Objective The objective of this study was to evaluate statistical evidence for relationships between flow event characteristics and annelid host distribution, an important and necessary step toward providing and justifying management actions. Declines in Klamath River salmon have been attributed to infection and disease that are caused by the myxozoan parasite Ceratonova shasta. Flow manipulation has been used to manage risk of C. shasta for juvenile salmonids in this system. One mechanism by which flow-related disturbance can reduce the risk of C. shasta infection for salmon is by reducing populations of the obligate invertebrate host Manayunkia occidentalis. We previously demonstrated that hydraulic conditions during peak discharge events drive the distribution of M. occidentalis and suggested that high-magnitude flow events would be effective for reducing the distribution of the annelid host. However, evidence of the effects of flow events on M. occidentalis was needed to support the use of flow management.Methods To address this knowledge gap, we leveraged a multiyear data set to estimate relationships between the distribution the annelid host and the characteristics of each discharge event. We measured the presence or absence of M. occidentalis at spatially georeferenced sampling locations, stratified across the range of hydraulic and substrate conditions in three reaches annually from 2012 to 2020.Results During the study period, the magnitude of peak discharge ranged from 52.4 to 314.3 m3/s. The inclusion of seven additional years' data in the base model (hierarchical; annual-level covariate + previous "single year" model) resulted in parameter estimates that were similar to those that were derived for the previous (2016) model, supporting that substrate, depth, and velocity during peak discharge predicted annelid distribution. The magnitude model (base model + annual-level covariate for peak discharge) showed evidence of a strong negative association with annelid presence (95% of all Markov chain-Monte Carlo draws were negative). Every 101.9 m3/s increase in peak discharge was associated with an estimated 39% decrease in the odds of annelid presence. The estimated effect of magnitude on the probability of annelid presence was illustrated in the contrast between low- and high-magnitude peak discharge scenarios. For both scenarios, the effects of depth, velocity at peak discharge, and substrate, consistent with the previous model (2016), were evident, as was the further reduction in probability of annelid presence at the higher peak discharge. However, under the low-magnitude discharge scenario, M. occidentalis were predicted to be present on smaller less stable substrates and at a wider range of depths and velocities than they were under the high-magnitude discharge scenario. In contrast to the covariate for magnitude, the inclusion of a duration covariate did not show a strong relationship with annelid distribution, which we attributed to the lack of variation in this covariate during the study period.Conclusions This work provides quantitative evidence that high-magnitude peak discharge flow events lead to lower probabilities of annelid host presence. Lower probabilities of annelid hosts are indicative of a reduced distribution of the M. occidentalis host and in turn reduced risk of C. shasta for salmon. This study investigates how water management can be used to disrupt the life cycle of a harmful parasite that affects salmon populations. We analyzed the relationship between flow event characteristics and the distribution of the parasite's obligate (required) worm host. Higher magnitude flow events significantly reduced the host's presence and distribution, in turn lowering risk of infection for juvenile salmon.

Ceratomyxosis due to the myxozoan parasite Ceratonova shasta affects salmonids, causing severe enteritis leading to hemorrhaging and necrosis. The waterborne parasite stages penetrate host gills and reach the fish intestine through the bloodstream. Steelhead trout (Oncorhynchus mykiss) populations from C. shasta endemic watersheds (sympatric) are less susceptible than populations from non-endemic watersheds (allopatric). We investigated the immune effectors behind these different susceptibilities. Both steelhead trout strains were exposed to C. shasta genotype 0. Intestinal tissue and serum samples of control and exposed sympatric and allopatric fish were taken at 8, 15, 22, 29, 57, and 183 days post exposure. Gills were taken at 1 and 8 days post exposure. Parasite abundance and histopathology were evaluated on tissue sections of fish that tested PCR+ for the parasite. Zap70+ T cells, IgT+ B cells, and IgD+ B cells were quantified, and the presence of specific IgM was evaluated from fish sera by immunohistochemistry. Parasite counts were significantly lower and limited to a shorter duration in sympatric fish. The initial intestinal inflammatory response in both fish strains was characterized by hyperplasia of the lamina propria-submucosa and epithelial infiltration of lymphocytes. Remarkably, hyperplasia was resolved earlier in sympatric fish, whereas in allopatric fish, hyperplasia was not resolved by the end of the experiment, coinciding with high intestinal parasite counts and sporogenesis. An increase of intestinal Zap70+ T cells occurred before IgT+ B cells peaked, earlier in sympatric than in allopatric fish. Low numbers of intestinal IgD+ B cells were detected in both strains. In gills, an early increase of Zap70+ T cells was observed in both fish strains at 1 day post exposure, and an increase of IgD+ B cells occurred only in the allopatric strain. Specific circulating IgM was detected much later in both fish strains, only at 57 and 183 days post exposure. In conclusion, sympatric steelhead trout restricted intestinal proliferation of C. shasta genotype 0 more effectively than allopatric fish, probably due to an earlier T cell response triggering a stronger IgT-based mucosal adaptive response in the intestine. Specific circulating IgM appeared later in both sympatric and allopatric fish, probably also contributing systemic protection.

IntroductionLittle is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae.MethodsOne group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection.ResultsIn the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated.DiscussionThe results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Myxosporeans are a diverse group of cnidarian endoparasites of aquatic animals, with more than 2,600 described species. The genus Ceratomyxa includes some 270 species that are mostly coelozoic and infect the gall bladders of mainly marine fish. During our survey in Tapajós River in the Brazilian Amazon, two Ceratomyxa species were discovered in the gallbladders of the anostomid fishes Schyzodon fasciatus and Schyzodon vittatus. Based on myxospore morphology and sequencing of small subunit ribosomal DNA, we propose that these are novel Ceratomyxa species. Ceratomyxa deformis n. sp. from S. fasciatus and Ceratomyxa anomala n. sp. from S. vittatus are similar in morphology, with mature myxospores slightly arcuate with rounded ends. Plasmodia are elongated, and can move by expanding and contracting the cytoplasm. This amoeboid-like motility is heretofore unreported in freshwater South American Ceratomyxa species. Ultrastructural analyses showed the plasmodial organization of both species comprised a cytoplasmic region harboring organelles and sporogonic stages and an internal large vacuole. Phylogenetic analysis corroborates monophyletic grouping of freshwater Ceratomyxa species from South America. Ceratomyxa deformis n. sp. and C. anomala n. sp., which are the first Ceratomyxa species described in anostomid fishes, grouped as sister taxa to C. gracillima and C. mandi, which infect bile in pimelodid fishes from the Amazon region.

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.

In the published publication [...]

We used a combination of morphological, molecular and biological data to characterize a novel Henneguya (Myxozoa) species infecting the Amazonian prochilodontid Semaprochilodus insignis or “kissing prochilodus”, a popular food fish and aquarium species in the Brazilian Amazon. Twenty-one S. insignis were caught live from the Tapajós river, Pará State, Brazil, then examined for myxozoan infections. Cysts of a novel Henneguya species were observed in the connective tissue of the fins. Myxospores measured 48 ± 4.9 (39.5–60.8) µm total length, of which caudal appendages were 33 ± 4.5 (26.4–45.2) µm and spore body was 15 ± 1.6 (12.4–20.5) µm. The spore body was 4.0 ± 0.6 (2.7–5.3) µm wide × 3.2 ± 0.4 (2.7–3.6) µm thick, with two unequal polar capsules (nematocysts) 7.2 ±0.8 (5.2–8.3) × 1.5 ± 0.3 (1.0–2.2) µm for the larger capsule and 5 ± 0.7 (4.0–6.3) × 1.4 ± 0.2 (1.0–1.8) µm for the smaller capsule. Polar tubules had 8–13 turns. Generative cells, immature and mature myxospores were observed within plasmodia. Ultrastructure showed plasmodia surrounded by collagen fibers, with the plasmodial membrane having pinocytotic channels. Phylogenetic analysis of small subunit ribosomal DNA sequences showed that the new Henneguya species clustered as a sister taxon to Henneguya tietensis, a parasite of the gills of the prochilodontid fish Prochilodus lineatus, from the geographically distant Paraná–Paraguai River basin.

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.

The health of fish populations and the river systems they inhabit have broad ecological, cultural, recreational, and economic relevance. This is exemplified by the iconic anadromous salmonid fishes native to the West Coast of North America. Salmon populations have been constrained since the mid nineteenth century by dam construction and water reallocation. In the Klamath River (Oregon and California, USA), a series of dams built in the early-mid 20th century cut the basin in two and blocked anadromous fish access to more than 600 river kilometers. This dramatic loss of habitat, coupled with infectious diseases and resulting epizootics, have impacted the wellbeing of these salmonid populations. In 2023-2024, the Klamath River will undergo the largest river restoration project in US history. Removal of the four lowermost dams will cause profound physical changes to the river, including flow, water temperature, and channel geomorphology. The dam removals will reconnect the lower and upper portions of the basin, and provide fish passage after a century of segregation. Reestablishment of upstream and downstream fish movements will also alter the occupancy and abundance of the salmonid hosts and their pathogens. The increased habitat availability and longer migration routes will increase duration of pathogen exposure and potential impacts on juvenile survival and adult pre-spawn mortality. However, restoration of more natural flow and sediment regimes will decrease overall fish disease risk by disrupting complex parasite life cycles. To better understand these multifarious, competing factors, we review the salmonid species in the Klamath River, and provide an overview of their historical pathogen challenges and associated diseases and use this as a framework to predict the effects of dam removals on disease dynamics. Our review and predictions are a synthesis of expertise from tribal biologists, fish health specialists and fish biologists, many of whom have lived and worked on the Klamath River for decades. We conclude with recommendations for expansion of current pathogen monitoring and research efforts to measure changes in host-pathogen dynamics basin-wide.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Myxozoans are widely distributed aquatic obligate endoparasites that were recently recognized as belonging within the phylum Cnidaria. They have complex life cycles with waterborne transmission stages: resistant, infectious spores that are unique to myxozoans. However, little is known about the processes that give rise to these transmission stages. To understand the molecular underpinnings of spore formation, we conducted proteomics on Ceratonova shasta , a highly pathogenic myxozoan that causes severe mortalities in wild and hatchery-reared salmonid fishes. We compared proteomic profiles between developmental stages from inside the fish host, and the mature myxospore, which is released into the water where it drifts passively, ready to infect the next host. We found that C. shasta contains 2,123 proteins; representing the first proteomic catalog of a myxozoan myxospore. Analysis of proteins differentially expressed between developing and mature spore stages uncovered processes that are active during spore formation. Our data highlight dynamic changes in the actin cytoskeleton, which provides myxozoan developmental stages with mobility through lamellipodia and filopodia, whereas in the mature myxospore the actin network supports F-actin stabilization that reinforces the transmission stage. These findings provide molecular insight into the myxozoan life cycle stages and, particularly, into the process of sporogenesis.

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.