as a junior synonym of C. fluviatile, is a valid species. The same method is used to display the distinction of Z. nova isolates from China and the Czech Republic. We show that C. cristatum is not an assemblage of more species, and our results support the synonymy of Chloromyxum cyprini with C. cristatum. We have developed a multiplex PCR as an effective tool for the detection and discrimination of Z. nova, C. cristatum, and C. fluviatile. It is especially advantageous for the distinction of the non-mature plasmodia of both Chloromyxum species. This method also helped to assess the exact prevalence of these parasites in examined samples and enabled to select single-infected host samples for the intended population studies.

Another chloromyxid lineage: molecular phylogeny and redescription of Chloromyxum careni from the Asian horned frog Megophrys nasuta.

Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 µm were localized in Bowman's space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0-8.5 × 5.0-6.5 µm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0-4.0 × 2.5-3.0 µm and a single sporoplasm. Each valve possesses 14-24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6-10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU-rich inserts. In all SSU and LSU rDNA-based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.

History of myxozoan character evolution on the basis of rDNA and EF-2 data.

BACKGROUND: Phylogenetic relationships among myxosporeans based on ribosomal DNA data disagree with traditional taxonomic classification: a number of myxosporeans with very similar spore morphology are assigned to the same genera even though they are phylogenetically distantly related. The credibility of rDNA as a suitable marker for Myxozoa is uncertain and needs to be proved. Furthermore, we need to know the history of myxospore evolution to understand the great diversity of modern species. RESULTS: Phylogenetic analysis of elongation factor 2 supports the ribosomal DNA-based reconstruction of myxozoan evolution. We propose that SSU rDNA is a reliable marker for inferring myxozoan relationships, even though SSU rDNA analysis markedly disagrees with the current taxonomy. The analyses of character evolution of 15 morphological and 5 bionomical characters show the evolution of individual characters and uncover the main evolutionary changes in the myxosporean spore morphology and bionomy. Most bionomical and several morphological characters were found to be congruent with the phylogeny. The summary of character analyses leads to the simulation of myxozoan ancestral morphotypes and their evolution to the current species. As such, the ancestor of all myxozoans appears to have infected the renal tubules of freshwater fish, was sphaerosporid in shape, and had a spore with polar capsules that discharged slightly sideways. After the separation of Malacosporea, the spore of the common myxosporean ancestor then changed to the typical sphaerosporid morphotype. This species inhabited the marine environment as a parasite of the gall bladder of marine fish and ultimately separated into the three main myxosporean lineages evident today. Two of these lineages re-entered the freshwater environment, one as a myxosporean with Chloromyxum and another with a primitive sphaerosporid morphotype. The common ancestor of all marine myxosporeans had a ceratomyxid shape of spore. CONCLUSIONS: We support rDNA based myxozoan phylogeny by the analysis of a protein coding gene and demonstrate the reliability of rDNA as a marker explaining myxozoan relationships. Our tracing the history of myxozoan character evolution discloses ancestral morphotypes and shows their development over the course of evolution. We point out several myxozoan characters that are to a certain extent congruent with the phylogeny and determined that the discrepancy between phylogeny and current taxonomy based on spore morphology is due to an extreme myxospore plasticity occurring during myxozoan evolution.

Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics.

Views on myxosporean phylogeny and systematics have recently undergone substantial changes resulting from analyses of SSU rDNA. Here, we further investigate the evolutionary trends within myxosporean lineages by using 35 new sequences of the LSU rDNA. We show a good agreement between the two rRNA genes and confirm the main phylogenetic split between the freshwater and marine lineages. The informative superiority of the LSU data is shown by an increase of the resolution, nodal supports and tree indexes in the LSU rDNA and combined analyses. We determine the most suitable part of LSU for the myxosporean phylogeny by comparing informative content in various regions of the LSU sequences. Based on this comparison, we propose the D5-3'-end part of the LSU rRNA gene as the most informative region which provides in concatenation with the complete SSU a well resolved and robust tree. To allow for simple amplification of the marker, we design specific primer set for this part of LSU rDNA.