Evolution of myxozoan mitochondrial genomes: insights from myxobolids.

BACKGROUND: Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms. RESULTS: Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene. CONCLUSIONS: Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

Identification of in vivo induced antigens of the malacosporean parasite Tetracapsuloides bryosalmonae (Cnidaria) using in vivo induced antigen technology.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that causes proliferative kidney disease (PKD) in wild and farmed salmonids in Europe and North America. The life cycle of T. bryosalmonae completes between invertebrate bryozoan and vertebrate fish hosts. Inside the fish, virulence factors of T. bryosalmonae are induced during infection or interactions with host cells. T. bryosalmonae genes expressed in vivo are likely to be important in fish pathogenesis. Herein, we identify in vivo induced antigens of T. bryosalmonae during infection in brown trout (Salmo trutta) using in vivo induced antigen technology (IVIAT). Brown trout were exposed to the spores of T. bryosalmonae and were sampled at different time points. The pooled sera were first pre-adsorbed with antigens to remove false positive results. Subsequently, adsorbed sera were used to screen a T. bryosalmonae cDNA phage expression library. Immunoscreening analysis revealed 136 immunogenic T. bryosalmonae proteins induced in brown trout during parasite development. They are involved in signal transduction, transport, metabolism, ion-protein binding, protein folding, and also include hypothetical proteins, of so far unknown functions. The identified in vivo induced antigens will be useful in the understanding of T. bryosalmonae pathogenesis during infection in susceptible hosts. Some of the antigens found may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against T. bryosalmonae in salmonids.

Ceratonova shasta: a cnidarian parasite of annelids and salmonids.

The myxozoan Ceratonova shasta was described from hatchery rainbow trout over 70 years ago. The parasite continues to cause severe disease in salmon and trout, and is recognized as a barrier to salmon recovery in some rivers. This review incorporates changes in our knowledge of the parasite's life cycle, taxonomy and biology and examines how this information has expanded our understanding of the interactions between C. shasta and its salmonid and annelid hosts, and how overarching environmental factors affect this host­parasite system. Development of molecular diagnostic techniques has allowed discrimination of differences in parasite genotypes, which have differing host affinities, and enabled the measurement of the spatio-temporal abundance of these different genotypes. Establishment of the C. shasta life cycle in the laboratory has enabled studies on host­parasite interactions and the availability of transcriptomic data has informed our understanding of parasite virulence factors and host defences. Together, these advances have informed the development of models and management actions to mitigate disease.

Using a mechanistic framework to model the density of an aquatic parasite Ceratonova shasta.

Ceratonova shasta is a myxozoan parasite endemic to the Pacific Northwest of North America that is linked to low survival rates of juvenile salmonids in some watersheds such as the Klamath River basin. The density of C. shasta actinospores in the water column is typically highest in the spring (March-June), and directly influences infection rates for outmigrating juvenile salmonids. Current management approaches require quantities of C. shasta density to assess disease risk and estimate survival of juvenile salmonids. Therefore, we developed a model to simulate the density of waterborne C. shasta actinospores using a mechanistic framework based on abiotic drivers and informed by empirical data. The model quantified factors that describe the key features of parasite abundance during the period of juvenile salmon outmigration, including the week of initial detection (onset), seasonal pattern of spore density, and peak density of C. shasta. Spore onset was simulated by a bio-physical degree-day model using the timing of adult salmon spawning and accumulation of thermal units for parasite development. Normalized spore density was simulated by a quadratic regression model based on a parabolic thermal response with river water temperature. Peak spore density was simulated based on retained explanatory variables in a generalized linear model that included the prevalence of infection in hatchery-origin Chinook juveniles the previous year and the occurrence of flushing flows (≥171 m3/s). The final model performed well, closely matched the initial detections (onset) of spores, and explained inter-annual variations for most water years. Our C. shasta model has direct applications as a management tool to assess the impact of proposed flow regimes on the parasite, and it can be used for projecting the effects of alternative water management scenarios on disease-induced mortality of juvenile salmonids such as with an altered water temperature regime or with dam removal.

A myxozoan genome reveals mosaic evolution in a parasitic cnidarian.

BACKGROUND: Parasite evolution has been conceptualized as a process of genetic loss and simplification. Contrary to this model, there is evidence of expansion and conservation of gene families related to essential functions of parasitism in some parasite genomes, reminiscent of widespread mosaic evolution-where subregions of a genome have different rates of evolutionary change. We found evidence of mosaic genome evolution in the cnidarian Myxobolus honghuensis, a myxozoan parasite of fish, with extremely simple morphology. RESULTS: We compared M. honghuensis with other myxozoans and free-living cnidarians, and determined that it has a relatively larger myxozoan genome (206 Mb), which is less reduced and less compact due to gene retention, large introns, transposon insertion, but not polyploidy. Relative to other metazoans, the M. honghuensis genome is depleted of neural genes and has only the simplest animal immune components. Conversely, it has relatively more genes involved in stress resistance, tissue invasion, energy metabolism, and cellular processes compared to other myxozoans and free-living cnidarians. We postulate that the expansion of these gene families is the result of evolutionary adaptations to endoparasitism. M. honghuensis retains genes found in free-living Cnidaria, including a reduced nervous system, myogenic components, ANTP class Homeobox genes, and components of the Wnt and Hedgehog pathways. CONCLUSIONS: Our analyses suggest that the M. honghuensis genome evolved as a mosaic of conservative, divergent, depleted, and enhanced genes and pathways. These findings illustrate that myxozoans are not as genetically simple as previously regarded, and the evolution of some myxozoans is driven by both genomic streamlining and expansion.

To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections.

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

Intestinal Transcriptomic and Histologic Profiling Reveals Tissue Repair Mechanisms Underlying Resistance to the Parasite Ceratonova shasta.

BACKGROUND: Myxozoan parasites infect fish worldwide causing significant disease or death in many economically important fish species, including rainbow trout and steelhead trout (Oncorhynchus mykiss). The myxozoan Ceratonova shasta is a parasite of salmon and trout that causes ceratomyxosis, a disease characterized by severe inflammation in the intestine resulting in hemorrhaging and necrosis. Populations of O. mykiss that are genetically fixed for resistance or susceptibility to ceratomyxosis exist naturally, offering a tractable system for studying the immune response to myxozoans. The aim of this study was to understand how steelhead trout that are resistant to the disease respond to C. shasta once it has become established in the intestine and identify potential mechanisms of resistance. RESULTS: Sequencing of intestinal mRNA from resistant steelhead trout with severe C. shasta infections identified 417 genes differentially expressed during the initial stage of the infection compared to uninfected control fish. A strong induction of interferon-gamma and interferon-stimulated genes was evident, along with genes involved in cell adhesion and migration. A total of 11,984 genes were differentially expressed during the late stage of the infection, most notably interferon-gamma, interleukin-6, and immunoglobulin transcripts. A distinct hardening of the intestinal tissue and a strong inflammatory reaction in the intestinal submucosa including severe hyperplasia and inflammatory cell infiltrates were observed in response to the infection. The massive upregulation of caspase-14 early in the infection, a protein involved in keratinocyte differentiation might reflect the rapid onset of epithelial repair mechanisms, and the collagenous stratum compactum seemed to limit the spread of C. shasta within the intestinal layers. These observations could explain the ability of resistant fish to eventually recover from the infection. CONCLUSIONS: Our results suggest that resistance to ceratomyxosis involves both a rapid induction of key immune factors and a tissue response that limits the spread of the parasite and the subsequent tissue damage. These results improve our understanding of the myxozoan-host dialogue and provide a framework for future studies investigating the infection dynamics of C. shasta and other myxozoans.

Data of de novo transcriptome assembly of the myxozoan parasite Tetracapsuloides bryosalmonae.

Tetracapsuloides bryosalmonae, a myxozoan endoparasite, causes proliferative kidney disease in salmonids. The life cycle of T. bryosalmonae occurs between invertebrate bryozoan and vertebrate fish hosts. T. bryosalmonae develops in the body cavity of colonial bryozoan and spores are released from mature spore sacs into the water likely through the vestibular pore and infect fish by attaching to their gills. However, very little is known about the transcriptome of this important parasite, which hampers studies into the molecular mechanisms of host-parasite interactions and understanding the parasite biology. In order to circumvent this limitation, we performed de novo transcriptome assembly on the sacs of T. bryosalmonae, collected from infected bryozoan Fredericella sultana. A total of 111.5 million filtered paired-end reads was obtained and assembled into 25,908 contigs corresponding to putative transcripts that were functionally annotated. More than 50% of the assembled transcripts (13,071 contigs) had a significant hit in NCBI non-redundant database. Based on Gene ontology annotation, the most highly scored categories of molecular function of the contigs were related to binding and catalytic activities in T. bryosalmonae. This study provides a global overview of the T. bryosalmonae transcriptome that will be a valuable resource for identifying virulence factors, gene discovery, genome annotation, and vaccine development applications. This data is accessible via NCBI BioProject (PRJNA680464).

A tale of two fish: Comparative transcriptomics of resistant and susceptible steelhead following exposure to Ceratonova shasta highlights differences in parasite recognition.

Diseases caused by myxozoan parasites represent a significant threat to the health of salmonids in both the wild and aquaculture setting, and there are no effective therapeutants for their control. The myxozoan Ceratonova shasta is an intestinal parasite of salmonids that causes severe enteronecrosis and mortality. Most fish populations appear genetically fixed as resistant or susceptible to the parasite, offering an attractive model system for studying the immune response to myxozoans. We hypothesized that early recognition of the parasite is a critical factor driving resistance and that susceptible fish would have a delayed immune response. RNA-seq was used to identify genes that were differentially expressed in the gills and intestine during the early stages of C. shasta infection in both resistant and susceptible steelhead (Oncorhynchus mykiss). This revealed a downregulation of genes involved in the IFN-γ signaling pathway in the gills of both phenotypes. Despite this, resistant fish quickly contained the infection and several immune genes, including two innate immune receptors were upregulated. Susceptible fish, on the other hand, failed to control parasite proliferation and had no discernible immune response to the parasite, including a near-complete lack of differential gene expression in the intestine. Further sequencing of intestinal samples from susceptible fish during the middle and late stages of infection showed a vigorous yet ineffective immune response driven by IFN-γ, and massive differential expression of genes involved in cell adhesion and the extracellular matrix, which coincided with the breakdown of the intestinal structure. Our results suggest that the parasite may be suppressing the host's immune system during the initial invasion, and that susceptible fish are unable to recognize the parasite invading the intestine or mount an effective immune response. These findings improve our understanding of myxozoan-host interactions while providing a set of putative resistance markers for future studies.

Myxobolus cerebralis Causes Presporogonic Mortality in Juvenile Mountain Whitefish.

Recent range expansions of whirling disease impelled us to understand the impacts of its causative agent, the myxozoan parasite Myxobolus cerebralis, on lesser-studied fish hosts. Mountain Whitefish Prosopium williamsoni overlap broadly with M. cerebralis across the western United States and Canada, and populations have experienced widespread declines since the 1990s. To evaluate effects of the parasite on Mountain Whitefish, we revisit formerly unpublished work of the Colorado Division of Wildlife (now Colorado Parks and Wildlife), comparing infection in age-matched Mountain Whitefish, Rainbow Trout Oncorhynchus mykiss, and Brown Trout Salmo trutta. To complement the original report, we reanalyze mortality data and include additional SEM imagery. Infection of M. cerebralis in juvenile Mountain Whitefish was characterized by a brief but heavy period of mortality in the first 2 weeks after exposure, with limited pathology. This clinical effect is unique among the known salmonid hosts of M. cerebralis.

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.

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection.

BACKGROUND: Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known. METHODS: We searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals. RESULTS: We identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish's gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a "venom" can have and represent targets for designing therapeutics against myxozoan infections.

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.

Myxosporea (Myxozoa, Cnidaria) Lack DNA Cytosine Methylation.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

Transcriptome Analysis Elucidates the Key Responses of Bryozoan Fredericella sultana during the Development of Tetracapsuloides bryosalmonae (Myxozoa).

Bryozoans are sessile, filter-feeding, and colony-building invertebrate organisms. Fredericella sultana is a well known primary host of the myxozoan parasite Tetracapsuloides bryosalmonae. There have been no attempts to identify the cellular responses induced in F. sultana during the T. bryosalmonae development. We therefore performed transcriptome analysis with the aim of identifying candidate genes and biological pathways of F. sultana involved in the response to T. bryosalmonae. A total of 1166 differentially up- and downregulated genes were identified in the infected F. sultana. Gene ontology of biological processes of upregulated genes pointed to the involvement of the innate immune response, establishment of protein localization, and ribosome biogenesis, while the downregulated genes were involved in mitotic spindle assembly, viral entry into the host cell, and response to nitric oxide. Eukaryotic Initiation Factor 2 signaling was identified as a top canonical pathway and MYCN as a top upstream regulator in the differentially expressed genes. Our study provides the first transcriptional profiling data on the F. sultana zooid's response to T. bryosalmonae. Pathways and upstream regulators help us to understand the complex interplay in the infected F. sultana. The results will facilitate the elucidation of innate immune mechanisms of bryozoan and will lay a foundation for further analyses on bryozoan-responsive candidate genes, which will be an important resource for the comparative analysis of gene expression in bryozoans.

A comparison of the structure and function of nematocysts in free-living and parasitic cnidarians (Myxozoa).

Myxozoans are obligate parasites that have complex life cycles requiring alternate vertebrate and invertebrate hosts, with transmission via microscopic waterborne spores. Unusually for parasites, they belong to the phylum Cnidaria, alongside thousands of free-living corals, sea anemones, jellyfish and hydrozoans. Their cnidarian affinity is affirmed by genetic relatedness and the presence of nematocysts, historically called "polar capsules" in myxozoan research. Free-living cnidarians utilise this cellular weaponry for defence, predation and adhesion, whereas myxozoans use it to anchor to their hosts as the first step in infection. Despite the ~650 million years of divergence between free-living cnidarians and myxozoans, their nematocysts retain many shared morphological and molecular characters. Both are intra-cellular capsules with a single opening, and contain a coiled, evertable tubule. They are composed of unique nematocyst proteins, nematogalectin and minicollagen, and both likely contain an internal matrix of metal cations covalently bound to the anionic polymer poly-gamma glutamate. The rapid dissociation of this matrix and the resulting increase in internal osmotic potential is the driving force behind tubule elongation during discharge. In this review, we compare the structure and function of nematocysts in Myxozoa and free-living Cnidaria, incorporating recent molecular characterizations. We propose that terminology for homologous myxozoan structures be synonymized with those from other Cnidaria, hence, "polar capsule" as a taxon-specific nematocyst morphotype and "polar filament" as "tubule." Despite taxonomic divergence, genome reduction and an evolution to parasitism, myxozoans maintain nematocysts that are structurally and functionally homologous to those of their free-living cnidarian relatives.

In vitro and in vivo assays reveal that cations affect nematocyst discharge in Myxobolus cerebralis (Cnidaria: Myxozoa).

Myxozoans are parasitic, microscopic cnidarians that have retained the phylum-characteristic stinging capsules called nematocysts. Free-living cnidarians, like jellyfish and corals, utilize nematocysts for feeding and defence, with discharge powered by osmotic energy. Myxozoans use nematocysts to anchor to their fish hosts in the first step of infection, however, the discharge mechanism is poorly understood. We used Myxobolus cerebralis, a pathogenic myxozoan parasite of salmonid fishes, and developed two assays to explore the nature of its nematocyst discharge. Using parasite actinospores, the infectious stage to fish, we stimulated discharge of the nematocysts with rainbow trout mucus in vitro, in solutions enriched with chloride salts of Na+, K+, Ca2+ and Gd3+, and quantified discharge using microscopy. We then used quantitative polymerase chain reaction to evaluate the in vivo effects of these treatments, plus Mg2+ and the common aquaculture disinfectant KMnO4, on the ability of M. cerebralis actinospores to infect fish. We found that Mg2+ and Gd3+ reduced infection in vivo, whereas Na+ and K+ over-stimulated nematocyst discharge in vitro and reduced infection in vivo. These findings align with nematocyst discharge behaviour in free-living Cnidaria, and suggest phylum-wide commonalties, which could be exploited to develop novel approaches for controlling myxozoan diseases in aquaculture.

Transcriptome-Wide Comparisons and Virulence Gene Polymorphisms of Host-Associated Genotypes of the Cnidarian Parasite Ceratonova shasta in Salmonids.

Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. Ceratonova shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the data sets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed single-nucleotide polymorphism-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.

First transcriptome analysis of bryozoan Fredericella sultana, the primary host of myxozoan parasite Tetracapsuloides bryosalmonae.

Bryozoans are aquatic invertebrate moss animals that are found worldwide. Fredericella sultana is a freshwater bryozoan and is the most common primary host of myxozoan parasite, Tetracapsuloides bryosalmonae. However, limited genomic resources are available for this bryozoan, which hampers investigations into the molecular mechanisms of host-parasite interactions. To better understand these interactions, there is a need to build a transcriptome dataset of F. sultana, for functional genomics analysis by large-scale RNA sequencing. Total RNA was extracted from zooids of F. sultana cultivated under controlled laboratory conditions. cDNA libraries were prepared and were analyzed by the Illumina paired-ends sequencing. The sequencing data were used for de novo transcriptome assembly and functional annotation. Approximately 118 million clean reads were obtained, and assembled into 85,544 contigs with an average length of 852 bp, an N50 of 1,085 bp, and an average GC content 51.4%. A total of 23,978 (28%) contigs were annotated using BLASTX analysis. Of these transcripts, 4,400 contigs had highest similarity to brachiopod species Lingula anatina. Based on Gene ontology (GO) annotation, the most highly scored categories of biological process were categorized into cellular process (27%), metabolic process (24%), and biological regulation (8%) in the transcriptome of F. sultana. This study gives first insights into the transcriptome of F. sultana and provides comprehensive genetic resources for the species. We believe that the transcriptome of F. sultana will serve as a useful genomic dataset to accelerate research of functional genomics and will help facilitate whole genome sequencing and annotation. Candidate genes potentially involved in growth, proteolysis, and stress/immunity-response were identified, and are worthy of further investigation.