Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Unraveling the mystery of a myxozoan parasite of the trout: redescription of Chloromyxum schurovi.

Myxozoans are microscopical parasites widely distributed in fish, with over 2,600 described species, but their actual diversity is still underestimated. Among salmonids, more than 70 myxozoan species have been identified. This study focuses on species of Chloromyxum Mingazzini, 1890 that infect salmonid kidneys, particularly C. majori Yasutake et Wood, 1957 and C. schurovi Shulman et Ieshko, 2003. Despite their similar spore morphology, they exhibit distinct host preferences, tissue affinities and geographical distributions. Chloromyxum schurovi predominantly infects the renal tubules of Salmo salar Linnaues and S. trutta Linnaeus in Europe, while C. majori targets the glomeruli of Oncorhynchus mykiss (Walbaum) and O. tshawytscha (Walbaum) in North America. The sequence data for C. majori and C. schurovi have been either missing or questionable. In our study, we examined the kidneys of two salmonid species for chloromyxid infections, using both morphological and molecular data to characterise Chloromyxum species in salmonids. The sequence of C. schurovi obtained in our study did not match the previously published parasite data. Instead, it clustered as an independent lineage sister to the Paramyxidium Freeman et Kristmundsson, 2018 clade gathering the species from various fish organs, including the urinary tract. Our findings clarified the taxonomic origin of the previous C. schurovi sequence as Myxidium giardi Cépède, 1906, highlighting the risks associated with the presence of myxozoan blood stages in the bloodstream of their fish host and the challenges of non-specific PCR amplification. We redescribe C. schurovi, thus contributing to a better understanding of the diversity and phylogeny of kidney-infecting species of Chloromyxum.

Evidence of striking morphological similarity and tissue tropism of phylogenetically distant myxozoan genera: Myxidium and Paramyxidium in the kidney of the European eel.

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.

Solving the Myxidium rhodei (Myxozoa) puzzle: insights into its phylogeny and host specificity in Cypriniformes.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.

Title: Résoudre le casse-tête de Myxidium rhodei (Myxozoa) : aperçu de sa phylogénie et de sa spécificité d'hôte chez les Cypriniformes. Abstract: Myxidium rhodei Léger, 1905 (Cnidaria : Myxozoa) est un Myxosporea infectant les reins qui a été décrit à l'origine chez la bouvière, Rhodeus amarus. Par la suite, il a été documenté, sur la base de la morphologie des spores, chez plus de 40 autres espèces de cypriniformes, le gardon Rutilus rutilus étant l'hôte le plus fréquemment signalé. Cette étude présente la première évaluation complète des données sur M. rhodei, réalisée par des méthodes morphologiques, écologiques et moléculaires. Les analyse morphologiques et phylogénétiques des séquences d'ADNr SSU des isolats de Myxidium obtenus à partir de bouvières et de gardons européens n'ont pas confirmé la conspécificité du parasite de ces poissons. En fait, les isolats infectant les gardons représentent trois espèces distinctes de parasites. Les deux premières, M. rutili n. sp. et M. rutilusi n. sp., sont des espèces cryptiques étroitement apparentées, regroupées avec d'autres Myxosporea du clade urinaire d'eau douce, partageant le même tropisme tissulaire. La troisième, M. batuevae n. sp., précédemment attribuée à M. cf. rhodei, appartient au clade biliaire hépatique, groupe-frère de M. rhodei infectant la bouvière. Notre examen de divers poissons cypriniformes, couplé à des analyses moléculaires et morphologiques, nous a permis de démêler la nature cryptique des espèces de M. rhodei et de découvrir l'existence de nouvelles espèces. Cela souligne la diversité largement méconnue des Myxozoaires et souligne la nécessité d'incorporer des données de séquence dans le diagnostic de nouvelles espèces.

Henneguya (Cnidaria: Myxobolidae) species infecting Oligosarcus jenynsii (Characiformes: Characidae) in a Neotropical shallow lake from Argentina: morphological and molecular characterisation.

Two previously undescribed myxozoan species, Henneguya sardellae sp. n. and H. margaritae sp. n., found infecting connective tissues of the Neotropical characid fish Oligosarcus jenynsii (Günther) from Argentina are morphologically and molecularly characterised. Mature spores of H. sardellae sp. n. are ellipsoid, with two, straight and visibly fused caudal appendages cleaved at its blunt terminal end; measuring 33.5 ± 1.2 (30.9-35.5) µm in total length, spore body 17.5 ± 0.6 (16.3-18.6) µm, 7.8 ± 0.4 (7.0-8.8) µm wide and 6.9 ± 0.2 (6.6-7.2) µm thick, with two elongated, unequally-sized polar capsules situated at anterior end, and 11-13 turns of polar tubules. Mature spores of H. margaritae sp. n. are pyriform, with two caudal appendages visible fused together and much longer than spore body, with unequal endings; measuring 35.9 ± 2.8 (29.2-40.7) µm in total length, spore body 11.5 ± 0.9 (9.2-13.0) µm long, 5.8 ± 0.4 (5.1-6.7) µm wide and 5.5 ± 0.2 (5.1-5.8) µm thick, with two polar capsules similar in size, pyriform polar capsules containing polar tubules with 4-5 coils. Both species showed a membraneous sheath surrounding the spore body and caudal appendages; in H. sardellae sp. n. this feature can deploy laterally. Phylogenetic analyses based on SSU rDNA sequences showed that H. sardellae sp. n. and H. margaritae sp. n. clustered with other myxobolids parasitising Characiformes in Brazil, Cichliformes in Mexico and Cyprinodontiformes in Mexico and the United States. The description of these two new species of Henneguya as the first described species of the genus that parasitise freshwater fish in Argentina highlights the importance of further research on the diversity and distribution of myxozoans in this region.

An ancient alliance: Matching evolutionary patterns of cartilaginous fishes (Elasmobranchii) and chloromyxid parasites (Myxozoa).

Myxozoa is a group of endoparasitic cnidarians covering almost 2600 species but merely 53 species, mostly from the genus Chloromyxum, have been reported from sharks, rays, and skates (Elasmobranchii). Elasmobranchs play a key role in the study of evolutionary trajectories of myxozoans as they represent ancestral vertebrate hosts. Our study provides new data on Chloromyxum spp. from 57 elasmobranchs, covering 20 species from geographical regions and host groups not previously investigated, such as Lamniformes and Hexanchiformes, the most basal phylogenetic shark lineage. In total, 28% of elasmobranchs were infected with Chloromyxum spp., indicating high diversity. Of the seven distinguished species, six are formally described based on morphological, morphometric, and genetic (18S rDNA) data. Comprehensive co-phylogenetic analyses and ancestral state reconstruction revealed that parasite and host phylogenies are clearly correlated, resulting in a distinct phylogenetic separation of chloromyxids from selachid (shark) vs. batoid (ray and skate) hosts. Species infecting the most ancient elasmobranchs formed a sublineage, branching off in the middle of the Chloromyxum sensu stricto clade. Our findings indicate that chloromyxids likely invaded an ancestral elasmobranch prior the time of divergence of shark and batoid lineages. Our analyses did not show a clear phylogeographic pattern of Chloromyxum parasites, probably due to the cosmopolitan distribution and migratory behaviour of many elasmobranch hosts, but geographical sampling must be extended to confirm or refute this observation. This study provides a complex view on species diversity, phylogeny, evolution, host-parasite co-phylogeny, and the phylogeographic origin of Chloromyxum species from elasmobranchs. Our results highlight the importance of adding missing data from previously un- or undersampled geographical regions and host species which results in a more accurate estimate of myxozoan biodiversity and a better understanding of the evolution of this parasite group in their hosts and in the different oceans of our planet.

Correlated evolution of fish host length and parasite spore size: a tale from myxosporeans inhabiting elasmobranchs.

Myxozoa represent a diverse group of microscopic cnidarian endoparasites alternating between invertebrate and vertebrate hosts. Of the approximately 2,600 species described predominantly from teleost fish, only 1.8% have been reported from cartilaginous fishes (Elasmobranchii). As ancestral vertebrate hosts of myxozoans, elasmobranchs may have played an important role in myxozoan evolution, however, they are also some of the largest vertebrate hosts known for this group of parasites. We screened 50 elasmobranchs belonging to nine species and seven families, from various geographical areas, for myxozoan infection. We found a 22% overall prevalence of myxozoans in elasmobranchs and describe five species new to science. We investigated, for the first known time, the evolution of spore size within three phylogenetic clades, Ceratomyxa, Sphaerospora sensu stricto and Parvicapsula. We found that spores from elasmobranch-infecting myxozoans were on average 4.8× (Ceratomyxa), 2.2× (Parvicapsula clade) and 1.8× (Sphaerospora sensu stricto except polysporoplasmic Sphaerospora spp.) larger than those from teleosts. In all analysed clades, spore size was correlated with phylogenetic position. In ceratomyxids, it was further strongly positively correlated with fish body size and habitat depth, independent of cellular composition of the spores and phylogenetic position in the tree. While in macroparasites a host size-correlated increase in parasite size occurs on a large scale and is often related to improved exploitation of host resources, in microscopic parasites size ranges vary at the scale of a few micrometres, disproportionate to the available additional space in a large host. We discuss the ecological role of these changes with regard to transmission under high pressure and an invertebrate fauna that is adapted to deeper marine habitats.

Survey of Kudoa spp. (Myxozoa, Cnidaria) in fishes from the Madeira Archipelago and the Portuguese mainland coast: detection of Kudoa thyrsites in new hosts Scomber colias and Micromesistius poutassou.

Myxozoan parasites of the genus Kudoa Meglitsch, 1947 are associated with post-mortem tissue degradation that causes great financial losses to commercial fisheries. Kudoa thyrsites (Gilchrist, 1924) is a species with a very wide host range including commercial tunas, mackerels, salmonids and flatfishes. A sample of 190 fishes of 18 species from the Madeira Archipelago and 30 Atlantic chub mackerel, Scomber colias Gmelin, and 30 blue whiting, Micromesistius poutassou (Risso), from the Portuguese mainland coast were examined for the presence of species of Kudoa. The prevalence of Kudoa spp. was 80% in M. poutassou and 60% in S. colias. No spore was detected in S. colias from Madeira, which was confirmed by specific PCR screening of the muscle from all individuals of S. colias. SSU rDNA analysis revealed that M. poutassou and S. colias from the Portuguese mainland coast were infected with K. thyrsites, an economically important myxozoan parasite. Both sequences were identical with sequences of the eastern Atlantic K. thyrsites genotype, including that from the type host of this parasite. This is the first report of K. thyrsites from M. poutassou and S. colias. The fact that spores of species of Kudoa were not detected in fishes screened in the Madeira Archipelago may be explained by various ecological factors, such as the absence of a continental shelf, a short insular shelf, and oceanic waters with low productivity, all resulting in reduced abundance of benthic organisms. Consequently, it is possible that as yet unknown annelid definitive hosts of Kudoa spp. are absent or very rare near Madeiran coasts.

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.

Mechanisms and Drivers for the Establishment of Life Cycle Complexity in Myxozoan Parasites.

It is assumed that complex life cycles in cnidarian parasites belonging to the Myxozoa result from incorporation of vertebrates into simple life cycles exploiting aquatic invertebrates. However, nothing is known about the driving forces and implementation of this event, though it fostered massive diversification. We performed a comprehensive search for myxozoans in evolutionary ancient fishes (Chondrichthyes), and more than doubled existing 18S rDNA sequence data, discovering seven independent phylogenetic lineages. We performed cophylogenetic and character mapping methods in the largest monophyletic dataset and demonstrate that host and parasite phylogenies are strongly correlated, and that tectonic changes may explain phylogeographic clustering in recent skates and softnose skates, in the Atlantic. The most basal lineages of myxozoans inhabit the bile of chondrichthyans, an immunologically privileged site and protective niche, easily accessible from the gut via the bile duct. We hypothesize that feed-integration is a likely mechanism of host acquisition, an idea supported by feeding habits of chimaeras and ancient sharks and by multiple entries of different parasite lineages from invertebrates into the new host group. We provide exciting first insights into the early evolutionary history of ancient metazoan parasites in a host group that embodies more evolutionary distinctiveness than most other vertebrates.

The kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan Sphaerospora molnari.

BACKGROUND: Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year. RESULTS: We have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (< 40 hours), an initial covert infection period with a moderate proinflammatory response for about 1-2 weeks, followed by a period of parasite multiplication in the blood which peaked at 28 days post-infection (dpi) and was associated with a massive lymphocyte response. Our data further revealed a switch to a massive anti-inflammatory response (up to 1456-fold expression of il-10), a strong increase in the expression of IgM transcripts and increased number of IgM+ B lymphocytes, which produce specific antibodies for the elimination of most of the parasites from the fish at 35 dpi. However, despite the presence of these antibodies, S. molnari invades the liver 42 dpi, where an increase in parasite cell number and indistinguishable outer cell membranes are indicative of effective exploitation and disguise mechanisms. From 49 dpi onwards, the acute infection changes to a chronic one, with low parasite numbers remaining in the fish. CONCLUSIONS: To our knowledge, this is the first time myxozoan early development and immune modulation mechanisms have been analyzed along with innate and adaptive immune responses of its fish host, in a controlled laboratory system. Our study adds important information on host-parasite interaction and co-evolutionary adaptation of early metazoans (Cnidaria) with basic vertebrate (fish) immune systems and the evolution of host adaptation and parasite immune evasion strategies.

Current view on phylogeny within the genus Flabellula Schaeffer, 1926 (Amoebozoa: Leptomyxida).

The molecular phylogeny of Flabellula Schaeffer, 1926 has been updated by analysing 18S rRNA and actin gene sequences of 19 new strains collected and characterised by the authors over the past ten years. The genus Flabellula Schaeffer, 1926 (Amoebozoa: Leptomyxida) is a taxon in which species delineation based on morphological data by themselves is insufficient or even misleading. The description of two novel species, F. schaefferi n. sp. and F. sawyeri n. sp., is justified by the congruence of morphological data with 18S rRNA and actin gene sequence phylogenies, in-silico secondary structure prediction of the V2 region in the 18S rRNA, and by recognition of species-specific sequential motifs within this region.

Life in a rock pool: Radiation and population genetics of myxozoan parasites in hosts inhabiting restricted spaces.

INTRODUCTION: Intertidal rock pools where fish and invertebrates are in constant close contact due to limited space and water level fluctuations represent ideal conditions to promote life cycles in parasites using these two alternate hosts and to study speciation processes that could contribute to understanding the roles of parasitic species in such ecosystems. MATERIAL AND METHODS: Gall bladder and liver samples from five clinid fish species (Blenniiformes: Clinidae) were morphologically and molecularly examined to determine the diversity, prevalence, distribution and host specificity of Ceratomyxa parasites (Cnidaria: Myxozoa) in intertidal habitats along the coast of South Africa. Phylogenetic relationships of clinid ceratomyxids based on the SSU rDNA, LSU rDNA and ITS regions were assessed additionally to the investigation of population genetic structure of Ceratomyxa cottoidii and subsequent comparison with the data known from type fish host Clinus cottoides. RESULTS AND DISCUSSION: Seven Ceratomyxa species including previously described Ceratomyxa dehoopi and C. cottoidii were recognized in clinids. They represent a diverse group of rapidly evolving, closely related species with a remarkably high prevalence in their hosts, little host specificity and frequent concurrent infections, most probably as a result of parasite radiation after multiple speciation events triggered by limited host dispersal within restricted spaces. C. cottoidii represents the most common clinid parasite with a population structure characterized by young expanding populations in the south west and south east coast and by older populations in equilibrium on the west coast of its distribution. Parasite and fish host population structures show overlapping patterns and are very likely affected by similar oceanographic barriers possibly due to reduced host dispersal enhancing parasite community differentiation. While fish host specificity had little impact on parasite population structure, the habitat preference of the alternate invertebrate host as well as tidal water exchange may be additional crucial variables affecting the dispersal and associated population structure of C. cottoidii.