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Infectious diseases stem from disrupted interactions among hosts, parasites, and the environment. Both abiotic and biotic factors can influence infection outcomes by shaping the abundance of a parasite's infective stages, as well as the host's ability to fight infection. However, disentangling these mechanisms within natural ecosystems remains challenging. Here, combining environmental DNA analysis and niche modelling at a regional scale, we uncovered the biotic and abiotic drivers of an infectious disease of salmonid fish, triggered by the parasite Tetracapsuloides bryosalmonae. We found that the occurrence and abundance of the parasite in the water-i.e., the propagule pressure- were mainly correlated to the abundances of its two primary hosts, the bryozoan Fredericella sultana and the fish Salmo trutta, but poorly to local abiotic environmental stressors. In contrast, the occurrence and abundance of parasites within fish hosts-i.e., proxies for disease emergence-were closely linked to environmental stressors (water temperature, agricultural activities, dams), and to a lesser extent to parasite propagule pressure. These results suggest that pathogen distribution alone cannot predict the risk of disease in wildlife, and that local anthropogenic stressors may play a pivotal role in disease emergence among wild host populations, likely by modulating the hosts' immune response. Our study sheds light on the intricate interplay between biotic and abiotic factors in shaping pathogen distribution and raises concerns about the effects of global change on pathogen emergence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Simon Blanchet reports financial support was provided by French Biodiversity Office. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The spotted seatrout, Cynoscion nebulosus, is a popular game fish in the southeastern USA. It is estimated that nearly 90% of the adult population in South Carolina estuaries are infected in their skeletal muscle by the myxosporean, Kudoa inornata. However, little is known about this parasite's biology, including the distribution and densities of myxospores within tissues of infected fish, which we expect affect the physiology of their hosts. In order to correlate densities with physiological parameters in future studies, we quantified the myxospores density in muscle and characterized the variation among individual fish. Naïve juvenile seatrout was experimentally infected via presumed K. inornata actinospores exposure to raw seawater. A plug of muscle was extracted from two bilaterally symmetrical regions in the epaxial fillet from fresh and frozen carcasses. Variation in density data was calculated both within and among individuals. Within individuals, density counts were compared between left- and right-side biopsies. There was no significant difference between fresh and frozen plugs, and variation among individuals accounted for the greatest proportion of variation at 68.8%, while variation within individuals was substantial at 25.6%. Simulation and correlation tests confirmed that bilaterally symmetrical replicates varied significantly within individuals. When sampled from areas surrounding the initial biopsies, myxospore density estimates were more similar than between sides. Our findings have important implications for sampling design, particularly for studies investigating physiological parameters at the cellular or molecular level in association with parasite infection., (© 2024. The Author(s).)

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

The thermotactic response of brown trout (Salmo trutta) was examined with the goal to investigate potential effects of the emerging temperature-dependent fatal trout disease PKD (proliferative kidney disease). First the differences in cold-water preferences of two forms of brown trout, lacustrine (migratory) and riverine, were determined. Second, it was studied whether this preference was changed in fish infected with PKD. The experiment involved a one-week habituation period at 14 °C in a two-chamber runway followed by a week of 3 °C temperature difference between the two runways. The fish could freely move between lanes via an opening at the end where food was provided. The temperature manipulation was repeated twice, and there were 3 trials per experimental group. All fish developed a clear spatial preference in the test. Lacustrine trout demonstrated a preference for warmer water, while riverine trout preferred cooler water. This may increase the risk to PKD in the lacustrine form. Most strikingly, riverine trout experimentally exposed to Tetracapsuloides bryosalmonae, the parasite that causes PKD, demonstrated stronger cold-seeking behaviour than control fish. Cold seeking behaviour suggests the occurrence of a disease-induced behavioural chill response, which may play an important role in disease recovery. This demonstrates the significance of protecting river connectivity and cold-water sanctuaries as management strategies for preserving salmonid populations in a warming climate., (© 2024. The Author(s).)

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1βeta (IL-1β), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramadan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Myxozoans are obligate parasites with complex life cycles, typically infecting fish and annelids. Here, we examined annelids from fish farm pond sediments in the Beit Shean Valley, in the Syrian-African Rift Valley, Israel, for myxozoan infections. We examined 1486 oligochaetes, and found 74 (5 %) were infected with actinospore stages. We used mitochondrial 16S sequencing to infer identity of 25 infected annelids as species of Potamothrix, Psammoryctides, Tubifex and Dero. We identified 7 myxozoan types from collective groups Neoactinomyxum and Sphaeractinomyxon, and characterized them by small subunit ribosomal DNA sequencing. The Neoactinomyxum type was genetically most similar (∼93 %) to cyprinid fish-infecting Myxobolus spp. The six Sphaeractinomyxon types were genetically similar (93-100 %) to Mugilid-infecting Myxobolus spp.; with one being the previously unknown actinospore stage of a myxospore that infects mullet from aquaculture from the Israeli coast of the Mediterranean Sea. As the farm pond system is artificial and geographically isolated from the Mediterranean, the presence of at least seven myxozoans in their annelid hosts demonstrates introduction and establishment of these parasites in a novel, brackish environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Members of the genus Ortholinea are among the worldwide distributed myxozoan parasites that mainly infect marine fish. In this study, a new myxosporean species, Ortholinea hamsiensis n. sp., was isolated from the urinary bladder of European anchovy Engraulis engrasicolus collected from the Sinop coasts of the Black Sea. The prevalence and density values of infection were 1.4% and 1–5 individuals in the field of view (1 + ), respectively. Mature myxospores are subspherical with slight tapering down to the less pronounced tip in the frontal view and subspherical in the sutural view. Myxospores measured 9.1 ± 0.25 (8.8–9.9) μ m in length, 9.2 ± 0.11 (8.9–9.4) μ m in thickness, and 8.4 ± 0.33 (8.2-9.1) μ m in width. Two polar capsules equal in size measured 3.1 ± 0.11 (3.0–3.3) μ m in length and 2.7 ± 0.11 (2.6–2.9) μ m in width. The polar tubule had 3–4 coils. Along with morphological peculiarities, the results of the 18S rDNA also revealed it to be a new species for science compared to the other species of the genus. In this study, another myxosporean species O. gobiusi was also detected in round goby Neogobius melanostomus with a prevalence of infection value of 4.8% and a density of 1–5 individuals in the field of view (1 + ). The present study also provided the first data of 18S rDNA of O. gobiusi from N. melanostomus and type species of the genus O. divergens from Gobius niger and the phylogenetic relationships of these species with other Ortholinea species have been revealed.

Metazoans with worm-like morphologies across diverse and disparate groups typically demonstrate motility generated by hydrostatic skeletons involving tissue layers (muscles and epithelia). Here we present representative morphological, behavioural and molecular data for parasitic cnidarians (myxozoans) that demonstrate unprecedented variation in form and function, developing as cellular hydrostats. Motile elongate plasmodia characterise a remarkable radiation of species in the genus Ceratomyxa. The vermiform plasmodia inhabit gall bladders of a range of South American freshwater fish and exhibit undulatory motility reminiscent of nematodes but achieved at the cellular level. Collective insights from ultrastructure, confocal and light microscopy along with videos depicting movements highlight key features that we propose explain the unique motility of the plasmodia. These features include cytoskeletal elements (net forming microfilaments and microtubules), a large internal vacuole, a relatively rigid outer glycocalyx and peripherally arranged mitochondria. These constituents provide collective evidence for repurposing of the cnidarian epitheliomuscular cell to support worm-like motility at the cellular level. The apparent restriction of vermiform ceratomyxids to South American freshwaters suggests an origination via Cretaceous or Miocene marine transgressions and subsequent radiation., (© 2021 Anatomical Society.)

The present study describes Hoferellus jutubensis n. sp., a myxozoan parasite found in the urinary bladder of the driftwood catfish Ageneiosus inermis, captured on Jutuba Island in the state of Pará, northern Brazil. A total of 30 A. inermis specimens were examined, of which 26 (86.7%) had myxospores and polysporic plasmodia of varying shapes and sizes dispersed in the lumen of the urinary bladder, either floating freely or attached to the epithelium. In the apical view, the myxospores of Hoferellus jutubensis n. sp. are rounded, 6.1 ± 0.2 (5.7-6.3) μm long and 5.5 ± 0.3 (5.2-6.0) μm wide, with two sub-spherical polar capsules, equal in size and shape, 2.5 ± 0.2 (2.3-2.7) μm long and 1.7 ± 0.2 (1.4-2.2) μm wide. The phylogenetic analysis of a partial sequence of the SSU rDNA gene, indicated that the new species is the sister taxon of Hoferellus azevedoi, with these two species forming a Brazilian lineage of Hoferellus. The comparison of the morphological and molecular data with those of the existing members of the genus confirmed the species status of Hoferellus jutubensis n. sp., which adds one further Hoferellus taxon to the known myxosporean diversity of the Amazon basin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enteromyxum leei is a causative agent of enteromyxosis, with a wide range of marine fish hosts. Recently, massive morbidity and mortality were caused by E. leei infection in cultured olive flounders in Korea. To reveal a relationship between E. leei abundance in culture water and the occurrence of parasite infection in host fish, we used a quantitative PCR assay targeting the 28S rDNA of E. leei in three fish farms (two where enteromyxosis had occurred and one where it did not) from April to November 2018. The gene of E. leei was detected at levels greater than 10 cells/L in the culture water where enteromyxosis occurred from July to September. Furthermore, 2 months after the detection in the water, the parasite gene (with more than 5,000 cells per 100 mg) was detected in fish intestine samples. However, in the fish farms where enteromyxosis had not occurred, the E. leei gene was detected at <10 cells in culture water (1 L) and fish intestine samples (100 mg). The quantification method used in this research provides a baseline of the infection timeline in olive flounder to develop effective management practices., (© 2021 John Wiley & Sons Ltd.)

The present study describes a new coelozoic, eukaryotic microparasite of the genus Ellipsomyxa Køie, 2003 (Ceratomyxidae: Myxozoa) found parasitizing the gallbladder of Satanoperca jurupari Heckel, 1840 collected in the Curiaú River Environmental Protection Area in Macapá, Amapá state, Brazil. The fish were collected using mesh cast net. The gallbladders were examined, preserved in 80% alcohol for molecular analysis (SSU rDNA gene), and fixed in Davidson for histological slide preparation. The new parasite had a prevalence of 81% in the gallbladder, asymmetric plasmodia, irregular free spores in the bladder fluid, with no cyst formation. The spores are elliptical, with characteristics of the genus Ellipsomyxa, and they had a mean length of 10.11 (8.56-10.5) μm, mean width of 7.81 (5.96-9.56) μm, and thick walls. The polar capsules are sub-spherical in shape, slightly asymmetrical, with a mean length of 3.12 (2.31-3.99) μm and mean width of 2.5 (2.22-2.95) μm, containing polar filament with five or six coils perpendicular to the longitudinal axis of the capsule. The Bayesian Inference assigned the new species to a subclade formed by a lineage of Ellipsomyxa species from the Amazon region. Ellipsomyxa tucujuensis n. sp. is the sixth species of this genus described in fish from the Amazon region, and the first for the state of Amapá., (Copyright © 2021. Published by Elsevier B.V.)

Turkey has more than 200 endemic freshwater fish species, one of which is the Ankara nase, Chondrostoma angorense Elvira, 1987 (Cypriniformes: Leuciscidae), a food fish in northern Turkey. Like most endemic fish species in Turkey, its myxosporean parasite fauna (Cnidaria: Myxosporea) are not yet described. We surveyed twenty C. angorense from Lâdik Lake in northern Turkey, and identified two myxosporean parasites from gills of these fish: Myxobolus arrabonensis Cech, Borzák, Molnár, Székely, 2015, and a co-infection of a novel species, Myxobolus polati sp. nov. We characterized both infections based on myxospore morphology, morphometry, tissue tropism, small subunit ribosomal DNA sequence and phylogenetic analysis. Plasmodia of both species were observed in gills, but had distinct tropism: M. arrabonensis is an intrafilamental vascular type, and M. polati sp. nov. is an intralamellar vascular type. We identified M. arrabonensis on the basis of myxospore characters and 100% similarity to the type DNA sequence from the closely-related host C. nasus. The small subunit ribosomal DNA sequence of M. polati sp. nov. (1946 base pairs; GenBank Accession number MH392318) had a maximum similarity of 98% with any Myxobolus sp. from other Eurasian cypriniforms. Phylogenetic analysis revealed that M. polati sp. nov. is most closely related to gill-infecting Myxobolus diversicapsularis from Rutilus rutilus (L.). The present study is the first record of myxosporean species infecting C. angorense comprising a novel species, M. polati sp. nov. and a known species M. arrabonensis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Proliferative kidney disease (PKD) is a widespread temperature-dependent disease in salmonids caused by the myxozoan parasite, Tetracapsuloides bryosalmonae (Canning, Curry, Feist, Longshaw et Okamura, 1999) (Tb). Tb has a two-host life cycle, involving fish as an intermediate host and freshwater bryozoans as the definitive host. Although salmonids are acknowledged as hosts for the parasite, it is less clear which fish species are active hosts in the life cycle of Tb. Differences in infection dynamics have been observed between some fish species, which are thought to be related to the existence of two main Tb-strains, the American and European. Iceland, having three species of indigenous salmonids and positioned geographically between Europe and North America, is an ideal location to study the natural development of Tb in wild fish. The main aim of this study was to determine the genetic origin of Tb in Iceland and confirm whether mature spores are produced in Icelandic salmonids. In this study, Icelandic salmonids were infected with the European Tb-strain. In situ hybridisation revealed that intraluminal sporogonic stages, including mature spores, were commonly observed in all three salmonid species. The presence of intraluminal stages has previously been confirmed in brown trout Salmo trutta Linnaeus and Atlantic salmon S. salar Linnaeus in Europe, but they have only been observed in Arctic charr Salvelinus alpinus (Linnaeus) in North America, infected by the local strain. This is, therefore, the first time that sporogonic stages have been observed in Arctic charr in Europe, where fish are infected with the European Tb-strain. Our data strongly suggest that all the three salmonid species inhabiting Icelandic waters serve as active hosts in the life cycle of Tb. However, for full confirmation, transmission trials are needed.

Proliferative kidney disease (PKD) is a disease found in salmonid fish that is widespread in Europe and North America. The dependency of the clinical signs on the water temperature is extensively reported in rainbow trout, but detailed information on brown trout is lacking. In this study, juvenile brown trout were exposed to the spores of Tetracapsuloides bryosalmonae and then kept at different ambient water temperatures (16°C, 19°C and 22°C) for 10 weeks along with recording of morbidity throughout the experiment. At 6, 8 and 10 weeks post-exposure, fish from each temperature group were sampled and underwent pathoanatomical examination to survey disease progression. At 16°C, brown trout showed a significantly higher survival probability compared to those kept in 19°C and 22°C water. Additionally, the parasitic burden (MSQ) was higher and the clinical signs were more pronounced in the brown trout kept at 19°C and 22°C compared with the ones kept at 16°C. This study highlights the correlation of PKD outbreaks and water temperature increases related to global climate change, which will impact the future distribution of brown trout in natural waters., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Global climate change is altering the abundance and spread of various parasites, which has important consequences not only for host-parasite interactions but also for the relationships between different host species. Here, we focus on the myxozoan endoparasite Tetracapsuloides bryosalmonae that causes temperature-dependent proliferative kidney disease (PKD) in salmonids. We characterized the temporal changes in the parasite load and the severity of PKD signs (renal hyperplasia, haematocrit) in two sympatric populations of wild brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). We found that both the parasite load and disease signs vary considerably between individuals, species, rivers and sampling periods. We showed that Atlantic salmon was able to slow down the initial parasite proliferation rate and subsequently tolerate high parasite burden without obvious disease signs. In contrast, the initial parasite proliferation rate was much higher in brown trout, which was followed by the development of severe PKD signs. Thus, the speed of parasite proliferation, rather than the absolute number of the parasites in the host kidney, may play an important role in interspecific variation in PKD susceptibility. To conclude, this study illustrates the usefulness of temporal perspective for understanding host defence mechanisms and climate change-mediated impacts in the wild., (© 2021 John Wiley & Sons Ltd.)

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 μm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) μm, body width 6.7 ± 0.3 (6.1-7.6) μm, thickness 5.1 ± 0.2 (4.8-5.4) μm, caudal appendage length 10.0 ± 1 (8.3-11.6) μm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) μm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) μm long and 1.9 ± 0.2 (1.5-2.3) μm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

Lumpfish ( Cyclopterus lumpus ), a North Atlantic "cleaner" fish, is utilized to biocontrol salmon louse ( Lepeophtheirus salmonis ) in Atlantic salmon ( Salmo salar ) farms. Lumpfish require excellent vision to scan for and eat louse on salmon skin. The lumpfish eye immune response to infectious diseases has not been explored. We examined the ocular response to a natural parasite infection in wild lumpfish and to an experimental bacterial infection in cultured lumpfish. Cysts associated with natural myxozoan infection in the ocular scleral cartilage of wild adult lumpfish harbored cells expressing cluster of differentiation 10 (CD10) and immunoglobulin M (IgM). Experimental Vibrio anguillarum infection, which led to exophthalmos and disorganization of the retinal tissues was associated with disruption of normal CD10 expression, CD10 + cellular infiltration and IgM expression. We further describe the lumpfish CD10 orthologue and characterize the lumpfish scleral skeleton in the context of myxozoan scleral cysts. We propose that lumpfish develop an intraocular response to pathogens, exemplified herein by myxozoan and V. anguillarum infection involving novel CD10 + cells and IgM + cells to contain and mitigate damage to eye structures. This work is the first demonstration of CD10 and IgM expressing cells in a novel ocular immune system component in response to disease in a teleost., (Copyright © 2020 Gendron, Paradis, Ahmad, Kao, Boyce, Good, Kumar, Vasquez, Cao, Hossain, Chakraborty, Valderrama and Santander.)

Proliferative kidney disease (PKD) is an emerging disease of salmonids, which is exacerbating with increasing water temperature. Its causative agent, the myxozoan parasite Tetracapsuloides bryosalmonae, exploits freshwater bryozoans as primary hosts and salmonids as intermediate hosts. Our experiments showed that the manipulation of exposure concentrations of infective malacospores had relatively minor impacts for the disease outcomes in the fish host. In this study, brown trout (Salmo trutta) were exposed to three different exposure concentrations of T. bryosalmonae malacospores: (a) a single low parasite concentration (LC), (b) a single high parasite concentration (HC) and (c) three times a low concentration (repeat exposure, RE). Parasite dynamics in the fish host and release of fish malacospores were quantified and fish kidney histopathology was evaluated to determine PKD pathogenesis. Infection prevalence was always lower in the LC group than in the other groups over the course of the study. While the parasite proliferation phase was slower in the LC group, the maximum parasite burden did not differ significantly amongst treatments. The onset of fish malacospore release (day 45 post-exposure), indicated by detection of T. bryosalmonae DNA in the tank water, occurred at the same time point for all groups. Reduced intensity of kidney pathological development was observed in the LC treatment indicating lower disease severity. While the LC treatment resulted in reduced outcomes across several infection parameters (infection prevalence, parasite proliferation, total fish malacospores released), the overall differences were small. The RE and HC treatment outcomes were for most parameters comparable. Our results suggest that repeated exposure, as is likely to occur in the wild during the summer months, might play a more important role in the dynamics of PKD as an emerging infectious disease than the actual concentration of spores., (© 2020 Blackwell Verlag GmbH.)

Transmission paths in the distribution of proliferative kidney disease (PKD) of salmonids are still largely unknown. In this study, the role of goosander (Mergus merganser) as possible transport host for Tetracapsuloides bryosalmonae through faeces was examined. Goosander fledglings were fed exclusively with diseased brown trout (Salmo trutta fario). In all trout used for feeding, intratubular sporogonic stage of the parasite was confirmed histologically. Between one to 10 hours post-feeding, the goosander faeces were sampled and tested for T. bryosalmonae DNA. In qPCR, only DNA fragments were found, and in conventional PCR, no amplification was confirmed. Therefore, we hypothesize that the role of goosander as transport hosts for T. bryosalmonae via their faeces can be neglected., (© 2020 Blackwell Verlag GmbH.)

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.

An infection model for sharpsnout seabream Diplodus puntazzo (Walbaum) challenged with the myxosporean Enteromyxum leei (Diamant, Lom et Dyková, 1994), resembling the natural infection conditions, was used to evaluate the antiparasitic efficacy of a functional diet. Fish of an average weight of 12.5 ± 1.2 g were delivered either a functional (included as feed supplement at 0.3% levels) or a control extruded diet. After four weeks of administration of the experimental diets, fish were challenged with the parasites (cohabitation with infected donors; donor: recipient ratio 1 : 1). The experiment was terminated four weeks after the start of the challenge. At the end of the experiment, growth and feeding (specific growth rate and feed efficiency), as well as immunological parameters (respiratory burst activity, antibacterial activities, hemoglobin concentration, anti-protease activity and ceruloplasmin activity) were measured along with cumulative mortality and total parasitic count in the gut. No significant difference was evident with regard to growth and feeding performance, mortality, gut parasitic load or immunological parameters as the parasitical challenge significantly affected both the performance of the control and functional diet fed fish. However, there was a less prominent impact on antibacterial, anti-protease and ceruloplasmin activity in fish fed with the functional diet. Overall, the present study validated the experimental cohabitation infection model and evaluated the efficacy of a functional ingredient as an antiparasitic agent, showing some potential effects on the fish immune response.

Proliferative kidney disease (PKD), caused by the myxozoan Tetracapsuloides bryosalmonae, is one of the most serious parasitic diseases of salmonids in which outbreaks cause severe economic constraints for the aquaculture industry and declines of wild species throughout Europe and North America. Given that rainbow trout (Oncorhynchus mykiss) is one of the most widely farmed freshwater fish and an important model species for fish immunology, most of the knowledge on how the fish immune response is affected during PKD is from this organism. Once rainbow trout are infected, PKD pathogenesis results in a chronic kidney immunopathology mediated by decreasing myeloid cells and increasing lymphocytes. Transcriptional studies have revealed the regulation of essential genes related to T-helper (Th)-like functions and a dysregulated B-cell antibody type response. Recent reports have discovered unique details of teleost B-cell differentiation and functionality and characterized the differential immunoglobulin (Ig)-mediated response. These studies have solidified the rainbow trout T. bryosalmonae system as a sophisticated disease model capable of feeding key advances into mainstream immunology and have contributed essential information to design novel parasite disease prevention strategies. In our following perspective, we summarize these efforts to evaluate the immune mechanisms of rainbow trout during PKD pathogenesis., (© 2020 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.)

Aims: As the most abundant cell population in the blood, erythrocytes represent an attractive source of nutrients and a protective niche to a number of pathogens. Previously, we observed the attachment of the myxozoan parasite Sphaerospora molnari to erythrocytes of its host, common carp (Cyprinus carpio), raising a number of questions about the nature of this interaction., Methods and Results: We elucidated the impact of S molnari on the number of erythrocytes in healthy and immunocompromised fish, over a period of 6 weeks. While we observed only a mild decrease in RBC numbers in healthy individuals, we witnessed gradual and finally severe haemolytic anaemia in immunosuppressed fish. Accompanying this overt loss was increased erythropoiesis as represented by an increase of erythroblasts in the blood. In vitro, we demonstrated the uptake of host proteins from CFSE-labelled erythrocytes, ultimately inducing death of host RBCs, likely for nutrient gain of the parasite. Nevertheless, the results do not exclude a possible role of erythrocyte-derived proteins in immune evasion., Conclusion: Overall, the obtained data provide first evidence for the previously unknown appetite of myxozoan parasites for host erythrocytes and create an important framework for future investigations into the molecular mechanisms underlining this interaction., (© 2019 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.)

Myxozoa (phylum Cnidaria) are a diverse group of metazoan parasites that predominately infect fish. Little is known regarding the composition and physiology of their myxospore life stage. The objective of this work was to investigate the composition of myxospores and extrasporogonic stages of nine myxozoan species infecting various teleost fish using histochemical staining techniques. Thirty histochemical stains were applied to formalin-fixed, paraffin-embedded tissues processed routinely for light microscopic evaluation. The polar capsules were the most consistent stain target across the taxa examined. Polar capsule staining with Alizarin red, von Kossa and methyl green-pyronin suggests the presence of intracapsular calcium and phosphate, which may contribute to polar filament discharge or pathogenesis of host invasion. The shell valves and suture lines of most myxozoans were stained with Luna and phosphotungstic acid haematoxylin stains, consistent with the presence of chitin and microfibrils, respectively. Vacuoles were consistently highlighted by diastase-susceptible periodic acid-Schiff and Grocott's methenamine silver staining, indicating glycogen. Other histochemical stains exhibited inconsistent staining across the taxa, suggesting differences in myxospore composition potentially reflective of physiologic variations and tissue tropisms. This work provides some information on conserved features and taxa-associated composition of myxospores and lends insight into myxozoan physiology and host-parasite interactions., (© 2020 John Wiley & Sons Ltd.)

Kudoa septempunctata , a myxosporean parasite infecting the trunk muscles of olive flounder ( Paralichthys olivaceus ), is reported to cause food poisoning in humans. The molecular mechanisms underlying the toxicity of K. septempunctata spores remain largely unknown. In the present study, we examine the molecular basis of such toxicity using DNA microarray analysis of K. septempunctata -inoculated human colon adenocarcinoma cells (Caco-2). We observed that the transepithelial resistance of the K. septempunctata -inoculated Caco-2 cell monolayers decreased markedly. DNA microarray analysis revealed that the mRNA expression profiles of control and inoculated cells clearly differed. Inflammatory and bacteria-related pathways, such as interleukin-8 (IL-8) production and MAPK/NF-kappa B pathway, were enriched. The concentrations of IL-8 and serotonin (5-HT) were higher in inoculated cells than in controls. K. septempunctata invasion damages the human intestinal epithelium, causing increased production of IL-8 and 5-HT, which likely results in the vomiting associated with K. septempunctata invasion. Abbreviations : AP-1: activator protein 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; ENS: enteric nervous system; FARMS: Factor Analysis for Robust Microarray Summarization; FDR: false discovery rate; GO: Gene Ontology; 5-HT: 5-hydroxytryptamine; IL-8: Interleukin-8; KEGG: Kyoto Encyclopedia of Genes and Genomes; K. septempunctata: Kudoa septempunctata ; NF-kappa B: nuclear factor-kappa B; TJ: tight junction; TER: transepithelial electrical resistance.

Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish-to-fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities., (© 2020 John Wiley & Sons Ltd.)

The myxozoan genus Unicapsula Davis, 1924 (Myxosporea: Multivalvulida: Trilosporidae) is characterized as having one functional polar capsule (PC) and two rudimentary PCs in a three-valved myxospore. The plasmodia of Unicapsula spp. grow either in the myofibres or in the gills, oesophageal walls and urinary organs of marine fish. Few studies have investigated the taxonomy of Unicapsula spp. including the type species Unicapsula muscularis. Accordingly, the taxonomy of the genus was explored in the present study by using 15 new isolates of seven Unicapsula spp. (U. muscularis, U. galeata, U. andersenae, U. pyramidata, U. pflugfelderi, and two new species) that had formed pseudocysts in the trunk myofibres of commercial fish collected in southern China and Japan from November 2015 to January 2019. Two new species Unicapsula trigona n. sp., and Unicapsula motomurai n. sp. exhibited unique myxospore morphologies (semi-triangular and spherical myxospores, respectively) and 18S and 28S rDNA sequences that were distinct from those of the other Unicapsula spp. Phylogenetic analysis of the 18S and 28S rDNA sequences confirmed the monophyletic status of Unicapsula.

The myxozoan parasite Ceratonova shasta is an important pathogen that infects multiple species of Pacific salmonids. Ongoing genetic surveillance has revealed stable host-parasite relationships throughout the parasite's endemic range. We applied Bayesian phylogenetics to test specific hypotheses about the evolution of these host-parasite relationships within the well-studied Klamath River watershed in Oregon and California, USA. The results provide statistical support that different genotypes of C. shasta are distinct lineages of one species, which is related to two other Ceratonova species in the same ecosystems; Ceratonova X in speckled dace and C. gasterostea in threespine stickleback. Furthermore, we found strong support for the hypothesis that C. shasta type 0 in native steelhead trout and type I in Chinook salmon each evolved with a specialist host adaptation strategy, while C. shasta type II in coho salmon resulted from a generalist host adaptation strategy. Inferred date and host species of the most recent common ancestor of extant Klamath basin types indicate that it occurred between 14,000 and 21,000 years ago, and most likely infected a native steelhead or rainbow trout host., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host-parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host-parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture., (© 2020 John Wiley & Sons Ltd.)

Myxozoans of the family Ceratomyxidae are common coelozoic parasites of marine, anadromous, and freshwater fish, and may also be found, less frequently, parasitizing the tissue of these hosts. The diversity and ecology of the freshwater species of the genus Ceratomyxa have been poorly investigated, leading to a knowledge gap that restricts the understanding of the distribution and prevalence of this group of parasites. In the present study, parasites were found inside vermiform plasmodia, characterised by oscillatory movements in the characiform species Hemiodus unimaculatus. The crescent-shaped and elongated spores, perpendicular to the suture line, have a mean length of 28.9 ± 2.7 μm and width of 2.6 ± 0.1 μm, with two symmetrical oval polar capsules, 1.9 ± 0.3 μm in length and 1.7 ± 0.2 μm in width, containing polar filaments with three or four coils, located near the central suture, with symmetrical lateral elongations 14.3 ± 1.1 μm in length and binucleate amoeboid sporoplasm. The integrated comparative analysis of the morphological characteristics and partial SSU rRNA sequences supported the identification of a new species of coelozoic Ceratomyxa, found in the gallbladder of H. unimaculatus, from the Tocantins basin, in the municipalities of Estreito and Imperatriz in eastern Brazilian Amazonia.The new species was denominated Ceratomyxa fonsecai n. sp.

Six types of sphaeractinomyxon are reported from the coelomic cavity of oligochaetes collected from the Minho River estuary in northern Portugal. Four new types are morphologically and molecularly described from freshwater species belonging to the genera Psammoryctides Hrabě and Potamothrix Vejdovský et Mrázek in the upper estuary, thus significantly increasing the number of known freshwater sphaeractinomyxon. In the lower estuary, sphaeractinomyxon types 8 and 10 of Rangel et al. (2016) are recorded infecting the marine oligochaete Tubificoides pseudogaster (Dahl). A single specimen of T. pseudogaster further displayed infection by one of the four new types found in the upper estuary, suggesting the involvement of sphaeractinomyxon in the life cycles of myxosporean species that infect migratory fish hosts. The acquisition of these second hosts is proposed to have allowed the myxosporean counterparts of sphaeractinomyxon to cross environmental barriers and conquer new habitats. Phylogenetic analyses of the SSU rRNA gene reveal the four new types clustering within the monophyletic clade of mugiliform-infecting myxobolids, strengthening the previously proposed involvement of the sphaeractinomyxon collective group in the life cycles of this specific group of myxosporeans. Endocapsa types also cluster within the latter clade, having actinospores that differ from those of sphaeractinomyxon only in the presence of valvular swellings that do not change when in contact with water. In this study, however, one type was found displaying actinospores with and without valvular swellings in the same oligochaete specimen. This overlap in actinospore morphology is given as grounds for the demise of the endocapsa collective group.

Infection of fish gills by Henneguya causes greater contact between the secondary gill lamellae, thereby giving rise to decreased absorption surface area at the end of the filaments. This ectoparasite can cause damages on the gills infected fish. In the present study, fresh gills of Metynnis lippincottianus were analyzed using optical microscopy techniques. The myxoporean Henneguya sp. was found to be infecting 80% of the gills of this host fish. Presence of this parasite caused hyperplasia and fusion of the gill lamellae, but without inflammation in the parasitized organ.

An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.

Freshwater fish are threatened by the cumulative impact of multiple stressors. The purpose of this study was to unravel the molecular and organism level reactions of rainbow trout, Oncorhynchus mykiss, to the combined impact of two such stressors that occur in the natural habitat of salmonids. Fish were infected with either the myxozoan parasite, Tetracapsuloides bryosalmonae, which causes proliferative kidney disease (PKD), or exposed to ethinylestradiol (EE2) an estrogenic endocrine disrupting compound, or to a combination of both (PKD × EE2). PKD is a slow progressive chronic disease here we focused on a later time point (130-day post-infection (d.p.i.)) when parasite intensity in the fish kidney has already started to decrease. At 130 d.p.i., RNA-seq technology was applied to the posterior kidney, the main target organ for parasite development. This resulted with 280 (PKD), 14 (EE2) and 444 (PKD × EE2) differentially expressed genes (DEGs) observed in the experimental groups. In fish exposed to the combination of stressors (PKD × EE2), a number of pathways were regulated that were neither observed in the single stressor groups. Parasite infection, alone and in combination with EE2, only resulted in a low intensity immune response that negatively correlated with an upregulation of genes involved in a variety of metabolic and inflammation resolution processes. This could indicate a trade-off whereby the host increases investment in recovery/resolution processes over immune responses at a later stage of disease. When PKD infection took place under simultaneous exposure to EE2 (PKD × EE2), parasite intensity decreased and pathological alterations in the posterior kidney were reduced in comparison to the PKD only condition. These findings suggest that EE2 modulated these response profiles in PKD infected fish, attenuating the disease impact on the fish., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1β, high il10 and low hsp90α expression)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate-buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease., (© 2018 John Wiley & Sons Ltd.)

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Proliferative kidney disease (PKD) of salmonids is a disease of economic and environmental concern caused by the myxozoan parasite Tetracapsuloides bryosalmonae. Finer details of the immune repertoire during T. bryosalmonae infection have been elucidated in rainbow trout (Oncorhynchus mykiss). In contrast, there remain many unanswered questions regarding the immune response of the wild fish host in Europe, the brown trout (Salmo trutta) to this parasite. The first aim of this study is to examine the brown trout immune response to T. bryosalmonae and compare it with the published information on rainbow trout as two species that have undergone a different coevolution with the parasite. According to ecoimmunology terminology, infected organisms may manage infection by reducing the damage caused by parasites (tolerance) or by limiting parasite burden (resistance). The second aim of this study is to investigate tolerance/resistance patterns of these species during PKD infection. Our results suggest subtle differences in sequential aspects of the immune response and of immune genes that correlate with parasite intensity for the brown trout, in contrast to rainbow trout, in terms of the B cell response and Th-like interplay that may be linked to PKD pathogenesis. These differences in the immune response also correlate with species-specific differences in tolerance/resistance patterns, in that brown trout had increased tolerance but rainbow trout had greater resistance to infection. The variance in tolerance/resistance investment resulted in a different evolutionary outcome for each host-parasite interaction. A greater exploration of these concepts and an association of immune mechanisms could open an additional gateway for interpreting fish host-parasite interactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Parasites are often hidden in their hosts and exhibit patchy spatial distributions. This makes them relatively difficult to detect and sample. Consequently we have poor knowledge of parasite diversities, distributions, and extinction. We evaluate our general understanding of parasite diversity and highlight the enormous bias in research on parasites such as helminths and arthropods that infect vertebrate hosts. We then focus on Myxozoa as an exemplary case for demonstrating uncharted parasite diversity. Myxozoans are a poorly recognized but speciose clade of endoparasitic cnidarians with complex life cycles that have radiated to exploit freshwater, marine, and terrestrial hosts by adopting strategies convergent to those of parasitic protists. Myxozoans are estimated to represent some 20% of described cnidarian species-greatly outnumbering the combined species richness of scyphozoans, cubozoans, and staurozoans. We summarize limited understanding of myxozoan diversification and geographical distributions, and highlight gaps in knowledge and approaches for measuring myxozoan diversity. We close by reviewing methods and problems in estimating parasite extinction and concerns about extinction risks in view of the fundamental roles parasites play in ecosystem dynamics and in driving host evolutionary trajectories.

The distribution of the freshwater myxozoan parasite Ceratonova shasta in the Pacific Northwest of North America is limited to overlap in the ranges of its 2 hosts: the polychaete Manyunkia sp., and Pacific salmonids. Studies in the Klamath River (Oregon/California) and Deschutes River (Oregon), showed that the parasite population is comprised of multiple sympatric genotypes, some of which correlate with particular salmonid host species and with differences in clinical disease in those hosts. The 3 primary genotypes O, I, and II are defined by the number of a specific tri-nucleotide repeat in the internal transcribed spacer-1 region. To understand the spatial extent of host-parasite genotype patterns, we sequenced the parasite from 448 salmonid fishes from river basins in California, Oregon, Washington, Idaho, and British Columbia, Canada. We sampled intestinal tissues from 6 species of salmon and trout, both those that exist naturally with the parasite (sympatric) and those that do not naturally co-occur with the parasite and were exposed artificially in cages (allopatric). In most river basins we detected the same primary C. shasta genotypes that were described from the Klamath and Deschutes rivers, and we did not detect any novel primary genotypes. Host-parasite genotype patterns were consistent with previous data: genotype O was found in sympatric trout only; genotype I predominantly in Chinook salmon, and genotype II in all 6 fish species but dominant in coho salmon. Our findings of widespread, consistent host-parasite genotype patterns support the hypothesis that C. shasta has a long evolutionary history with salmonid fishes in the Pacific Northwest, and impels additional studies to determine if these parasite genotypes should be considered different species.

Myxozoans are endoparasites with complex life cycles that alternate between invertebrate and vertebrate hosts. Though considered protozoans for over 150 years, they are now recognized as metazoans, given their multicellularity and ultrastructural features. In recognition of synapomorphies and cnidarian-specific genes, myxozoans were placed recently within the phylum Cnidaria. Although they have lost genetic and structural complexity on the path to parasitism, myxozoans have retained characteristic cnidarian cnidocysts, but use them for initiating host infection. Myxozoans represent at least 20% of phylum Cnidaria, but as a result of rapid evolution, extensive diversification and host specialization, they are probably at least as diverse as their free-living relatives. The ability of myxozoans to infect freshwater, marine and terrestrial hosts implies that Cnidaria are no longer constrained to the aquatic environment., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Backgroud: Enteromyxum leei is a myxozoan parasite that produces a slow-progressing intestinal disease. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestine. The aim of the present study was to gain insights into fish T cell responses in the gilthead sea bream-E. leei infection model using a PCR-array with 30 signature molecules for different leukocyte responses in head kidney, spleen, anterior and posterior intestine., Results: The PCR-array results suggest that E. leei induced migration of T cells from head kidney to intestines where T H1 , CTL and T H17 profiles were activated and kept in balance by the upregulation of regulatory cytokines. These results were partially validated by the use of cross-reacting antibodies and BrdU immunostaining to monitor proliferation. Zap70 immunostaining supported the increased number of T cells in the anterior intestine detected by gene expression, but double staining with BrdU did not show active proliferation of this cell type at a local level, supporting the migration from lymphohaematopoietic tissues to the site of infection. Global analyses of the expression profiles revealed a clear separation between infected and exposed, but non-infected fish, more evident in the target organ. Exposed, non-infected animals showed an intermediate phenotype closer to the control fish., Conclusions: These results evidence a clear modulation of the T cell response of gilthead sea bream upon E. leei infection. The effects occurred both at local and systemic levels, but the response was stronger and more specific at the site of infection, the intestine. Altogether, this research poses a promising basis to understand the response against this important parasite and establish effective preventive or palliative measures.

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

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site., (© 2017 John Wiley & Sons Ltd.)

The relationships between parasites and their hosts are intimate, dynamic and complex; the evolution of one is inevitably linked to the other. Despite multiple origins of parasitism in the Cnidaria, only parasites belonging to the Myxozoa are characterized by a complex life cycle, alternating between fish and invertebrate hosts, as well as by high species diversity. This inspired us to examine the history of adaptive radiations in myxozoans and their hosts by determining the degree of congruence between their phylogenies and by timing the emergence of myxozoan lineages in relation to their hosts. Recent genomic analyses suggested a common origin of Polypodium hydriforme, a cnidarian parasite of acipenseriform fishes, and the Myxozoa, and proposed fish as original hosts for both sister lineages. We demonstrate that the Myxozoa emerged long before fish populated Earth and that phylogenetic congruence with their invertebrate hosts is evident down to the most basal branches of the tree, indicating bryozoans and annelids as original hosts and challenging previous evolutionary hypotheses. We provide evidence that, following invertebrate invasion, fish hosts were acquired multiple times, leading to parallel cospeciation patterns in all major phylogenetic lineages. We identify the acquisition of vertebrate hosts that facilitate alternative transmission and dispersion strategies as reason for the distinct success of the Myxozoa, and identify massive host specification-linked parasite diversification events. The results of this study transform our understanding of the origins and evolution of parasitism in the most basal metazoan parasites known., (© 2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

A suspected new enteric Sphaerospora species was believed to be directly associated with the mass mortality of yearling groupers of Epinephelus spp. in South China. The epizootic generally emerged from late September to late April of the following year. The infection prevalence and mortality rate were significantly negatively correlated with fish size. Clinical signs included anorexia, cachexia and extrusion of white pulp-like substance from anus after gentle pressure on the abdomen. Upon necropsy, severe intestinal oedema, thin and transparent intestinal wall, swollen spleen, kidney and gall bladder could be observed. Wet preparation of the infected samples showed large amount of typical disporous plasmodia of the genus Sphaerospora, but no mature spores were observed. Epidemiological investigation showed that this parasite exclusively infected Epinephelus groupers. Histopathologically, this species mainly infected the epithelium of intestine and kidney tubules and caused severe epithelia sloughing and the collapse of intestinal villus. Interestingly, this enteric myxosporidiosis did not cause severe emaciation of infected fish for mass mortality usually emerged within 2-3 days after appearance of clinical signs. The species was most genetically related to Sphaerospora fugu (89% sequence identity) and phylogenetically positioned within marine Sphaerospora lineage. This is the first report of enteric sphaerosporosis of groupers., (© 2017 John Wiley & Sons Ltd.)

Climate change, in particular rising temperature, is suspected to be a major driver for the emergence of many wildlife diseases. Proliferative kidney disease of salmonids, caused by the myxozoan Tetracapsuloides bryosalmonae, was used to evaluate how temperature dependence of host-parasite interactions modulates disease emergence. Brown trout (Salmo trutta fario) kept at 12 and 15 °C, were experimentally infected with T. bryosalmonae. Parasite development in the fish host and release of spores were quantified simultaneously to unravel parasite transmission potential from the vertebrate to the invertebrate host. A change to a stable plateau in infection intensity of the kidney coincided with a threshold at which spore shedding commenced. This onset of parasite release was delayed at the low temperature in accordance with reaching this infection intensity threshold, but the amount of spores released was irrespective of temperature. The production of parasite transmission stages declined with time. In conclusion, elevated temperature modifies the parasite transmission opportunities by increasing the duration of transmission stage production, which may affect the spread and establishment of the parasite in a wider range of rivers.

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature-dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set-up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post-exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post-exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species., (© 2017 John Wiley & Sons Ltd.)

Tetracapsuloides bryosalmonae is a malacosporean parasite and the causative agent of proliferative kidney disease (PKD) that seriously impacts farmed and wild salmonids. The parasite's life cycle includes an invertebrate host, the bryozoan Fredericella sultana, and a vertebrate host, salmonid fish. The persistence of T. bryosalmonae in brown trout Salmo trutta for up to 2 yr following exposure is well documented. Results from the present study confirmed that one brown trout that had recovered from PKD did not completely clear the parasite from its tissues and that T. bryosalmonae could persist in brown trout for up to 5 yr post exposure. Furthermore, recovered infected brown trout can release viable T. bryosalmonae spores that are able to infect specific pathogen-free F. sultana colonies. T. bryosalmonae DNA was detected by PCR in every organ, and parasite stages were observed in the kidney, spleen and liver following immunohistochemistry. This finding indicates that T. bryosalmonae-infected brown trout can act as asymptomatic carriers and release the parasite for several years after the initial infection, acting as a reservoir of infection, and contributing to the dissemination of the parasite to new areas.