Molecular studies on the seasonal occurrence and development of five myxozoans in farmed Salmo trutta L.

Five myxozoan species, Tetracapsuloides bryosalmonae, Sphaerospora truttae, Chloromyxum schurovi, Chloromyxum truttae and a Myxobolus species were detected in farmed brown trout, Salmo trutta L. from Central Scotland. Using PCR and in situ hybridization, this study investigated the seasonal occurrence and tissue location of these species in young of the year brown trout. C. schurovi, C. truttae and Myxobolus sp. were first detected in brown trout in April, 2 months before T. bryosalmonae and S. truttae. T. bryosalmonae and S. truttae showed proliferation in the blood with intravascular stages of T. bryosalmonae accumulating in the heart. In contrast, only small amounts of PCR products of C. schurovi and C. truttae were obtained from the blood, suggesting that these species use the vascular system for transport but proliferate only in their target tissues from which large amounts of PCR product were obtained and where parasites were visible in histological sections. Large amounts of PCR product were obtained for T. bryosalmonae, S. truttae and both Chloromyxum species from the gills of brown trout, suggesting the gills as entry locus for these species. The neurotropic Myxobolus species formed plasmodia predominantly in the peripheral nerves, possibly indicating an entry route through the skin. Presporogonic stages of all other species had disappeared by September and mature spores were present from August onwards

Transcriptome of Sphaerospora molnari (Cnidaria, Myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp

Background Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such asSphaerospora molnarireplicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of aS. molnarigenome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identifyS. molnariproteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. Results An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. Conclusions In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study ofS. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change

The myxozoan minicollagen gene repertoire was not simplified by the parasitic lifestyle: computational identification of a novel myxozoan minicollagen gene

Background Lineage-specific gene expansions represent one of the driving forces in the evolutionary dynamics of unique phylum traits. Myxozoa, a cnidarian subphylum of obligate parasites, are evolutionarily altered and highly reduced organisms with a simple body plan including cnidarian-specific organelles and polar capsules (a type of nematocyst). Minicollagens, a group of structural proteins, are prominent constituents of nematocysts linking Myxozoa and Cnidaria. Despite recent advances in the identification of minicollagens in Myxozoa, the evolutionary history and diversity of minicollagens in Myxozoa and Cnidaria remain elusive. Results We generated new transcriptomes of two myxozoan species using a novel pipeline for filtering of closely related contaminant species in RNA-seq data. Mining of our transcriptomes and published omics data confirmed the existence of myxozoan Ncol-4, reported only once previously, and revealed a novel noncanonical minicollagen, Ncol-5, which is exclusive to Myxozoa. Phylogenetic analyses support a close relationship between myxozoan Ncol-1-3 with minicollagens of Polypodium hydriforme, but suggest independent evolution in the case of the myxozoan minicollagens Ncol-4 and Ncol-5. Additional genome- and transcriptome-wide searches of cnidarian minicollagens expanded the dataset to better clarify the evolutionary trajectories of minicollagen. Conclusions The development of a new approach for the handling of next-generation data contaminated by closely related species represents a useful tool for future applications beyond the field of myxozoan research. This data processing pipeline allowed us to expand the dataset and study the evolution and diversity of minicollagen genes in Myxozoa and Cnidaria. We identified a novel type of minicollagen in Myxozoa (Ncol-5). We suggest that the large number of minicollagen paralogs in some cnidarians is a result of several recent large gene multiplication events. We revealed close juxtaposition of minicollagens Ncol-1 and Ncol-4 in myxozoan genomes, suggesting their common evolutionary history. The unique gene structure of myxozoan Ncol-5 suggests a specific function in the myxozoan polar capsule or tubule. Despite the fact that myxozoans possess only one type of nematocyst, their gene repertoire is similar to those of other cnidarians

Severe glomerular disease in juvenile grey snapper Lutjanus griseus L. in the Gulf of Mexico caused by the myxozoan Sphaerospora motemarini n. sp.

AbstractIn the eastern Gulf of Mexico, off the coast of Florida, grey snapper, Lutjanus griseus was found to be infected with the myxozoan parasite Sphaerospora motemarini n. sp., with high prevalence (83%) and intensity of infection occuring in age-0 fish, and with parasite levels decreasing with age (age-1 snapper 40%; age-2 snapper 0%). The morphological, molecular and phylogenetic characterisation of the myxozoan showed that it is a member of the typically marine, polysporoplasmid Sphaerospora spp. which form a subclade within the Sphaerospora sensu stricto clade of myxozoans, which is characterised by large expansion segments in their SSU rDNA sequences. Presporogonic stages of S. motemarini n. sp. were detected in the blood, using PCR. Pseudoplasmodia and spores were found to develop in the renal corpuscles of the host, causing their massive expansion. Macroscopic and histopathological changes were observed in age-0 fish and show that S. motemarini n. sp. causes severe glomerulonephritis in L. griseus leading to a compromised host condition, which makes it more susceptible to stress (catch-and-release, predators, water quality) and can result in mortalities. These results are discussed in relation to the exploitation of grey snapper populations by commercial and recreational fisheries and with the observed increased mortalities with temperature along the coast of Florida. In the future, we would like to determine prevalence and intensity of infection with S. motemarini n. sp. in juvenile L. griseus in different areas of the Gulf of Mexico in order to be able to estimate the temperature dependence of S. motemarini n. sp. proliferation and to be able to predict its distribution and severity during climatic changes in the Gulf

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

Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. Ceratonova shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the data sets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed single-nucleotide polymorphism-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.Fil: Alama Bermejo, Gema. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; Argentina. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. State University of Oregon; Estados UnidosFil: Meyer, Eli. Oregon State University; Estados UnidosFil: Atkinson, Stephen D.. Oregon State University; Estados UnidosFil: Holzer, Astrid S.. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Wiśniewska, Monika M.. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Kolísko, Martin. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. University of South Bohemia; República ChecaFil: Bartholomew, Jerri. Oregon State University; Estados Unido

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians

Acknowledgments: We thank to Baveesh Pudhuvai (BC CAS, Budweis, Czech Republic) for help in the PCR verification of Buddenbrockia stefin. We also thank Ivan Fiala (BC CAS, Budweis, Czech Republic) for providing suggestions to improve the manuscript and for sharing M. lieberkuehni and N. pickii transcriptomic data. We are grateful to Hanna Hartikainen (ETH Zurich, Switzerland) for sharing T. bryosalmonae genome data. Funding: This research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, grant number LTAUSA17201; by the European Commission under the H2020 Programme— ParaFishControl, grant number 634429; by the Czech Science Foundation, grant number 19-28399X (to A. S. Holzer, G. Alama-Bermejo, and J. Kyslík) and 21-16565S and by the Czech Academy of Sciences and Hungarian Academy of Sciences, grant number MTA 19-07. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein.Peer reviewedPublisher PD

‘Who’s who’ in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish – molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.Keywords: Ribosomal DNA, Cryptic speciation, Cyprinid, Multi-species infection, Molecular identification, Myxozoa, Morphometry, Sphaerospora, Blood stage

The acute effects of a lunch containing capsaicin on energy and substrate utilisation, hormones, and satiety

BACKGROUND: Addition of capsaicin to the diet has been shown to increase satiety and thermogenesis. The effects of capsaicin on ghrelin, peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), in relation to changes in hunger and satiety are unknown. AIM: To test the acute effects of a lunch containing capsaicin on gut derived hormones (GLP-1, ghrelin, and PYY), energy expenditure (EE), substrate oxidation and satiety at lunch in the postprandial state. METHODS: Thirty subjects (age: 31 +/- 14 years, BMI: 23.8 +/- 2.8 kg/m(2)) were studied twice in a crossover design. After 30 min resting on a bed, resting metabolic rate was measured by a ventilated hood system. Subsequently lunch (35% of daily energy intake) was served. The two lunch conditions were: (1) lunch without capsaicin and (2) lunch with capsaicin (CAPS). The macronutrient composition (energy percentage) of the lunches was 60% carbohydrates, 10% protein and 30% fat. During 3 h after the lunch diet-induced thermogenesis was measured. Furthermore, anchored 100 mm visual analogue scales on the appetite profile were collected (t = 0, 30, 60, 120, 150, 180 and 240) and blood samples were taken for analysis of GLP-1, PYY, and ghrelin concentrations (t = 0, 45, 60, 120, and 180). RESULTS: Satiety and EE were not different after CAPS lunch as compared to the control lunch. Fifteen minutes after lunch CAPS lunch increased GLP-1 (p < 0.05) and tended to decrease ghrelin (p = 0.07) as compared to the control lunch. PYY responses were not different between the CAPS lunch and the control lunch. CONCLUSIONS: An acute lunch containing capsaicin had no effect on satiety, EE, and PYY, but increased GLP-1 and tended to decrease ghrelin

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari

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

Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa)

Abstract 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