A new freshwater Ceratomyxa species (Myxozoa: Ceratomyxidae) parasitizing a sciaenid fish from the Amazon Basin, Brazil.

Myxozoans of Ceratomyxidae Doflein, 1899 are common coelozoic parasites of marine life, and are also found less frequently in freshwater fish. The present study describes Ceratomyxa ranunculiformis n. sp. as a new freshwater myxosporean species infecting the gall bladder of the Amazonian sciaenid Plagioscion squamosissimus. The new Ceratomyxa was described based on its host, myxospore morphology, ribosomal rDNA gene sequencing, parasite distribution, and phylogenetic analysis. Immature and mature plasmodia were tadpole-shaped or pyriform, and exhibited slow undulatory motility. The myxospores were elongated and crescent-shaped in the frontal view, with a sutural line between two valves, which had rounded ends. The measurements of the formalin-fixed myxospores were: average length 4.9 (4.0-6.6) µm, average thickness 37.6 (32.4-43.9) µm, average posterior angle 165° (154°-173°). Two ovoid polar capsules of equal size, average length 2.0 (1.4-3.0) µm and average width 1.9 (1.4-2.4) µm, were located adjacent to the suture and contained polar filaments with 2-3 coils. The integrated comparative analysis of the morphological characteristics and molecular analyses of the ribosomal rDNA genes supported the identification of a new species of coelozoic Ceratomyxa. Maximum likelihood analyses showed the new species clustering within a well-supported clade, together with all the other Amazonian freshwater ceratomyxids.

Integrative Taxonomy Reveals a Panmictic Population of Henneguya longisporoplasma n. sp. (Cnidaria: Myxozoa) in the Amazon Basin.

PURPOSE: Henneguya Thélohan, 1892 is one of the most species-rich genera of myxosporean parasites and infects fish around the world. The present study describes a new species infecting the gill filaments, fins, and kidneys of Plagioscion squamosissimus (Heckel, 1840), an economically important freshwater fish distributed in watersheds in the north of South America. METHODS: A total of 108 P. squamosissimus specimens were examined from three geographic localities in the Amazon basin: the Lago Grande do Curuai, a marginal lake of the Amazon River; the Tapajós River, in the state of Pará; and the Solimões River, in the state of Amazonas, Brazil. The analyses were based on the myxospore morphology, ribosomal DNA sequencing, phylogeny, prevalence, and geographic distribution of the host and its parasite. RESULTS: Parasite prevalences were 50% in both the Tapajós and Solimões rivers, and 35.4% in the Lago Grande do Curuai. In terms of the site of infection, the prevalence total was 23.1% in the gill filament, 29.6% in the fins, and 1.8% in the kidney. Regarding gender, the prevalence was 59.5% for males, 32.5% for females, and 21.7% for undetermined sex. The specimens found here were both morphologically and molecularly identical regardless of the infected organ and geographic locality, but distinct from all other Henneguya species, revealing that the parasite reported represents a novel species named Henneguya longisporoplasma n. sp. Despite the sampling being carried out in three different geographic localities of the Amazon basin, no population-level genetic variation was observed, even in the typically more variable ITS-1 region, revealing a panmictic population of H. longisporoplasma n. sp. in this large watershed. Maximum likelihood and Bayesian analyses showed the novel Henneguya clustered as a sister branch of the subclade formed of Henneguya that infect fish belonging to the family Cichlidae. CONCLUSIONS: A novel Henneguya species was identified parasitizing P. squamosissimus. The parasite presented wide geographic distribution in the Amazon basin and genetic analyses showed it as revealing a panmictic population.

Occurrence of the host-parasite system Rhaphiodon vulpinus and Ceratomyxa barbata n. sp. in the two largest watersheds in South America.

While around world, species of the genus Ceratomyxa parasite majority marine hosts, growing diversity has been reported in South American freshwater fish. The present study reports Ceratomyxa barbata n. sp. parasitizing the gallbladder of the Rhaphiodon vulpinus fish from the Amazon and La Plata basins. Morphological (light and transmission electron microscopy), molecular (sequencing of small subunit ribosomal DNA - SSU rDNA), and phylogenetic analyses were used to characterize the new species. Worm-like plasmodia endowed with motility were found swimming freely in the bile. The myxospores were elongated, lightly arcuate, with rounded ends and had polar tubules with 3 coils in the polar capsules. Ultrastructural analysis revealed plasmodia composed of an outer cytoplasmic region, where elongated tubular mitochondria, a rough endoplasmic reticulum, sporogonic stages, and a large vacuole occupying the internal area were observed. Phylogenetic analysis, based on SSU rDNA, found that among all South America freshwater Ceratomyxa species, C. barbata n. sp. arises as an earlier divergent species. The present study reveals the occurrence of this host-parasite system (R. vulpinus/C. barbata n. sp.) in the two largest watersheds on the continent.

A novel Ceratomyxa species (Myxozoa: Cnidaria) infecting an Amazonian catfish.

Parasites are important organisms for the health of ecosystems. While the Amazon Basin is home to a great diversity of ichthyofauna, our knowledge of myxozoan diversity in the biome remains relatively limited. The present study describes a new myxozoan species, Ceratomyxa mandii n. sp., parasitizing the gallbladder of the Amazonian catfish Pimelodina flavipinnis (Pimelodidae) from the Solimões River, in the region of Manaus, Brazil. Light and electron microscopy,  small subunit ribosomal DNA (SSU rDNA) sequencing and phylogenetic analysis were performed. The new species exhibited worm-like plasmodia with undulatory motility. The SSU rDNA based phylogenetic analysis revealed it to be a sister taxon of C. gracillima, which also parasitizes an Amazonian pimelodid fish, possibly reflecting a host-parasite co-speciation process. This study contributes to our understanding of this little sampled group of organisms.

Expanding the geographic distribution of the freshwater parasite Ceratomyxa (Cnidaria: Myxozoa) with vermiform-type plasmodia.

Although species of the genus Ceratomyxa Thélohan, 1892 mostly parasitize marine fish around the world, a surprising diversity of the genus has recently been reported from Amazonian freshwater fish. In this study, we report a freshwater Ceratomyxa species parasitizing Hemiodus orthonops (Hemiodontidae) from the Paraná River (La Plata Basin) in a watershed flowing into the southern part of South America, which expands the geographic distribution of this fish parasite in the freshwater resources of the continent. We applied a combination of morphological, small subunit ribosomal DNA (SSU rDNA), and phylogenetic analyses, and vermiform-shaped plasmodia endowed with motility were found swimming in the bile of the fish. The characteristics of the plasmodia and myxospores of the Ceratomyxa species found in the Paraná River resembled those of Ceratomyxa fonsecai, a parasite of the congeneric host Hemiodus unimaculatus from the Tocantins River basin in northern Brazil. Due to the close morphological and morphometric resemblances and the impossibility of genetic comparison, the parasite found in H. orthonops from the Paraná River was designated as Ceratomyxa cf. fonsecai, and the definition of its taxonomic status was left for further study. Maximum likelihood and Bayesian analyses showed Ceratomyxa cf. fonsecai clustering within a well-supported clade, together with other Amazonian freshwater ceratomyxids. The present study suggests that shifts of the complex host/parasite between marine and freshwater environments were facilitated by marine incursions into South America in the Early Miocene.

How to build single-celled cnidarians with worm-like motility: Lessons from Myxozoa.

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

Growing diversity supports radiation of an Ellipsomyxa lineage into the Amazon freshwater: Description of two novel species parasitizing fish from Tapajós and Amazon rivers.

Species of the genus Ellipsomyxa Køie, 2003, parasitize mostly marine and brackish fish around the world. In the present study, we describe two novel species of Ellipsomyxa: Ellipsomyxa plagioscioni n. sp. parasitizing the gall bladder of Plagioscion squamosissimus (Sciaenidae), a freshwater fish but commonly found in brackish water in the Amazonian estuarine environment; and Ellipsomyxa paraensis n. sp. infecting Cichla monoculus (Cichlidae), a strictly freshwater fish. The host specimens were caught from the Amazon and Tapajós rivers, in the municipal region of Santarém, in the State of Pará, Brazil. The study was performed using a combination of morphological, biological, and SSU rDNA-based phylogeny, which suggested that marine transgressions of the Miocene epoch, in the central region of South America, were a pathway for the adaptation and radiation of these cnidarian parasites in the freshwater environment. Both disporic plasmodia and mature myxospores were found floating freely in the bile. Mature myxospores from both species were ellipsoidal in the valvular and sutural views, with thin smooth valves elongated in the direction perpendicular to the plane of the transverse sutural line. Ellipsomyxa plagioscioni n. sp. myxospores measured 11.1 (10.2-12.8) µm in length and 6.6 (5.6-7.6) µm in width. Two pyriform polar capsules discharging on opposite sides, some distance from both the sutural line and the spore ends, measured 3.8 (3.2-4.4) µm in length and 2.8 (2.3-3.3) µm in width, with 5-6 coil polar tubules. Ellipsomyxa paraensis n. sp. myxospores measured 11.5 (10.5-12.4) µm in length and 7.5 (6.6-8.6) µm in width. Two pyriform polar capsules which discharged on opposite sides some distance from both the sutural line and spore ends, measured 3.2 (2.1-3.9) µm in length and 2.6 (2.0-3.3) µm in width, with 2-3 coil polar tubules. Valvular protrusions were observed, associated with the tips of the polar capsules. Molecular analysis based on the SSU rDNA sequences indicated that the two novel Ellipsomyxa species were distinct from all other sequences deposited in the GenBank database. The phylogenetic trees clustered E. plagioscioni n. sp. as a basal species of a lineage of the marine/estuarine Ellipsomyxa, while E. paraensis n. sp. clustered together with other Amazonian species.

Morphological, ultrastructural, and phylogenetic analysis of two novel Myxobolus species (Cnidaria: Myxosporea) parasitizing bryconid fish from São Francisco River, Brazil.

Twelve Myxobolus species have been previously described to parasitize Bryconidae fish in South America. Here, we describe two novel myxosporean species that parasitize economically important Bryconidae from the São Francisco River basin in Brazil. Myxospores morphometry, morphology, small-subunit ribosomal DNA - ssrDNA sequences, and other biological traits were used in the taxonomic analysis. Phylogenetic analysis was performed to assess the position of the new Myxobolus species among the closest Myxobolus/Henneguya. Myxobolus iecoris n. sp. was found infecting the liver of Salminus franciscanus (dourado). Myxospores were oval with the anterior region aculiform in frontal view and biconvex in lateral view and measured 11.4-14.2 (12.8 ±â€¯0.8) µm long, 7.7-9.9 (8.7 ±â€¯0.6) µm wide, 6.5-7.5 (6.9 ±â€¯0.4) µm thick. Two pyriform and equal-sized polar capsules measuring 4.9-7.4 (5.9 ±â€¯0.5) µm long and 2.3-3.5 (3.0 ±â€¯0.2) µm wide contained polar tubules with 8-9 turns. Myxobolus lienis n. sp. was found infecting the spleen of Brycon orthotaenia (matrinxã). Myxospores were round to oval in frontal view and biconvex in lateral view and measured 10.3-13.8 (12 ±â€¯0.6) µm long, 6.8-9.3 (8.3 ±â€¯0.5) µm wide, and 6.9-7.0 (7.0 ±â€¯0.6) µm thick. Two oval and equal-sized polar capsules measured 3.9-5.8 (4.6 ±â€¯0.5) µm long and 2.0-3.5 (2.8 ±â€¯0.3) µm wide contained polar tubules with 5-6 turns. Ultrastructural analysis revealed asynchronous sporogenesis with germinative cells and young sporogonic stages in the periphery of the plasmodia. A connective tissue capsule was observed surrounding Myxobolus lienis n. sp., but it was absent for Myxobolus iecoris n. sp. Maximum likelihood and Bayesian inferences showed the two novel species clustering in a well-supported subclade composed by Myxobolus spp. of bryconids. Myxobolus iecoris n. sp. appeared as a sister species of M. aureus and Myxobolus lienis n. sp. as sister to M. umidus.

Novel Myxobolus and Ellipsomyxa species (Cnidaria: Myxozoa) parasiting Brachyplatystoma rousseauxii (Siluriformes: Pimelodidae) in the Amazon basin, Brazil.

We describe two novel myxosporean parasites from Brachyplatystoma rousseauxii, an economically important freshwater catfish from the Amazon basin, Brazil. Myxobolus tapajosi n. sp., was found in the gill filaments of 23.5% of 17 fish, with myxospores round to oval in frontal view and biconvex in lateral view: length 15 (13.5-17) µm and width 10.7 (9.6-11.4) µm; polar capsules equal, length 5.8 (4.6-7.1) µm and width 3 (2.3-3.8) µm containing polar tubules with 6-7 turns. Ellipsomyxa amazonensis n. sp. myxospores were found floating freely or inside plasmodia in the gall bladder of 23.5% of fish. The myxospores were ellipsoidal with rounded extremities: length 12.8 (12.3-13.6) µm and width 7.6 (6.7-8.7) µm; with two equal, slightly pyriform polar capsules, length 3.8 (3.8-4.0) µm and width 3.1 (2.5-3.4) µm, containing polar tubules with 2-3 turns. We combined spore morphometry, small-subunit ribosomal DNA data, specific host, and phylogenetic analyses, to identify both of these parasites as new myxozoan species. Maximum likelihood and Bayesian analyses showed that Myxobolus tapajosi n. sp. clustered in a basal branch in a subclade of parasites from exclusively South American pimelodid fishes. Ellipsomyxa amazonensis n. sp. clustered within the marine Ellipsomyxa lineage, but we suspect that although the parasite was collected in freshwater, its hosts perform a large migration throughout the Amazon basin and may have become infected from a brackish/marine polychaete host during the estuary phase of its life.

Ceratomyxa gracillima n. sp. (Cnidaria: Myxosporea) provides evidence of panmixia and ceratomyxid radiation in the Amazon basin.

We describe a new freshwater myxosporean species Ceratomyxa gracillima n. sp. from the gall bladder of the Amazonian catfish Brachyplatystoma rousseauxii; the first myxozoan recorded in this host. The new Ceratomyxa was described on the basis of its host, myxospore morphometry, ssrDNA and internal transcribed spacer region (ITS-1) sequences. Infected fish were sampled from geographically distant localities: the Tapajós River, Pará State, the Amazon River, Amapá State and the Solimões River, Amazonas State. Immature and mature plasmodia were slender, tapered at both ends, and exhibited vermiform motility. The ribosomal sequences from parasite isolates from the three localities were identical, and distinct from all other Ceratomyxa sequences. No population-level genetic variation was observed, even in the typically more variable ITS-1 region. This absence of genetic variation in widely separated parasite samples suggests high gene flow as a result of panmixia in the parasite populations. Maximum likelihood and maximum parsimony analyses placed C. gracillima n. sp. sister to Ceratomyxa vermiformis in a subclade together with Ceratomyxa brasiliensis and Ceratomyxa amazonensis, all of which have Amazonian hosts. This subclade, together with other Ceratomyxa from freshwater hosts, formed an apparently early diverging lineage. The Amazonian freshwater Ceratomyxa species may represent a radiation that originated during marine incursions into the Amazon basin that introduced an ancestral lineage in the late Oligocene or early Miocene.

Amazonian waters harbour an ancient freshwater Ceratomyxa lineage (Cnidaria: Myxosporea).

A new species of Ceratomyxa parasitizing the gall bladder of Cichla monoculus, an endemic cichlid fish from the Amazon basin in Brazil, is described using morphological and molecular data. In the bile, both immature and mature myxospores were found floating freely or inside elongated plasmodia: length 304 (196-402) µm and width 35.7 (18.3-55.1) µm. Mature spores were elongated and only slightly crescent-shaped in frontal view with a prominent sutural line between two valve cells, which had rounded ends. Measurements of formalin-fixed myxospores: length 6.3±0.6 (5.1-7.5) µm, thickness 41.2±2.9 (37.1-47.6) µm, posterior angle 147°. Lateral projections slightly asymmetric, with lengths 19.3±1.4µm and 20.5±1.3µm. Two ovoid, equal size polar capsules, length 2.6±0.3 (2-3.3) µm, width 2.5±0.4 (1.8-3.7) µm, located adjacent to the suture and containing polar filaments with 3-4 turns. The small subunit ribosomal DNA sequence of 1605 nt was no more than 97% similar to any other sequence in GenBank, and together with the host, locality and morphometric data, supports diagnosis of the parasite as a new species, Ceratomyxa brasiliensis n. sp. Maximum parsimony and maximum likelihood analyses showed that C. brasiliensis n. sp. clustered within the marine Ceratomyxa clade, but was in a basally divergent lineage with two other freshwater species from the Amazon basin. Our results are consistent with previous studies that show Ceratomyxa species can cluster according to both geography and host ecotype, and that the few known freshwater species diverged from marine cousins relatively early in evolution of the genus, possibly driven by marine incursions into riverine environments.

Ultrastructural, ssrDNA sequencing of Myxobolus prochilodus and Myxobolus porofilus and details of the interaction with the host Prochilodus lineatus.

Myxobolus prochilodus and Myxobolus porofilus are parasites of Prochilodus lineatus, an economically important South American fish found in La Plata and Paraiba do Sul river basins. This study focusing on parasite-host interaction provides an ultrastructural and phylogenetic analysis, the latter based on ssrDNA sequencing of these parasites respectively infecting the gill filaments and fins of P. lineatus taken from the Mogi Guaçu River, São Paulo, Brazil. A total of 13 adult specimens were examined in this study. The prevalence of infection was 7.69 % for M. prochilodus and 15.38 % for M. porofilus. Phylogenetic analysis showed M. prochilodus and M. porofilus clustered in a subclade composed of parasites of the Prochilodontidae family. In M. prochilodus infecting gill filaments, where cellular degeneration in the epithelium was observed, the plasmodia were surrounded by a capsule composed of layers of fibrocyte-like cells, with cellular projections joined to the projections of other fibrocyte-like cells by desmosomes, and more externally typical fibroblast layers. Some granular leukocytes were seen interspersed among these layers. In M. porofilus infecting the fins, the capsule of connective tissue was represented only by loosely arranged collagen fibers, and no granular leucocytes were observed. Finally, several unusual vacuoles with filamentous content and some characteristics usually described as degenerative alterations, as myelin figure, were noted in plasmodia and pansporoblasts of both myxosporean species. The possible influence of inflammatory response and xenobiotics was considered to be the explanation for the alterations observed in Myxobolus species and its host.

Morphology, ultrastructure and phylogeny of Myxobolus curimatae n. sp. (Myxozoa: Myxosporea) a parasite of Prochilodus costatus (Teleostei: Prochilodontidae) from the São Francisco River, Brazil.

Myxobolus curimatae n. sp. has been found infecting the gill filaments of Prochilodus costatus (Prochilodontidae) from the São Francisco River in the state of Minas Gerais, Brazil. The prevalence of the species was 18.7%. Mature spores were rounded from a frontal view, with elongated polar capsules of equal size, and had polar filaments with 9-10 turns. Ultrastructural analysis revealed that sporogenesis patterns followed those of other Myxobolus species. The plasmodium walls had numerous invaginations and protrusions, and few pinocytic channels. Numerous mitochondria, generative cells and young pansporoblasts were observed in the peripherical areas of the plasmodia, and mature spores were found in deeper layers. A layer of collagenic fibrils surrounded the plasmodia. The morphological data and molecular analysis of the 18S rDNA identified this parasite as a new species. The maximum likelihood phylogenetic tree showed M. curimatae n. sp., as a sister species of Thelohanellus marginatus, in a basal branch of the subclade composed by parasites with tropism to different organs and host families.

Host-parasite and phylogenetic relationships of Myxobolus filamentum sp. n. (Myxozoa: Myxosporea), a parasite of Brycon orthotaenia (Characiformes: Bryconidae) in Brazil.

Myxobolus filamentum sp. n. was found infecting gill filaments of three of 39 Brycon orthotaenia Günther specimens examined (8%), which were taken from the river São Francisco in Minas Gerais state, Brazil. Plasmodia of the parasite were white and long, measuring 5 mm in lenght. Mature spores of M. filamentum sp. n. were oval from the frontal view and biconvex from the lateral view, measuring 7.5-9.7 µm (9.0 ± 0.3 µm) in length and 5.2-7.3 µm (6.2 ± 0.4 µm) in width. The polar capsules were elongated and equal in size, measuring 3.8-5.5 µm (4.7 ± 0.3 µm) in length and 1.3-2.2 µm (1.7 ± 0.1 µm) in width. The development of the parasite led to compression of the adjacent tissues and inflammatory infiltrate with granulocytic cells. Ultrastructural observation revealed that the plasmodia were delimited by two membranes, which had numerous and extensive pinocytotic channels extending into the wide ectoplasm zone. The plasmodial wall exhibited abundant villi-like projections and a thin layer of granular material prevented direct contact between the plasmodial wall and the host tissue. Phylogenetic analysis, based on 18S rDNA, showed M. filamentum sp. n. as a sister species of Myxobolus oliveirai Milanin, Eiras, Arana, Maia, Alves, Silva, Carriero, Ceccarelli et Adriano, 2010, a parasite of other fish species of the genus Brycon Müller et Troschel from South America.