Serine protease inhibitors of the whirling disease parasite Myxobolus cerebralis (Cnidaria, Myxozoa): Expression profiling and functional predictions.

Here, we studied the expression pattern and putative function of four, previously identified serine protease inhibitors (serpins) of Myxobolus cerebralis, a pathogenic myxozoan species (Cnidaria: Myxozoa) causing whirling disease of salmonid fishes. The relative expression profiles of serpins were determined at different developmental stages both in fish and in annelid hosts using serpin-specific qPCR assays. The expression of serpin Mc-S1 was similar throughout the life cycle, whereas a significant decrease was detected in the relative expression of Mc-S3 and Mc-S5 during the development in fish, and then in the sporogonic stage in the worm host. A decreasing tendency could also be observed in the expression of Mc-S4 in fish, which was, however, upregulated in the worm host. For the first time, we predicted the function of M. cerebralis serpins by the use of several bioinformatics-based applications. Mc-S1 is putatively a chymotrypsin-like inhibitor that locates extracellularly and is capable of heparin binding. The other three serpins are caspase-like inhibitors, and they are probably involved in protease and cell degradation processes during the early stage of fish invasion.

A new species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) infecting stratum spongiosum of the imperiled sicklefin redhorse, Moxostoma sp. (Cypriniformes: Catostomidae) from the Little Tennessee River, North Carolina, USA.

The sicklefin redhorse, Moxostoma sp. (Cypriniformes: Catostomidae), is an innominate imperiled catostomid endemic to the Hiwassee and Little Tennessee river basins, which has been restricted to a few tributaries of these systems by impoundments. During collections to propagate sicklefin redhorse for reintroduction, a myxozoan, described herein, was observed infecting sicklefin redhorse in the Little Tennessee River Basin, North Carolina. Myxobolus naylori Ksepka et Bullard sp. n. infects the stratum spongiosum covering the scales of sicklefin redhorse. Myxospores of the new species differ from all congeners by the combination of having a mucous envelope, intercapsular process, and sutural markings as well as lacking an iodinophilic vacuole in the sporoplasm. A phylogenetic analysis of the 18S rDNA gene recovered the new species in a polytomy with Myxobolus marumotoi Li et Sato, 2014 and a clade comprised of species of Myxobolus Bütschli, 1882; Thelohanellus Kudo, 1933, and Dicauda Hoffman et Walker, 1973. Histological sections of infected sicklefin redhorse skin revealed myxospores within a plasmodium in the stratum spongiosum dorsal to scales, encapsulated in collagen fibres, and associated with focal erosion of scales directly beneath the plasmodium; in some instances, the scale was perforated by the plasmodium. The specificity of the new species to sicklefin redhorse may make it a useful biological tag to differentiate sicklefin redhorse from morphologically similar species. The new species is the first parasite reported from sicklefin redhorse, a species of concern to the United States Fish and Wildlife Service. No species of Myxobolus has been reported from species of Moxostoma in the Southeast United States. As it was observed that Myxobolus minutus Rosser, Griffin, Quiniou, Alberson, Woodyard, Mischker, Greenway, Wise et Pote, 2016 is a primary junior homonym of Myxobolus minutus Nemeczek, 1911, we propose the replacement name Myxobolus diminutus (Rosser, Griffin, Quiniou, Alberson, Woodyard, Mischker, Greenway, Wise et Pote, 2016).

Myxozoan hidden diversity: the case of Myxobolus pseudodispar Gorbunova, 1936.

Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa) was originally described as a parasite of common roach, Rutilus rutilus (Linnaeus), with developing stages in muscles and spores disseminated in macrophage centres of different organs and tissues. Later, this parasite was described from several other cyprinids, but with relatively large intraspecific differences based on SSU rDNA gene sequences. Within our long-term study on myxozoan biodiversity, we performed a broad microscopic and molecular screening of various freshwater fish species (over 450 specimens, 36 species) from different localities. We investigated the cryptic species status of M. pseudodispar. Our analysis revealed four new unique SSU rDNA sequences of M. pseudodispar as well as an infection in new fish host species. Myxobolus pseudodispar sequence analysis showed clear phylogenetic grouping according to fish host criterion forming 13 well-recognised clades. Using 1% SSU rDNA-based genetic distance criterion, at least ten new species of Myxobolus Bütschli, 1882 may be recognised in the group of M. pseudodispar sequences. Our analysis showed the paraphyletic character of M. pseudodispar sequences and the statistical tests rejected hypothetical tree topology with the monophyletic status of the M. pseudodispar group. Myxobolus pseudodispar represents a species complex and it is a typical example of myxozoan hidden diversity phenomenon confirming myxozoans as an evolutionary very successful group of parasites with a great ability to adapt to a new hosts with subsequent speciation events.

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

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

Transcriptomic analysis of oligochaete immune responses to myxosporeans infection: Branchiura sowerbyi infected with Myxobolus cultus.

The Myxozoa are endoparasites characterized by a two-host life cycle that typically involves invertebrates and vertebrates as definitive and intermediate hosts, respectively. However, little is known about invertebrate-myxosporean interactions, particularly about patterns of host immune defense. We used RNA-sequencing to identify genes that are possibly involved in the immune responses of the oligochaete Branchiura sowerbyi naturally infected with Myxobolus cultus. De novo assembly of the B. sowerbyi transcriptome yielded 119,031 unigenes, with an average length of 896 bp and an N50 length of 1754 bp. Comparative transcriptome analysis revealed 4059 differentially expressed genes (DEGs) between M. cultus-infected and uninfected B. sowerbyi groups, including 3802 upregulated genes and 257 downregulated genes. Among the B. sowerbyi immune factors implicated in the responses to M. cultus infection, DEGs related to lectins, ubiquitin-mediated proteolysis, phagocytosis, oxidative-antioxidative responses, proteases, and protease inhibitors were upregulated. The expression of some immune-related molecules such as calmodulin, heat shock proteins, antimicrobial peptides, lysenin, and serum amyoid A protein were also significantly upregulated. The expression patterns of 14 immune-related DEGs identified by RNA-seq were validated by quantitative real-time polymerase chain reaction. This study is the first attempt to characterize the B. sowerbyi transcriptome and identify immune-related molecules possibly associated with M. cultus infection. It is also the first report of invertebrate host-myxosporean interactions at the transcriptomic level. Our results will facilitate the elucidation of adaptive evolution mechanisms of myxosporean parasites in the definitive host and the genetic basis for differences in resistance of invertebrate hosts of different genotypes to a myxosporean species.

RNA-seq analysis of local tissue of Carassius auratus gibelio with pharyngeal myxobolosis: Insights into the pharyngeal mucosal immune response in a fish-parasite dialogue.

The lack of practical control measures for pharyngeal myxobolosis is becoming an important limiting factor for the sustainable development of the gibel carp (Carassius auratus gibelio) culture industry in China. Myxobolus honghuensis has been identified as the causative agent of this pandemic disease, which exclusively infects the pharynx of gibel carp, a potential important mucosal lymphoid-associated tissue (MLAT). Myxozoa generally initiate invasion through the mucosal tissues of fish, where some of them also complete their sporogonial stages. However, the pharynx-associated immune responses of teleost against myxosporeans infection remain unknown. Here, a de novo transcriptome assembly of the pharynx of gibel carp naturally infected with M. honghuensis was performed for the first time, using RNA-seq. Comparative analysis of severely infected and mildly infected pharyngeal tissues (SI group and MI group) from the same fish individuals and control pharyngeal tissues (C group) from the uninfected fish was carried out to investigate the potential mucosal immune function of the fish pharynx, and characterize the panoramic picture of pharynx local mucosal immune responses of gibel carp against the M. honghuensis infection. A total of 242,341 unigenes were obtained and pairwise comparison resulted in 13,009 differentially-expressed genes (DEGs) in the SI/C group comparison, 6014 DEGs in the MI/C group comparison, and 9031 DEGs in the SI/MI group comparison. Comprehensive analysis showed that M. honghuensis infection elicited a significant parasite load-dependent alteration of the expression of numerous innate and adaptive immune-related genes in the local lesion tissue. Innate immune molecules, including mucins, toll-like receptors, C-type lectin, serum amyloid A, cathepsins and complement components were significantly up-regulated in the SI group compared with the C group. Up-regulation of genes involved in apoptosis signaling pathway and the IFN-mediated immune system were found in the SI group, suggesting these two pathways played a crucial role in innate immune response to M. honghuensis infection. Up-regulation of chemokines and chemokine receptors and the induction of the leukocyte trans-endothelial migration pathways in the severely and mildly infected pharynx suggested that many leucocytes were recruited to the local infected sites to mount a strong mucosal immune responses against the myxosporean infection. Up-regulation of CD3D, CD22, CD276, IL4/13A, GATA3, arginase 2, IgM, IgT and pIgR transcripts provided strong evidences for the presence of T/B cells and specific mucosal immune responses at local sites with M. honghuensis infection. Our results firstly demonstrated the mucosal function of the teleost pharynx and provided evidences of intensive local immune defense responses against this mucosa-infecting myxosporean in the gibel carp pharynx. Pharyngeal myxobolosis was shaped by a prevailing anti-inflammatory response pattern during the advanced infection stages. Further understanding of the functional roles of fish immune molecules involved in the initial invasion and/or final sporogony site may facilitate future development of control strategies for this myxobolosis.

Distinctive site preference of the fish parasite Myxobolus cerebralis (Cnidaria, Myxozoa) during host invasion.

Here, we experimentally studied the site preference of Myxobolus cerebralis, one of the most pathogenic myxozoan (Cnidaria, Myxozoa) fish parasites, which causes whirling disease in salmonids. Parasite invasion was examined in three fish species with various susceptibility levels: the type host brown trout, the highly susceptible rainbow trout, and the non-susceptible gibel carp, in which parasite spores do not develop. We investigated the first two hours of fish invasion, and measured the site preference of triactinomyxons (TAMs) during attachment and penetration of fish in three body parts (gills, fins, skin). Infection prevalence and intensity were estimated using a species-specific nested PCR, optimised in the present study. The highest infection prevalence was detected in the most susceptible fish species, rainbow trout. Interestingly, higher prevalence was observed in gibel carp than in the type host, brown trout (95.2% vs. 85.7%). Considering body locations, remarkable differences were detected in infection intensities. The highest intensity was observed in fins, whereas skin was the least infected body part in every fish species examined. Infection prevalence and intensity did not differ significantly among fish species. Thus, we confirmed that M. cerebralis TAMs cannot discern fish species. Furthermore, we proved experimentally that fish fin is significantly more attractive to fish-invading parasite TAMs than gills or skin.

Susceptibility-related differences in the quantity of developmental stages of Myxobolus spp. (Myxozoa) in fish blood.

Here, we investigated the early development of two closely related myxozoan parasites, the highly pathogenic Myxobolus cerebralis, the causative agent of the whirling disease in salmonids, and Myxobolus pseudodispar, a common, non-pathogenic parasite of cyprinids. The aim of our study was to examine under in vivo laboratory conditions whether fish blood is involved in the intrapiscine development of the two parasite species and investigate if there is dissimilarity between the parasite infection intensity in blood and if it varies in terms of host susceptibility and parasite pathogenicity. Highly susceptible, less susceptible and non-susceptible hosts were involved. Blood samples were taken 1 day, 1 week and 1 month post exposure to M. cerebralis and M. pseudodispar, respectively. The prevalence and infection intensity was estimated by parasite-specific quantitative real-time PCR. Although previous findings assumed that M. cerebralis might escape from host immune system by migrating via peripheral nerves, our experimental results demonstrated that M. cerebralis is present in blood during the early stage of intrapiscine development. For the non-pathogenic M. pseudodispar, the highest infection prevalence was found in the original host, common roach Rutilus rutilus, whereas the highest infection intensity was detected in rudd Scardinius erythrophthalmus, a "dead-end" host of the parasite. The presence of M. pseudodispar developmental stages in the blood of both susceptible and non-susceptible cyprinids suggests that the susceptibility differences remain hidden during the early stage of infection. Our findings supply further evidence that host specificity is not determined during the early, intrapiscine development involving the vascular system. Furthermore, we found remarkable differences in the infection dynamics of the two parasite species examined, possibly due to their distinct pathogenicity or variations in adaptive capabilities to immune components in host blood.

Spatial and temporal variability of myxozoan parasite, Myxobolus inornatus, prevalence in young of the year smallmouth bass in the Susquehanna River Basin, Pennsylvania.

A myxozoan parasite, Myxobolus inornatus, is one disease agent identified in young of the year (YOY) smallmouth bass in the Susquehanna River Basin, Pennsylvania. We investigated spatial and temporal variability in M. Inornatus prevalence across the Susquehanna River Basin and at several out-of-basin sites. We examined potential land use drivers of M. Inornatus prevalence including agricultural and developed land use. In 1,267 YOY smallmouth bass collected from 32 sites during 2013-2016, M. Inornatus was documented in 43.6% of samples. Among-site variability in parasite prevalence was greater than among-year variability. The effect of agricultural land use on M. Inornatus prevalence had a high probability of being positively correlated at multiple spatial scales (probability of positive effect > 0.80). The effect of developed land use on M. Inornatus prevalence had a relatively high probability of being negatively correlated at multiple spatial scales (probability of negative effect > 0.70). Our results suggest that land use practices could be related to M. Inornatus infection of smallmouth bass. Further study will be necessary to determine whether disease dynamics are a consequence of effects on the host, alterations of instream habitat mediating invertebrate host dynamics and/or survival and dispersal of the parasite infective stage.

Histopathological and ultrastructural assessment of two mugilid species infected with myxozoans and helminths.

The histopathology and ultrastructure of the intestine of mullets, Liza ramada and Liza saliens, from Comacchio lagoons (northern Italy) naturally infected with myxozoans and helminths were investigated and described. Sixty-two (80.5%) of 77 mullets harboured one or more of the following parasites species: Myxobolus mugchelo (Myxozoa), Neoechinorhynchus agilis (Acanthocephala), Haplosplanchnus pachysomus and Dicrogaster contractus (Digenea). Co-occurrence of helminths with myxozoans was common. The main damage caused by digeneans was destruction of the mucosal epithelium of the villi, necrosis and degeneration of intestinal epithelial cells. More severe intestinal damage was caused by acanthocephalans which reach the submucosa layer with their proboscis. At the site of helminths infection, several mast cells (MCs), rodlet cells (RCs), mucous cells and few neutrophils and macrophages were observed in the epithelium. RCs and mucous cells exhibited discharge activity in close vicinity to the worm's tegument. M. mugchelo conspicuous plasmodia were encysted mainly in muscle and submucosa layers of the intestine. Indeed, spores of M. mugchelo were documented within the epithelial cells of host intestine and in proximity to MCs. Degranulation of the MCs near the myxozoans was very frequent.

The impact of Tetracapsuloides bryosalmonae and Myxobolus cerebralis co-infections on pathology in rainbow trout.

BACKGROUND: Myxozoan parasites pose emerging health issues for wild and farmed salmonid fish. Rainbow trout (Oncorhynchus mykiss) is a particularly susceptible species to Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of Proliferative Kidney Disease (PKD), and to Myxobolus cerebralis (Myxosporea), the etiological agent of Whirling Disease (WD). The objective of this study was to investigate the impact of myxozoan co-infections on the pathogenesis of PKD and WD in the rainbow trout. METHODS: Two groups of rainbow trout (96 fish each) were primarily infected with T. bryosalmonae and triactinomyxons of M. cerebralis; after 30 days half of the fish in each group were co-infected with these parasites vice versa and remaining half was continued as single infection. Mortalities and clinical signs were recorded at different time points. Histopathology and immunohistochemistry were performed to assess the extent of each infection and estimate the parasite burden between groups. RESULTS: Fish firstly infected with M. cerebralis and co-infected with T. bryosalmonae exhibited exacerbated pathological changes of both parasitic diseases and elicited a higher mortality rate. A higher kidney swelling index (grade 4) appeared together with more severe cartilage destruction and displacement, when compared to the pathological changes in fish upon single infections with T. bryosalmonae or M. cerebralis. Conversely, fish firstly infected with T. bryosalmonae and co-infected with M. cerebralis also exhibited typical pathological changes of both parasitic diseases, but with a lower mortality rate, similar as caused by the single T. bryosalmonae or M. cerebralis infection. WD clinical signs were milder, without skeletal deformities, while kidney swelling index was similar to single infection with T. bryosalmonae (grade 2 to 3). CONCLUSIONS: In this study, a co-infection with myxozoan parasites was for the first time successfully achieved in the laboratory under controlled conditions. The impact of co-infections in concurrent myxozoan infections mainly depends on the primary pathogen infecting the host, which could alter the outcomes of the secondary pathogen infection. The primary M. cerebralis infection followed by T. bryosalmonae had a much more serious impact and elicited a synergistic interaction. Contrasting results were instead seen in rainbow trout primarily infected with T. bryosalmonae and then co-infected with M. cerebralis.

A RNAi-based therapeutic proof of concept targets salmonid whirling disease in vivo.

Myxobolus cerebralis is a cnidarian-myxozoan parasite that causes salmonid whirling disease. M. cerebralis alternates between two hosts: (1) a vertebrate salmonid and (2) an invertebrate oligochaete, Tubifex tubifex. There is no successful treatment for salmonid whirling disease. MyxSP-1 is a M. cerebralis serine protease implicated in whirling disease pathogenesis. We hypothesized that short-interfering RNA (siRNA)-induced RNA interference (RNAi) can silence MyxSP-1 in the invertebrate host and abrogate the M. cerebralis life cycle. This would preclude whirling disease infection in the salmonid host. To test this hypothesis, we first developed a siRNA delivery protocol in T. tubifex. Second, we determined the effective dose for siRNA treatment of M. cerebralis-infected T. tubifex. M. cerebralis-infected T. tubifex were treated with different concentrations of MyxSP-1 or negative control siRNAs (1µM, 2µM, 5µM or 7µM) at 15°C for 24h, 48h, 72h and 96h, respectively. We monitored MyxSP-1 knockdown using real-time quantitative PCR (qPCR). siRNA treatment with MyxSP-1 siRNA at 2µM concentration for 24h at 15°C showed maximum significant MyxSP-1 knockdown in T. tubifex. Third, we determined the time points in the M. cerebralis life cycle in T. tubifex at which siRNA treatment was most effective. M. cerebralis-infected T. tubifex were treated with MyxSP-1 or negative control siRNAs (2µM concentration for 24h at 15°C) at 24 hours post-infection (24hpi), 48hpi, 72hpi, 96hpi, 1 month post-infection (1mpi), 2mpi and 3mpi, respectively. We observed that siRNA treatment of T. tubifex was most effective at 1mpi, 2mpi and 3mpi. Fourth, we immersed specific-pathogen-free rainbow trout fry in water inhabited by MyxSP-1 siRNA-treated T. tubifex (at 1mpi, 2mpi and 3mpi). The salmonids did not develop whirling disease and showed significant MyxSP-1 knockdown. We also observed long-term RNAi in T. tubifex. Together these results demonstrate a novel RNAi-based therapeutic proof of concept in vivo against salmonid whirling disease.

Myxobolus chushi n. sp. (Myxozoa:Myxosporea) parasitizing Schizothorax niger (Heckel), a native cyprinid fish from Wullar Lake in Kashmir Himalayas.

In the study, a new species, Myxobolus chushi n. sp. infecting gills of wild specimens of Schizothorax niger (Heckel) inhabiting Wullar Lake in Kashmir Himalayas, (J&K) India has been described based on morphology of the myxospore and using partial 18S rDNA sequencing. Pathological changes in the gills have been studied with the help of histological sections stained with Luna's method. Twenty fish specimens were examined, out of which four had oval, white plasmodia in gills measuring 2.0×0.5mm. The myxospores were spherical to ovoidal in shape with slightly attenuated posterior end, measuring 11.17±0.23 (10.60-11.40)µm in length and 9.14±0.06 (8.80-9.20)µm in width, having a prominent pore at the anterior end. The polar capsules were pyriform in shape, measuring 4.25±0.15 (4.00-4.40)µm in length and 2.38±0.27 (2.00-2.65)µm in width having polar filaments forming coils up to 5 in number. Parietal folds 9 in number present on the posterior part of the shell. The intensity of infection was recorded to be moderate as indicated by gill plasmodial index (GPI=2). The plasmodium was located in the vascular network occupying whole of the gill lamella therefore typed as intralamellar vascular type, LV3. Analysis of 18S small subunit (SSU) rDNA sequence of the isolate demonstrated 90% homogeneity with M. sp. KLT-2014 infecting scales of Labeo rohita from Myanmar and 89% with M. dermiscalis infecting scales of Labeo rohita from India.

Morphological and ultrastructural characteristics of Myxobolus ridibundae n. sp. (Myxosporea: Bivalvulida) infecting the testicular tissue of the marsh frog Rana ridibunda (Amphibia: Ranidae) in Egypt.

Myxozoans are one of the most economically important groups of protozoan parasites causing many serious diseases of their hosts. In the present study, a total of 60 live adult male specimens of the marsh frog Rana ridibunda have been randomly captured during the period of January-December 2015 in different areas at Kafr El-Sheikh Governorate, Egypt and were examined for infection by myxosporidian parasites. A total of 48 (80.0 %) out of 60 frog specimens were found to be infected with Myxobolus species. Parasitic infection was restricted to the testicular tissue of the examined frogs. Macroscopic cysts (plasmodia) which heavily infested different parts of the testes were recovered. Morphological and ultrastructural characteristics of these myxosporidian species were carried out using light and transmission electron microscopy. Plasmodia measured 0.16-0.53 (0.34 ± 0.01) mm in diameter. Mature spores appeared oval in frontal view, measuring 8.9-11.5 (9.6 ± 0.1) µm in length and 7.5-9.1 (8.4 ± 0.1) µm in width containing 5-6 turns of polar filaments. Morphometric characterization revealed that the very small size of the present Myxobolus species was the most distinctive feature that separates them from all previously described Myxobolus species. Ultrastructural analysis showed that the plasmodia are surrounded by a plasma membrane with numerous pinocytotic protrusions extending toward the host cell. The generative cells and the different developmental stages are arranged at the periphery of the plasmodia, while immature and mature spores are centrally located. Sporogenesis, capsulogenesis, valvogenesis, and spore maturation of the present parasite are also described. The present species is described as Myxobolus ridibundae and represents a new species.

Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift?

Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa) is capable of infecting and developing mature myxospores in several cyprinid species. However, M. pseudodispar isolates from different fish show up to 5% differences in the SSU rDNA sequences. This is an unusually large intraspecific difference for myxozoans and only some of the muscle-dwelling myxozoan species possess such a high genetic variability. We intended to study the correlation between the host specificity and the phylogenetic relationship of the parasite isolates, and to find experimental proof for the putatively wide host range of M. pseudodispar with cross-infection experiments and phylogenetic analyses based on SSU rDNA. The experimental findings distinguished 'primary' and less-susceptible 'secondary' hosts. With some exceptions, M. pseudodispar isolates showed a tendency to cluster according to the fish host on the phylogenetic tree. Experimental and phylogenetic findings suggest the cryptic nature of the species. It is likely that host-shift occurred for M. pseudodispar and the parasite speciation in progress might explain the high genetic diversity among isolates which are morphologically indistinguishable.

Assessment of the Long-Term Viability of the Myxospores of Myxobolus cerebralis as Determined by Production of the actinospores by Tubifex tubifex.

While whirling disease was first observed in Rainbow Trout Oncorhynchus mykiss in 1893, the complete life cycle of Myxobolus cerebralis (Mc), the causative agent of the disease, was not understood until 1984, when it was shown to involve two obligate hosts, a salmonid fish and the aquatic oligochaete Tubifex tubifex (Tt). The viability of the triactinomyxon (TAM) actinospores produced by Tt has been well studied, and is known to be temperature dependent and measured in days and weeks. Assertions that Mc myxospores produced by infected fish remain viable for years or even decades were made during the mid-20th century, decades before the Mc life cycle was described. Moreover, the duration of myxospore viability has not been well studied since the life cycle was elucidated. In a series of time-delay treatments, we assessed the long-term viability of Mc myxospores by exposure to Mc-susceptible Tt oligochaetes and quantified TAM production. As the time delay between inoculation and incubation of Mc myxospores in sand and water and exposure to Tt oligochaetes increased, TAM production decreased exponentially. Production among the 15-d time-delay replicates was reduced 74.7% compared with the 0-d treatment. Likewise, total TAM production was reduced 94.5, 99.4, and 99.9%, respectively, in the 90-, 120-, and 180-d time-delay treatments. Linear regression analysis of our data and the absence of TAM production among replicates of Mc myxospores held at 5°C for 365 d prior to exposure to Mc-susceptible Tt oligochaetes indicate that the long-term viability of Mc myxospores is less than 1 year under the conditions of this study.

Whirling disease revisited: pathogenesis, parasite biology and disease intervention.

Whirling disease (WD) is an ecologically and economically debilitating disease of rainbow trout Oncorhynchus mykiss caused by the actinosporean spores of the parasite Myxobolus cerebralis. M. cerebralis has a complex, 2-host life cycle alternating between salmonid fish and the oligochaete host Tubifex tubifex. The parasite alternates between 2 spore forms as transmission stages: an actinosporean triactinomyxon spore that is produced in the oligochaete host and a myxosporean spore that develops in the salmonid host. Waterborne triactinomyxon spores released from infected T. tubifex oligochaetes attach to the salmonid host by polar filament extrusion elicited by chemical (nucleoside) and mechanical (thigmotropy) stimuli-a process which is rapidly followed by active penetration of the sporoplasms into the fish epidermis. Upon penetration, sporoplasms multiply and migrate via peripheral nerves and the central nervous system to reach the cartilage where they form trophozoites which undergo further multiplication and subsequent sporogenesis. M. cerebralis myxospores are released into the aquatic environment when infected fish die and autolyse, or when they are consumed and excreted by predators. Myxospores released into the water are ingested by susceptible T. tubifex where they develop intercellularly in the intestine over a period of 3 mo through 4 developmental stages to give rise to mature actinospores. In this article, we review our current understanding of WD-the parasite and its alternate hosts, life cycle and development of the parasite in either host, disease distribution, susceptibility and resistance mechanisms in salmonid host and strategies involved in diagnosis, prevention and control of WD.

Parental genetic diversity of brown trout (Salmo trutta m. fario) brood stock affects offspring susceptibility to whirling disease.

BACKGROUND: Whirling disease, caused by the myxozoan parasite Myxobolus cerebralis, has high economical and ecological importance worldwide. Susceptibility to the disease varies considerably among salmonid species. In brown trout (Salmo trutta) the infection is usually subclinical with low mortality, which increases the risk of parasite dissemination, especially when farm fish are used for stocking natural habitats. The influence of intraspecific genetic differences (especially the level of homozygosity) on susceptibility is unknown. Therefore, we examined the possible correlations between parental genetic diversity and offspring susceptibility of brown trout stocks to whirling disease. METHODS: Two brown trout brood stocks from a German and a Hungarian fish farm were genetically characterized using microsatellite and lineage-specific genetic markers. The individual inbreeding coefficient f and pairwise relatedness factor r were estimated based on eight microsatellite markers. Brood stock populations were divided into groups according to low and high f and r value estimates and subjected to selective fertilization. The offspring from these separate groups were exposed to M. cerebralis actinospores, and the infection prevalence and intensity was measured and statistically analysed. RESULTS: The analysis of phylogeographic lineage heritage revealed high heterogeneity in the Hungarian brood stock since > 50% of individuals were Atlantic-Danubian hybrids, while only pure Atlantic-descending specimens were detected in the German population. Based on f msat and r msat estimations, classified non-inbred (NIB), inbred (IB) and a group of closely related fish (REL) were created. The susceptibility of their offspring varied considerably. Although there was no significant difference in the prevalence of M. cerebralis infection, the mean intensity of infection differed significantly between NIB and IB groups. In REL and IB groups, a high variability was observed in infection intensity. No external clinical signs were observed in the exposed brown trout groups. CONCLUSIONS: Our findings indicate that the allelic diversity of brown trout brood stock may constitute a significant factor in disease susceptibility, i.e. the intensity of parasite infection in the subsequent generation.

[THE DESCRIPTION OF MYXOBOLUS PELECICOLA SP. NOV. (MYXOZOA: MYXOBOLIDAE) FROM PELECUS CULTRATUS (CYPRINIFORMES, CYPRINIDAE)].

Morphological study of myxosporean parasites Myxobolus pelecicola sp. n. from Pele- cus cultratus (L., 1758), is presented. Presporogenic stage not observed. Polysporous, el- lipsoidal. plasmodia up to 1.2 mm in length and 0.1 to 0.4 mm in width develop in the ske- letal musculature. Mature spores elongate oval or oval form in frontal view, lens-shaped in sutural view. Length of spores 16.5 ± 0.83 (14.9-18.0), width 12.1 ± 0.63 (11.6-13.3) (n = 50), thickness 7.8 ± 0.3 (7.4-8.3) µm (n = 10). Polar capsules pyriform, unequal, length of large 8.0 ± 0.44 (7.5-8.8), small 7.6 ± 0.26 (7.2-7.8), width of large 4.5 ± 0.22 (4.2-4.7), small 4.0 ± 0.21 (3.8-4.3) µm. Five to 6 distinct filament coils oriented obli- quely to capsule length, found in polar capsule. The length of extruded (in water) filament up to 130 µm. Large, but indistinct triangular intercapsular appendix present in spores. Single rodlet inclusion is very often observed in the posterior part of spore. Mucous enve- lope is absent. The type host of Myxobolus pelecicola sp. n. is sabrefish Pelecus cultratus (L., 1758). Type locality: Finnish Bay (60°05' N 29°55' E) and Ladoga lake (60°07' N 32°19' E), Russia.

Myxobolus cerebralis (Hofer) infection risk in native cutthroat trout Oncorhynchus clarkii (Richardson) and its relationships to tributary environments in the Yellowstone Lake Basin.

Conservation of native species is challenged by the introduction of non-native pathogens and diseases into aquatic and terrestrial environments worldwide. In the Yellowstone Lake basin, Yellowstone National Park, the invasive parasite causing salmonid whirling disease Myxobolus cerebralis (Hofer) has been identified as one factor contributing to population declines of native Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri (Jordan & Gilbert). In 2002 and 2003, we examined relationships between the stream environment and severity of M. cerebralis infection in native trout. Coefficients of variation of environmental features were calculated to examine variability. Ten years later, we reassessed infection levels at 22 tributaries broadly across the system. Results of principal component analysis (PCA) of physical features (2003) were negatively correlated with infection severity, mostly in lower jaw cartilage of cutthroat trout, and PCA of chemical features (and temperature) correlated with infection severity in cranial cartilage. Pelican Creek, where M. cerebralis prevalence and severity was high 2002-2003, remained high in 2012. We did not find evidence that the parasite had dispersed further within the system. Variable environmental features (physiological stress) across short spatiotemporal scales within a stream or season may possibly predispose salmonids to infection in the wild and facilitate parasite establishment.