Unraveling the mystery of a myxozoan parasite of the trout: redescription of Chloromyxum schurovi.

Myxozoans are microscopical parasites widely distributed in fish, with over 2,600 described species, but their actual diversity is still underestimated. Among salmonids, more than 70 myxozoan species have been identified. This study focuses on species of Chloromyxum Mingazzini, 1890 that infect salmonid kidneys, particularly C. majori Yasutake et Wood, 1957 and C. schurovi Shulman et Ieshko, 2003. Despite their similar spore morphology, they exhibit distinct host preferences, tissue affinities and geographical distributions. Chloromyxum schurovi predominantly infects the renal tubules of Salmo salar Linnaues and S. trutta Linnaeus in Europe, while C. majori targets the glomeruli of Oncorhynchus mykiss (Walbaum) and O. tshawytscha (Walbaum) in North America. The sequence data for C. majori and C. schurovi have been either missing or questionable. In our study, we examined the kidneys of two salmonid species for chloromyxid infections, using both morphological and molecular data to characterise Chloromyxum species in salmonids. The sequence of C. schurovi obtained in our study did not match the previously published parasite data. Instead, it clustered as an independent lineage sister to the Paramyxidium Freeman et Kristmundsson, 2018 clade gathering the species from various fish organs, including the urinary tract. Our findings clarified the taxonomic origin of the previous C. schurovi sequence as Myxidium giardi Cépède, 1906, highlighting the risks associated with the presence of myxozoan blood stages in the bloodstream of their fish host and the challenges of non-specific PCR amplification. We redescribe C. schurovi, thus contributing to a better understanding of the diversity and phylogeny of kidney-infecting species of Chloromyxum.

Evidence of striking morphological similarity and tissue tropism of phylogenetically distant myxozoan genera: Myxidium and Paramyxidium in the kidney of the European eel.

European eel, Anguilla anguilla (Linnaeus) (Elopomorpha: Anguilliformes), is a critically endangered fish of ecological and economic importance, hosting numerous parasites, including myxozoans (Cnidaria). Since its initial discovery in the kidney of European eel, Myxidium giardi Cépède, 1906 has been reported with numerous spore sizes and shapes from various tissues of multiple anguillid species. Morphological variability, wide host and tissue spectrum, and lack of sequence data raised doubts about the conspecificity of reported isolates. Subsequent studies provided 18S rDNA sequences of several isolates from anguillids and other elopiform fish, and demonstrated a split of parasite data into two distinct phylogenetic lineages, one comprising the M. giardi sequence, and the other all species infecting elopiform fishes classified under the recently established genus Paramyxidium Freeman et Kristmundsson, 2018. Myxidium giardi was, however, transferred to this genus as Paramyxidium giardi n. comb. and designated as the type species of the genus. In line with this change, the sequence originally identified as M. giardi was considered to have been incorrectly associated with this species. To shed light on the status of M. giardi originally described by Cépède (1906), we conducted microscopic and molecular examinations of various organs of 24 individuals of European eel, originating from diverse Czech habitats. Through morphometric and molecular analyses, we demonstrated that spore and polar capsule morphology, morphometry and tissue tropism of our European eel kidney parasite isolates matched the features of the original M. giardi description. Our isolates clustered in the lineage encompassing the first published M. giardi sequence. Thus, the originally described M. giardi indeed represents an existing species within the genus Myxidium Bütschli, 1882, which we formally resurrect and redescribe. Due to the morphological and molecular differences between M. giardi and P. giardi of Freeman et Kristmundsson (2018), we additionally rename the latter species as Paramyxidium freemani nom. nov.

Solving the Myxidium rhodei (Myxozoa) puzzle: insights into its phylogeny and host specificity in Cypriniformes.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.

Title: Résoudre le casse-tête de Myxidium rhodei (Myxozoa) : aperçu de sa phylogénie et de sa spécificité d'hôte chez les Cypriniformes. Abstract: Myxidium rhodei Léger, 1905 (Cnidaria : Myxozoa) est un Myxosporea infectant les reins qui a été décrit à l'origine chez la bouvière, Rhodeus amarus. Par la suite, il a été documenté, sur la base de la morphologie des spores, chez plus de 40 autres espèces de cypriniformes, le gardon Rutilus rutilus étant l'hôte le plus fréquemment signalé. Cette étude présente la première évaluation complète des données sur M. rhodei, réalisée par des méthodes morphologiques, écologiques et moléculaires. Les analyse morphologiques et phylogénétiques des séquences d'ADNr SSU des isolats de Myxidium obtenus à partir de bouvières et de gardons européens n'ont pas confirmé la conspécificité du parasite de ces poissons. En fait, les isolats infectant les gardons représentent trois espèces distinctes de parasites. Les deux premières, M. rutili n. sp. et M. rutilusi n. sp., sont des espèces cryptiques étroitement apparentées, regroupées avec d'autres Myxosporea du clade urinaire d'eau douce, partageant le même tropisme tissulaire. La troisième, M. batuevae n. sp., précédemment attribuée à M. cf. rhodei, appartient au clade biliaire hépatique, groupe-frère de M. rhodei infectant la bouvière. Notre examen de divers poissons cypriniformes, couplé à des analyses moléculaires et morphologiques, nous a permis de démêler la nature cryptique des espèces de M. rhodei et de découvrir l'existence de nouvelles espèces. Cela souligne la diversité largement méconnue des Myxozoaires et souligne la nécessité d'incorporer des données de séquence dans le diagnostic de nouvelles espèces.

Inside the Host: Understanding the Evolutionary Trajectories of Intracellular Parasitism.

This review explores the origins of intracellular parasitism, an intriguing facet of symbiosis, where one organism harms its host, potentially becoming deadly. We focus on three distantly related groups of single-celled eukaryotes, namely Kinetoplastea, Holomycota, and Apicomplexa, which contain multiple species-rich lineages of intracellular parasites. Using comparative analysis of morphological, physiological, and molecular features of kinetoplastids, microsporidians, and sporozoans, as well as their closest free-living relatives, we reveal the evolutionary trajectories and adaptations that enabled the transition to intracellular parasitism. Intracellular parasites have evolved various efficient mechanisms for host acquisition and exploitation, allowing them to thrive in a variety of hosts. Each group has developed unique features related to the parasitic lifestyle, involving dedicated protein families associated with host cell invasion, survival, and exit. Indeed, parallel evolution has led to distinct lineages of intracellular parasites employing diverse traits and approaches to achieve similar outcomes.

Blood-feeding adaptations and virome assessment of the poultry red mite Dermanyssus gallinae guided by RNA-seq.

Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Expression profiling and cellular localization of myxozoan minicollagens during nematocyst formation and sporogenesis.

In free-living cnidarians, minicollagens are major structural components in the biogenesis of nematocysts. Recent sequence mining and proteomic analysis demonstrate that minicollagens are also expressed by myxozoans, a group of evolutionarily ancient cnidarian endoparasites. Nonetheless, the presence and abundance of nematocyst-associated genes/proteins in nematocyst morphogenesis have never been studied in Myxozoa. Here, we report the gene expression profiles of three myxozoan minicollagens, ncol-1, ncol-3, and the recently identified noncanonical ncol-5, during the intrapiscine development of Myxidium lieberkuehni, the myxozoan parasite of the northern pike, Esox lucius. Moreover, we localized the myxozoan-specific minicollagen Ncol-5 in the developing myxosporean stages by Western blotting, immunofluorescence, and immunogold electron microscopy. We found that expression of minicollagens was spatiotemporally restricted to developing nematocysts within the myxospores during sporogenesis. Intriguingly, Ncol-5 is localized in the walls of nematocysts and predominantly in nematocyst tubules. Overall, we demonstrate that despite being significantly reduced in morphology, myxozoans retain structural components associated with nematocyst development in free-living cnidarians. Furthermore, our findings have practical implications for future functional and comparative studies as minicollagens are useful markers of the developmental phase of myxozoan parasites.

An ancient alliance: Matching evolutionary patterns of cartilaginous fishes (Elasmobranchii) and chloromyxid parasites (Myxozoa).

Myxozoa is a group of endoparasitic cnidarians covering almost 2600 species but merely 53 species, mostly from the genus Chloromyxum, have been reported from sharks, rays, and skates (Elasmobranchii). Elasmobranchs play a key role in the study of evolutionary trajectories of myxozoans as they represent ancestral vertebrate hosts. Our study provides new data on Chloromyxum spp. from 57 elasmobranchs, covering 20 species from geographical regions and host groups not previously investigated, such as Lamniformes and Hexanchiformes, the most basal phylogenetic shark lineage. In total, 28% of elasmobranchs were infected with Chloromyxum spp., indicating high diversity. Of the seven distinguished species, six are formally described based on morphological, morphometric, and genetic (18S rDNA) data. Comprehensive co-phylogenetic analyses and ancestral state reconstruction revealed that parasite and host phylogenies are clearly correlated, resulting in a distinct phylogenetic separation of chloromyxids from selachid (shark) vs. batoid (ray and skate) hosts. Species infecting the most ancient elasmobranchs formed a sublineage, branching off in the middle of the Chloromyxum sensu stricto clade. Our findings indicate that chloromyxids likely invaded an ancestral elasmobranch prior the time of divergence of shark and batoid lineages. Our analyses did not show a clear phylogeographic pattern of Chloromyxum parasites, probably due to the cosmopolitan distribution and migratory behaviour of many elasmobranch hosts, but geographical sampling must be extended to confirm or refute this observation. This study provides a complex view on species diversity, phylogeny, evolution, host-parasite co-phylogeny, and the phylogeographic origin of Chloromyxum species from elasmobranchs. Our results highlight the importance of adding missing data from previously un- or undersampled geographical regions and host species which results in a more accurate estimate of myxozoan biodiversity and a better understanding of the evolution of this parasite group in their hosts and in the different oceans of our planet.

Correlated evolution of fish host length and parasite spore size: a tale from myxosporeans inhabiting elasmobranchs.

Myxozoa represent a diverse group of microscopic cnidarian endoparasites alternating between invertebrate and vertebrate hosts. Of the approximately 2,600 species described predominantly from teleost fish, only 1.8% have been reported from cartilaginous fishes (Elasmobranchii). As ancestral vertebrate hosts of myxozoans, elasmobranchs may have played an important role in myxozoan evolution, however, they are also some of the largest vertebrate hosts known for this group of parasites. We screened 50 elasmobranchs belonging to nine species and seven families, from various geographical areas, for myxozoan infection. We found a 22% overall prevalence of myxozoans in elasmobranchs and describe five species new to science. We investigated, for the first known time, the evolution of spore size within three phylogenetic clades, Ceratomyxa, Sphaerospora sensu stricto and Parvicapsula. We found that spores from elasmobranch-infecting myxozoans were on average 4.8× (Ceratomyxa), 2.2× (Parvicapsula clade) and 1.8× (Sphaerospora sensu stricto except polysporoplasmic Sphaerospora spp.) larger than those from teleosts. In all analysed clades, spore size was correlated with phylogenetic position. In ceratomyxids, it was further strongly positively correlated with fish body size and habitat depth, independent of cellular composition of the spores and phylogenetic position in the tree. While in macroparasites a host size-correlated increase in parasite size occurs on a large scale and is often related to improved exploitation of host resources, in microscopic parasites size ranges vary at the scale of a few micrometres, disproportionate to the available additional space in a large host. We discuss the ecological role of these changes with regard to transmission under high pressure and an invertebrate fauna that is adapted to deeper marine habitats.

Plasmepsin-like Aspartyl Proteases in Babesia.

Apicomplexan genomes encode multiple pepsin-family aspartyl proteases (APs) that phylogenetically cluster to six independent clades (A to F). Such diversification has been powered by the function-driven evolution of the ancestral apicomplexan AP gene and is associated with the adaptation of various apicomplexan species to different strategies of host infection and transmission through various invertebrate vectors. To estimate the potential roles of Babesia APs, we performed qRT-PCR-based expressional profiling of Babesia microti APs (BmASP2, 3, 5, 6), which revealed the dynamically changing mRNA levels and indicated the specific roles of individual BmASP isoenzymes throughout the life cycle of this parasite. To expand on the current knowledge on piroplasmid APs, we searched the EuPathDB and NCBI GenBank databases to identify and phylogenetically analyse the complete sets of APs encoded by the genomes of selected Babesia and Theileria species. Our results clearly determine the potential roles of identified APs by their phylogenetic relation to their homologues of known function-Plasmodium falciparum plasmepsins (PfPM I-X) and Toxoplasma gondii aspartyl proteases (TgASP1-7). Due to the analogies with plasmodial plasmepsins, piroplasmid APs represent valuable enzymatic targets that are druggable by small molecule inhibitors-candidate molecules for the yet-missing specific therapy for babesiosis.

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians.

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts.

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

Genetic Diversity of Serine Protease Inhibitors in Myxozoan (Cnidaria, Myxozoa) Fish Parasites.

We studied the genetic variability of serine protease inhibitors (serpins) of Myxozoa, microscopic endoparasites of fish. Myxozoans affect the health of both farmed and wild fish populations, causing diseases and mortalities. Despite their global impact, no effective protection exists against these parasites. Serpins were reported as important factors for host invasion and immune evasion, and as promising targets for the development of antiparasitic therapies. For the first time, we identified and aligned serpin sequences from high throughput sequencing datasets of ten myxozoan species, and analyzed 146 serpins from this parasite group together with those of other taxa phylogenetically, to explore their relationship and origins. High intra- and interspecific variability was detected among the examined serpins. The average sequence identity was 25-30% only. The conserved domains (i.e., motif and signature) showed taxon-level differences. Serpins clustered according to taxonomy rather than to serpin types, and myxozoan serpins seemed to be highly divergent from that of other taxa. None of them clustered with their closest relative free-living cnidarians. The genetic distinction of myxozoan serpins further strengthens the idea of an independent origin of Myxozoa, and may indicate novel protein functions potentially related to parasitism in this animal group.

Mechanisms and Drivers for the Establishment of Life Cycle Complexity in Myxozoan Parasites.

It is assumed that complex life cycles in cnidarian parasites belonging to the Myxozoa result from incorporation of vertebrates into simple life cycles exploiting aquatic invertebrates. However, nothing is known about the driving forces and implementation of this event, though it fostered massive diversification. We performed a comprehensive search for myxozoans in evolutionary ancient fishes (Chondrichthyes), and more than doubled existing 18S rDNA sequence data, discovering seven independent phylogenetic lineages. We performed cophylogenetic and character mapping methods in the largest monophyletic dataset and demonstrate that host and parasite phylogenies are strongly correlated, and that tectonic changes may explain phylogeographic clustering in recent skates and softnose skates, in the Atlantic. The most basal lineages of myxozoans inhabit the bile of chondrichthyans, an immunologically privileged site and protective niche, easily accessible from the gut via the bile duct. We hypothesize that feed-integration is a likely mechanism of host acquisition, an idea supported by feeding habits of chimaeras and ancient sharks and by multiple entries of different parasite lineages from invertebrates into the new host group. We provide exciting first insights into the early evolutionary history of ancient metazoan parasites in a host group that embodies more evolutionary distinctiveness than most other vertebrates.

Author Correction: Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites.

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

The description of two new species of Chloromyxum from skates in the Argentine Sea reveals that a limited geographic host distribution causes phylogenetic lineage separation of myxozoans in Chondrichthyes.

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.

Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa).

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.

Life in a rock pool: Radiation and population genetics of myxozoan parasites in hosts inhabiting restricted spaces.

INTRODUCTION: Intertidal rock pools where fish and invertebrates are in constant close contact due to limited space and water level fluctuations represent ideal conditions to promote life cycles in parasites using these two alternate hosts and to study speciation processes that could contribute to understanding the roles of parasitic species in such ecosystems. MATERIAL AND METHODS: Gall bladder and liver samples from five clinid fish species (Blenniiformes: Clinidae) were morphologically and molecularly examined to determine the diversity, prevalence, distribution and host specificity of Ceratomyxa parasites (Cnidaria: Myxozoa) in intertidal habitats along the coast of South Africa. Phylogenetic relationships of clinid ceratomyxids based on the SSU rDNA, LSU rDNA and ITS regions were assessed additionally to the investigation of population genetic structure of Ceratomyxa cottoidii and subsequent comparison with the data known from type fish host Clinus cottoides. RESULTS AND DISCUSSION: Seven Ceratomyxa species including previously described Ceratomyxa dehoopi and C. cottoidii were recognized in clinids. They represent a diverse group of rapidly evolving, closely related species with a remarkably high prevalence in their hosts, little host specificity and frequent concurrent infections, most probably as a result of parasite radiation after multiple speciation events triggered by limited host dispersal within restricted spaces. C. cottoidii represents the most common clinid parasite with a population structure characterized by young expanding populations in the south west and south east coast and by older populations in equilibrium on the west coast of its distribution. Parasite and fish host population structures show overlapping patterns and are very likely affected by similar oceanographic barriers possibly due to reduced host dispersal enhancing parasite community differentiation. While fish host specificity had little impact on parasite population structure, the habitat preference of the alternate invertebrate host as well as tidal water exchange may be additional crucial variables affecting the dispersal and associated population structure of C. cottoidii.