Molecular studies on Sphaerospora truttae and other freshwater myxozoans

This study investigates the life cycle of Sphaerospora truttae, a myxozoan parasite of the Atlantic salmon, using molecular methods based on the 185 rONA. DNA sequencing showed that the 185 rONA of S. truttae differs substantially from the sequence obtained from its proposed alternate actinosporean life cycle stage, Echinactinomyxon type 5. With more than 90% sequence identity Echinactinomyxon type 5 is closely related to Myxobolus portucalensis whereas S. truttae with an extraordinary long 185 sequence (2541 bp), with inserts in the variable regions of the gene, does not relate closely to any myxozoans. On the basis of the obtained sequence for S. truttae, a single round nested peR assay was developed which allows low-level detection and specific identification of S. truttae in all life cycle stages. Furthermore, two of the primers from the peR assay were successfully used on tissue sections in an optimised in situ hybridisation (ISH) protocol. ISH experimentally identified the gills as the predominant entry locus of S. trottse into the fish host and it detected the spatiotemporal migration of the parasite via the vascular system into the target organ, the kidney. The ISH protocol and the peR assay were also used to screen oligochaetes and other co-occurring invertebrates for S. truttae infection but an alternate host for S. truttse could not be identified. However, 12 actinosporean stages were found and they were characterized on the basis of their 185 rONA, together with 9 further myxosporean species from wild fish in the same riverine habitat. Three actinosporeans were found to be genetically identical with three myxosporeans (Myxidium truttae, Chloromyxum truttse and Chloromyxum sp.) and thus represent alternate life cycle stages of these species. Phlyogenetic analysis of the myxozoans identified a very basal position of S. truttae and S. elegans, as a sister group to the marine species. All other species were nested in the freshwater clades and clustered according to host tissue localization, but independent from host species or myxozoan spore taxonomy

The description of two new species of Chloromyxum from skates in the Argentine Sea reveals that a limited geographic host distribution causes phylogenetic lineage separation of myxozoans in Chondrichthyes

During a survey on the myxosporean fauna of Rajiformes from the Atlantic coast of Argentina, in waters off Buenos Aires Province (34°-42°S; 53°-62°W), the gall bladders of 217 specimens belonging to seven species of skates, representatives of two families, were examined. As a result, three species of Chloromyxum Mingazzini, 1890, namely C. atlantoraji n. sp., C. zearaji n. sp. and C. riorajum Azevedo, Casal, Garcia, Matos, Teles-Grilo and Matos, 2009 were found infecting three endemic host species, the spotback skate Atlantoraja castelnaui (Arhynchobatidae), the yellownose skate Zearaja chilensis (Rajidae) and the Rio skate Rioraja agassizii (Arhynchobatidae), respectively. These species were described based on myxospore morphology and morphometry characterization, as well as by providing their small subunit ribosomal DNA (SSU rDNA) sequences. The SSU rDNA-based phylogenetic analyses showed that these three species constituted a well-established monophyletic subclade within the marine Chloromyxum clade, while branches subtending the other Chloromyxum species were poorly resolved or unresolved, independently of the host taxonomic identities (Carchariniformes, Myliobatiformes, Orectolobiformes, Pristiophoriformes, Rajiformes, Squaliformes and Torpediniformes) and/or host geographic distribution (Atlantic coast of Portugal, Atlantic coast of the USA, Australian waters or Mediterranean Sea). The possible causes of these discrepancies are discussed, providing new insights into the phylogeny of the marine Chloromyxum clade.Fil: Cantatore, Delfina María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: Irigoitia, Manuel Marcial. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: Holzer, Astrid Sibylle. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Bartošová Sojková, Pavla. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Pecková, Hana. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Fiala, Ivan. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Timi, Juan Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas,CCT Mar del Plata,Instituto de Investigaciones Marinas y Costeras;Universidad Nacional de Mar del Plata, FCEN, IIMYC; Argentin

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

© 2021 by the authors.The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.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

‘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

3D Morphology, Ultrastructure and Development of Ceratomyxa puntazzi Stages: First Insights into the Mechanisms of Motility and Budding in the Myxozoa

Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream

Motility and budding of <i>Ceratomyxa puntazzi</i> in the bile of <i>Diplodus puntazzo</i>.

<p>A–D: LM, E–G: SEM, H–K: CLSM (DAPI and Phalloidin stained). A) Small ellipsoidal stage. B) Pyriform stage with a wide hyaline area and refractive granules at rounded, anterior end of parasite. C) Pyriform stage showing large filopodia and abundant refractive bodies at rounded end and a large, rigid cytoplasm extension at posterior end. D) Pyriform stage with abundant vacuoles present in almost the whole body. Refractive bodies were concentrated at anterior end. E–G) Exogenous budding with several stages dividing by plasmotomy. Arrows indicate cytoplasm constrictions. Some filopodia and blebs can be seen on the surface of the stages. H) Three stages, a small ellipsoidal stage with 4 nuclei and two larger stages with 10 and 12 nuclei. I) Pyriform stage with abundant filopodia at round side, where F-actin (green stain) is accumulated, and rigid cytoplasmic extension at the posterior end. Four nuclei are visible. J) Several stages with a clear pattern of accumulation of F-actin in the hyaline area at the anterior end of the parasites where the filopodia are located. Upper parasite: exogenous budding of a round stage with three nuclei (arrow head) and an F-actin rich surface at opposite end from the “mother” parasite it is emerging from. K) Two stages showing exogenous budding with still attached buds moving in opposite directions. Abbreviations: HA: hyaline area; CE: cytoplasmic extension; FP: filopodia; RG: refractive granules; V: vacuole; B: bleb; Nu: Nuclei. Scale Bar: A = 3 µm; B–D = 10 µm; E–K = 4 µm.</p

To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections

© 2021 Holzer, Piazzon, Barrett, Bartholomew and Sitjà-Bobadilla.Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/10.13039/501100011033 co-funded by the European Social Fund (ESF) & ACOND/2020 Generalitat Valenciana). JLB and DB were funded by the Bureau of Reclamation, U.S. Department of Interior through Interagency Agreement #R19PG00027. The funders had no role in study. AH and the open access publication of the present article was funded by the Czech Science Foundation EXPRO grant #19-28399X (AQUAPARA-OMICS; 2019-2023). Part of the information gathered in this review was obtained with financial support from the European Commission under the project #634429 (ParaFishControl)

Schematic drawing of the locomotive action of an active pyriform stage.

<p>Projection of filopodia from the anterior, median part radially to most posterior part of the hyaline area, allowing active parasite movement.</p

Sporogenesis of <i>Ceratomyxa puntazzi</i> from the bile of <i>Diplodus puntazzo</i> (TEM).

<p>A) Initial sporoblast with two capsulogenic cells developing the external tube. Both capsulogenic cells are enveloped by a sporoplasmogenic cell harbouring two sporoplasmic nuclei. Laterally, a valvogenic cell and its nucleus. Lipid droplets and vacuoles are abundant in the cytoplasm of the P cell. B) Sporoblast with two capsulogenic cells, a sporoplasmogenic cell with two nuclei and two valvogenic cells. The nucleus of the valvogenic cell is connected by a cytoplasmic bridge (*). Notice formation of suture (arrows). C) Detail of a sporoblast, showing the binucleate sporoplasm, with two eccentric nucleoli. Abundant rough endoplasmic reticulum is present in the cytoplasm of the sporoplasmogenic cell. D) Capsulogenic cell with a prominent external tube and a capsular primordium. Note vesicular body associated to the membrane of the capsulogenic cell. E) Detail of the vesicular body of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032679#pone-0032679-g006" target="_blank">Figure 6D</a>. F) Detail of a sporoblast with vesicular body between the membranes of the two capsulogenic cells and the sporoplasmogenic cell. Suture forming between the two valvogenic cells (arrows). G) Detail of vesicular body shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032679#pone-0032679-g006" target="_blank">Figure 6F</a>. Abbreviations: P: primary cell; PNu: primary nucleus; CC: capsulogenic cell; CNu: capsulogenic nucleus; SP: sporoplasmogenic cell; SpNu: sporoplasmogenic nuclei; VC: valvogenic cell; VNu: Valvogenic nucleus; ET: external tube; rER: rough endoplasmic reticulum; LD: lipid droplets; M: mitochondria; V: vacuole; Cp: capsular primordium; N: nucleoli; VB: vesicular body. Scale Bar: A = 2 µm; B = 5 µm; C = 2 µm; D = 1 µm; E = 0.2 µm; F–G = 1 µm.</p

Diagrams of some representative developmental stages of <i>Ceratomyxa puntazzi</i> in the bile of <i>Diplodus puntazzo</i>.

<p>A) Early, active ellipsoidal stage with a few filopodia at anterior part, showing a primary cell with two primary nuclei and two secondary cells with a nucleus each, and refractive granules. B) Budding of a small stage from the “mother” parasite. Both stages show accumulation of F-actin in opposite poles allowing separation. “Daughter” parasite shows our suggestion of a secondary-tertiary cell doublets as proliferative stages. C) Active, pyriform stage, with many filopodia, at anterior part, some of them ramified. This stage possesses a primary cell with a primary nucleus, three single secondary cells and a secondary cell with two tertiary cells, abundant lipid droplets and refractive granules. D) Sporogonic stage with two forming spores, still showing motility and abundant lipid droplets. E) Sporogonic stage close to the end of spore development, with loss of motility and reduced size and number of lipid droplets. F) Mature spore with a binucleate sporoplasm, showing two capsulogenic cells with their nuclei and two polar capsules.</p