BACKGROUND: Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such as Sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of a S. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify S. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. RESULTS: An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. CONCLUSIONS: In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study of S. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

• Keywords
• Aquaculture, Drug targets, In silico screening, Myxozoa, Parasite, Proteases,
• MeSH
• Antiparasitic Agents pharmacology   therapeutic use   MeSH
• Virulence Factors MeSH
• Phylogeny MeSH
• Carps microbiology   MeSH
• Myxozoa genetics   growth & development   MeSH
• Fish Diseases parasitology   therapy   MeSH
• Drug Discovery MeSH
• Parasitic Diseases, Animal parasitology   therapy   MeSH
• Peptide Hydrolases genetics   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiparasitic Agents MeSH
• Virulence Factors MeSH
• Peptide Hydrolases MeSH

During a survey on the myxosporean fauna of Rajiformes from the Atlantic coast of Argentina, in waters off Buenos Aires Province (34°-42°S; 53°-62°W), the gall bladders of 217 specimens belonging to seven species of skates, representatives of two families, were examined. As a result, three species of Chloromyxum Mingazzini, 1890, namely C. atlantoraji n. sp., C. zearaji n. sp. and C. riorajum Azevedo, Casal, Garcia, Matos, Teles-Grilo and Matos, 2009 were found infecting three endemic host species, the spotback skate Atlantoraja castelnaui (Arhynchobatidae), the yellownose skate Zearaja chilensis (Rajidae) and the Rio skate Rioraja agassizii (Arhynchobatidae), respectively. These species were described based on myxospore morphology and morphometry characterization, as well as by providing their small subunit ribosomal DNA (SSU rDNA) sequences. The SSU rDNA-based phylogenetic analyses showed that these three species constituted a well-established monophyletic subclade within the marine Chloromyxum clade, while branches subtending the other Chloromyxum species were poorly resolved or unresolved, independently of the host taxonomic identities (Carchariniformes, Myliobatiformes, Orectolobiformes, Pristiophoriformes, Rajiformes, Squaliformes and Torpediniformes) and/or host geographic distribution (Atlantic coast of Portugal, Atlantic coast of the USA, Australian waters or Mediterranean Sea). The possible causes of these discrepancies are discussed, providing new insights into the phylogeny of the marine Chloromyxum clade.

Lors d’une étude de la faune des Myxozoaires des Rajiformes de la côte atlantique argentine, dans les eaux situées au large de la province de Buenos Aires (34°–42°S; 53°–62°O), les vésicules biliaires de 217 spécimens appartenant à sept espèces, représentants deux familles, ont été examinés. En conséquence, trois espèces de Chloromyxum Mingazzini, 1890, à savoir C. atlantoraji n. sp., C. zearaji n. sp. et C. riorajum Azevedo, Casal, Garcia, Matos, Teles-Grilo et Matos, 2009 ont été trouvées, infectant trois espèces hôtes endémiques, Atlantoraja castelnaui (Arhynchobatidae), Zearaja chilensis (Rajidae) et Rioraja agassizii (Arhynchobatidae), respectivement. Ces espèces sont décrites sur la base de la morphologie et de la morphométrie des myxospores, ainsi qu’en fournissant leurs petites séquences d’ADN ribosomal (SSU ADNr). Les analyses phylogénétiques basées sur l’ADNr SSU ont montré que ces trois espèces constituaient un sous-clade monophylétique bien établi dans le clade des Chloromyxum marins, tandis que les branches sous-jacentes aux autres espèces de Chloromyxum étaient mal ou non résolues, indépendamment des identités taxonomiques hôtes (Carchariniformes, Myliobatiformes, Orectolobiformes, Pristiophoriformes, Rajiformes, Squaliformes et Torpediniformes) et/ou de la répartition géographique de l’hôte (côte atlantique du Portugal, côte atlantique des États-Unis, eaux australiennes ou mer Méditerranée). Les causes possibles de ces divergences sont discutées, fournissant de nouvelles informations sur la phylogénie du clade des Chloromyxum marins.

• MeSH
• Species Specificity MeSH
• Phylogeny * MeSH
• Microscopy, Electron, Scanning MeSH
• Myxozoa classification   genetics   isolation & purification   MeSH
• Fish Diseases epidemiology   parasitology   MeSH
• Oceans and Seas MeSH
• Parasitic Diseases, Animal epidemiology   parasitology   MeSH
• Skates, Fish parasitology   MeSH
• DNA, Ribosomal genetics   MeSH
• Base Sequence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Argentina epidemiology   MeSH
• Oceans and Seas MeSH
• Names of Substances
• DNA, Ribosomal MeSH

BACKGROUND: Myxozoa are extremely diverse microscopic parasites belonging to the Cnidaria. Their life-cycles alternate between vertebrate and invertebrate hosts, predominantly in aquatic habitats. Members of the phylogenetically well-defined Sphaerospora (sensu stricto) clade predominantly infect the urinary system of marine and freshwater fishes and amphibians. Sphaerosporids are extraordinary due to their extremely long and unique insertions in the variable regions of their 18S and 28S rDNA genes and due to the formation of motile proliferative stages in the hosts' blood. To date, DNA sequences of only 19 species have been obtained and information on the patterns responsible for their phylogenetic clustering is limited. METHODS: We screened 549 fish kidney samples from fish of various geographical locations, mainly in central Europe, to investigate sphaerosporid biodiversity microscopically and by 18S rDNA sequences. We performed multiple phylogenetic analyses to explore phylogenetic relationships and evolutionary trends within the Sphaerospora (s.s.) clade, by matching host and habitat features to the resultant 18S rDNA trees. The apparent co-clustering of species from related fish hosts inspired us to further investigate host-parasite co-diversification, using tree-based (CoRE-PA) and distance-based (ParaFit) methods. RESULTS: Our study considerably increased the number of 18S rDNA sequence data for Sphaerospora (s.s.) by sequencing 17 new taxa. Eight new species are described and one species (Sphaerospora diminuta Li & Desser, 1985) is redescribed, accompanied by sufficient morphological data. Phylogenetic analyses showed that sphaerosporids cluster according to their vertebrate host order and habitat, but not according to geography. Cophylogenetic analyses revealed a significant congruence between the phylogenetic trees of sphaerosporids and of their vertebrate hosts and identified Cypriniformes as a host group of multiple parasite lineages and with high parasite diversity. CONCLUSIONS: This study significantly contributed to our knowledge of the biodiversity and evolutionary history of the members of the Sphaerospora (s.s.) clade. The presence of two separate phylogenetic lineages likely indicates independent historical host entries, and the remarkable overlap of the larger clade with vertebrate phylogeny suggests important coevolutionary adaptations. Hyperdiversification of sphaerosporids in cypriniform hosts, which have undergone considerable radiations themselves, points to host-driven diversification.

• Keywords
• Host-parasite codiversification, Myxozoa, Phylogeny, Sphaerospora sensu stricto, Taxonomy, Teleost,
• MeSH
• Biodiversity * MeSH
• Biological Evolution MeSH
• Cnidaria MeSH
• Phylogeny * MeSH
• Myxozoa classification   genetics   isolation & purification   physiology   MeSH
• Fish Diseases parasitology   MeSH
• Parasitic Diseases, Animal genetics   parasitology   MeSH
• DNA, Ribosomal genetics   MeSH
• Fishes classification   genetics   parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal MeSH

BACKGROUND: Myxozoans are metazoan parasites whose traditional spore morphology-based taxonomy conflicts DNA based phylogenies. Freshwater species of the genus Hoferellus are parasites of the excretory system, with several members infecting food and ornamental fish species, as well as amphibians. This study aims to increase our understanding of their molecular diversity and development, aspects about which little is known, and to generate a molecular diagnostic tool to discriminate between different pathogenic and non-pathogenic Hoferellus spp. METHODS: SSU and ITS rDNA phylogeny, along with morphological descriptions using light and electron microscopy were used to identify and characterize Hoferellus species collected from the urinary system of fishes and frogs. A PCR-based diagnostic assay was designed to differentiate between cryptic Hoferellus spp in cyprinid fishes commonly cultured in Central Europe. RESULTS: Our phylogenetic results separate the species of Hoferellus into two phylogenetic sublineages which are indistinguishable on the basis of generic morphological traits: 1) The Hoferellus sensu stricto sublineage, which is composed of the type species Hoferellus cyprini, Hoferellus carassii and a cryptic species, Hoferellus sp. detected only molecularly in common carp. 2) The Hoferellus sensu lato sublineage into which the new species we described in this study, Hoferellus gnathonemi sp. n. from the kidney of the elephantnose fish and Hoferellus anurae from reed frogs, are placed together with Hoferellus gilsoni previously sequenced from European eel. Apart from phylogenetic analyses, we also provide novel ultrastructural data on the phagocytotic nature of some Hoferellus plasmodia and on the elusive intracellular stages ascribed to the presporogonic development of this genus. CONCLUSIONS: We provide molecular evidence of the polyphyly of the genus Hoferellus and provide novel morphological details of its members. Based on the presented data, we revise and propose emendation of the genus Hoferellus.

• MeSH
• Cyprinidae MeSH
• Phylogeny MeSH
• Kidney pathology   MeSH
• Molecular Sequence Data MeSH
• Myxozoa classification   genetics   isolation & purification   ultrastructure   MeSH
• Fish Diseases parasitology   MeSH
• Parasitic Diseases, Animal parasitology   MeSH
• DNA, Ribosomal chemistry   genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA veterinary   MeSH
• Anura parasitology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH

BACKGROUND: Swim bladder inflammation (SBI) is an important disease of common carp fingerlings in Central Europe. In the 1980s, its etiology was ascribed to multicellular proliferative stages of the myxozoan parasite Sphaerospora dykovae (formerly S. renicola). S. dykovae was reported to proliferate in the blood and in the swim bladder prior to the invasion of the kidney, where sporogony takes place. Due to the presence of emerging numbers of proliferative myxozoan blood stages at different carp culture sites in recent years we analysed cases of SBI, for the first time, using molecular diagnostics, to identify the myxozoan parasites present in diseased swim bladders. METHODS: We amplified myxozoan SSU rDNA in a non-specific approach and compared the species composition in swim bladders at culture sites where carp demonstrated 1. No signs of SBI, 2. Minor pathological changes, and 3. Heavy SBI. Based on DNA sequences, we determined the localisation and distribution of the most frequent species by in situ hybridisation, thereby determining which myxozoans are involved in SBI. RESULTS: Large multicellular myxozoan swim bladder stages characterised heavy SBI cases and were identified as S. dykovae, however, blood stages were predominantly represented by Sphaerospora molnari, whose numbers were greatly increased in carp with mild and heavy SBI, compared with SBI-free fish. S. molnari was found to invade different organs and cause inflammatory changes also in the absence of S. dykovae. One site with mild SBI cases was characterised by Buddenbrockia sp. infection in different organs and a general granulomatous response. CONCLUSIONS: We provide evidence that the etiology of SBI can vary in relation to culture site and disease severity and that emerging numbers of S. molnari in the blood represent an important co-factor or precondition for SBI.

• MeSH
• Time Factors MeSH
• Carps MeSH
• Cloning, Molecular MeSH
• Myxozoa classification   genetics   MeSH
• Fish Diseases parasitology   pathology   MeSH
• Parasitic Diseases, Animal parasitology   pathology   MeSH
• Prevalence MeSH
• Seasons MeSH
• Air Sacs parasitology   pathology   MeSH
• Inflammation parasitology   pathology   veterinary   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In the eastern Gulf of Mexico, off the coast of Florida, grey snapper, Lutjanus griseus was found to be infected with the myxozoan parasite Sphaerospora motemarini n. sp., with high prevalence (83%) and intensity of infection occuring in age-0 fish, and with parasite levels decreasing with age (age-1 snapper 40%; age-2 snapper 0%). The morphological, molecular and phylogenetic characterisation of the myxozoan showed that it is a member of the typically marine, polysporoplasmid Sphaerospora spp. which form a subclade within the Sphaerospora sensu stricto clade of myxozoans, which is characterised by large expansion segments in their SSU rDNA sequences. Presporogonic stages of S. motemarini n. sp. were detected in the blood, using PCR. Pseudoplasmodia and spores were found to develop in the renal corpuscles of the host, causing their massive expansion. Macroscopic and histopathological changes were observed in age-0 fish and show that S. motemarini n. sp. causes severe glomerulonephritis in L. griseus leading to a compromised host condition, which makes it more susceptible to stress (catch-and-release, predators, water quality) and can result in mortalities. These results are discussed in relation to the exploitation of grey snapper populations by commercial and recreational fisheries and with the observed increased mortalities with temperature along the coast of Florida. In the future, we would like to determine prevalence and intensity of infection with S. motemarini n. sp. in juvenile L. griseus in different areas of the Gulf of Mexico in order to be able to estimate the temperature dependence of S. motemarini n. sp. proliferation and to be able to predict its distribution and severity during climatic changes in the Gulf.

• Keywords
• Fisheries, Glomerulonephritis, Juvenile pathology, Mangrove snapper, Mortality, Myxozoa,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Bayes Theorem MeSH
• Peptide Elongation Factor 2 genetics   MeSH
• Phylogeny * MeSH
• Evolution, Molecular * MeSH
• Myxozoa anatomy & histology   classification   genetics   MeSH
• Likelihood Functions MeSH
• DNA, Ribosomal genetics   MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptide Elongation Factor 2 MeSH
• DNA, Ribosomal MeSH