RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

• MeSH
• Actins genetics   MeSH
• Cnidaria * genetics   MeSH
• Phylogeny MeSH
• Myxozoa * genetics   MeSH
• Fish Diseases * genetics   MeSH
• Parasites * MeSH
• Cell Movement MeSH
• RNA Interference MeSH
• Fishes MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Actins MeSH

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 host–parasite 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 host–parasite 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.

• Keywords
• Actinospore, Myxozoa, disease, enteronecrosis, environmental factors, epidemiology, fish immunity, intra-specific parasite diversity, management, monitoring, myxospore,
• MeSH
• Cnidaria * MeSH
• Myxozoa * MeSH
• Fish Diseases * parasitology   MeSH
• Oncorhynchus mykiss * parasitology   MeSH
• Parasitic Diseases, Animal * parasitology   MeSH
• Parasites * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

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.

• Keywords
• Cnidaria, Myxospore, Myxozoa, Nematocyst, Proteome, Toxin, Transcriptome, Venom,
• Publication type
• Journal Article MeSH

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.

• Keywords
• B lymphocytes, RNAseq, T lymphocytes, adaptive immunity, immune evasion, immunoglobulin, parasite, teleost,
• MeSH
• Adaptive Immunity * MeSH
• Antiparasitic Agents pharmacology   MeSH
• B-Lymphocytes immunology   metabolism   parasitology   MeSH
• Immune Evasion MeSH
• Immunoglobulins immunology   metabolism   MeSH
• Host-Parasite Interactions MeSH
• Myxozoa drug effects   immunology   pathogenicity   MeSH
• Fish Diseases immunology   metabolism   parasitology   prevention & control   MeSH
• Parasitic Diseases, Animal immunology   metabolism   parasitology   prevention & control   MeSH
• Immunity, Innate * MeSH
• Fishes immunology   metabolism   parasitology   MeSH
• T-Lymphocytes immunology   metabolism   parasitology   MeSH
• Vaccines pharmacology   MeSH
• Aquaculture MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Antiparasitic Agents MeSH
• Immunoglobulins MeSH
• Vaccines MeSH

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.

• Keywords
• 3D protein structure, aminopeptidase, aspartic protease, cysteine protease, gene expression, homologous search, myxozoa, stefin,
• MeSH
• Cnidaria * MeSH
• Fish Diseases * parasitology   MeSH
• Oncorhynchus mykiss * parasitology   MeSH
• Parasitic Diseases, Animal * MeSH
• Peptide Hydrolases MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Peptide Hydrolases MeSH

Two genotypes of the intestinal parasite Ceratonova shasta infect Oncorhynchus mykiss: genotype 0 results in a chronic infection with low mortality while genotype IIR causes disease with high mortality. We determined parasite load and the relative expression of six immune factors (IgT, IgM, IL-6, IL-8, IL-10, IFNG) in fish infected with either genotype over 29 days post-exposure. In genotype IIR infections the host responded with upregulation of inflammatory and regulatory cytokines. In contrast, genotype 0 infection did not elicit an inflammatory response and expression of IFNG and IL-10 was lower. Antibody expression was upregulated in both infections but appeared to have limited efficacy in the virulent genotype IIR infections. Histologically, in genotype 0 infections the parasite migrated through the tissue layers causing inflammation but minimal damage to the mucosal epithelium, which contrasts with the severe pathology found in genotype IIR infections.

• Keywords
• Cytokine, Fish disease, Immunoglobulin, Intestine, Salmonid,
• MeSH
• Cytokines genetics   metabolism   MeSH
• Genotype * MeSH
• Immunoglobulin M blood   MeSH
• Immunoglobulins blood   MeSH
• Host-Parasite Interactions MeSH
• Myxozoa genetics   pathogenicity   MeSH
• Fish Diseases immunology   MeSH
• Oncorhynchus mykiss immunology   MeSH
• Parasitic Diseases, Animal immunology   MeSH
• Parasite Load MeSH
• Cell Movement MeSH
• Fish Proteins blood   MeSH
• Mucous Membrane immunology   MeSH
• Virulence MeSH
• Inflammation immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Cytokines MeSH
• immunoglobulin T, teleost MeSH Browser
• Immunoglobulin M MeSH
• Immunoglobulins MeSH
• Fish Proteins MeSH

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.

• Keywords
• conserved domains, free-living Cnidaria, microscopic parasite, phylogeny, serpins, signature, therapeutic target,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Myxozoa, SNPs, aquaculture, cell migration/motility, proteases,
• MeSH
• Virulence Factors genetics   MeSH
• Phylogeny MeSH
• Genotype MeSH
• Host-Parasite Interactions genetics   MeSH
• Polymorphism, Single Nucleotide MeSH
• DNA Contamination MeSH
• Myxozoa genetics   pathogenicity   MeSH
• Oncorhynchus mykiss parasitology   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Comparative Study MeSH
• Names of Substances
• Virulence Factors MeSH