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.

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.

Morphological and molecular characterization of Ameloblastella pirarara sp. n. (Monogenoidea: Dactylogyridae) parasitizing the large Amazonian catfish Phractocephalus hemioliopterus.

In this study, integrative taxonomy is applied to describe a new dactylogyrid species, Ameloblastella pirarara sp. n. from the gills of Phractocephalus hemioliopterus, a commercially and ecologically important Amazonian catfish. Ameloblastella pirarara sp. n. can be distinguished from its congeners mainly by the morphology of the male copulatory organ (MCO), accessory piece, and anchors. The new species most resembles Ameloblastella unapi, from the Peruvian Amazon, but differs from it by the number of MCO rings, morphology of the vaginal canal and sclerotized structures of the haptor. Phylogenetic analyses based on sequences of the partial 28S rDNA (D1-D2 domains) gene placed the new species in a well-supported subclade of Ameloblastella spp. parasites of Neotropical siluriform fish, as a sister taxon to Ameloblastella unapioides. Thus, the new species described herein expands our knowledge of the diversity of monogenoid parasites from Amazonian freshwater fish.

Morphostructural data and phylogenetic relationships of a new cnidarian myxosporean infecting spleen of an economic and ecological important bryconid fish from Brazil.

A new cnidarian myxosporean infecting the spleen of an economic and ecological important bryconid fish (Salminus franciscanus) is described based on integrative taxonomic approach including morphological, ultrastructural, biological traits, geography, molecular data and phylogenetic analysis. In a total of thirty specimens examined, nineteen (63.3%) were infected by an undescribed parasite species belonging to the genus Myxobolus. Plasmodial development was asynchronous, with young development in the periphery and mature myxospores in the central area and without projections and microvilli in the plasmodial wall. Mature myxospores were ovoid in shape and measured 7.9 ± 0.2 µm (7.6-8.1 µm) in length and 5.4 ± 0.1 µm (5.0-5.6 µm) in width. The two polar capsules were equal in size, occupying a little more than half of the myxospore body, measuring 4.0 ± 0.2 µm (3.9-4.1 µm) in length and 1.7 ± 0.1 µm (1.5-1.8 µm) in width. The polar tubules coiled in six turns, perpendicular to the long axis of polar capsule. Phylogenetic analysis placed the new species within a clade containing nine myxobolid species from South American characiforms fish and appears as a close species of Myxobolus pantanalis. Nevertheless, the sequences of the new species and M. pantanalis have a large genetic divergence of 13.5% in their SSU rDNA. In light of the differences observed from the integrative taxonomy, we confidently considered that this isolate is a new species of cnidarian myxosporean, M. douradae n. sp., increasing the knowledge of diversity of this enigmatic group of cnidarians.

Increasing the known biodiversity of cnidarian parasites of bryconid fishes from South America: two novel Myxobolus species with ultrastructure and ssrDNA-based phylogeny.

This study increases the known biodiversity of cnidarian parasites in neotropical bryconid fishes. Two novel Myxobolus species are described based on morphology, ultrastructure and small subunit ribosomal DNA (ssrDNA) sequencing: Myxobolus vetuschicanus n. sp. infecting fins of Salminus franciscanus and Myxobolus mineirus n. sp. infecting the mesentery of Brycon orthotaenia from the São Francisco River basin, Minas Gerais State, Brazil. Ultrastructural analysis of the two species revealed an asynchronous sporogenesis process, with germinative cells and young developmental stages of myxospores in the periphery of the plasmodia. In M. vetuschicanus n. sp., the plasmodia were surrounded by a layer of fibroblasts and in M. mineirus n. sp., the plasmodial membrane had direct contact with the host tissue. The phylogenetic analysis based on the ssrDNA of Henneguya/Myxobolus species showed that the two novel Myxobolus species grouped in subclades together with other parasite species of bryconid fishes.

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.

Morphology and molecular data of two novel cnidarian myxosporean (Myxobolidae) infecting Piaractus brachypomus from the Amazon basin.

The objective of this study was reports, through morphological and small subunit ribosomal DNA (SSU rDNA) sequencing, two novel myxobolid myxosporeans infecting Piaractus brachypomus, an economicaly important Amazonian fish popularly known as "pirapitinga". Of a total of 25 specimens of P. brachypomus examined 68% had the gill filament parasitized by Henneguya tapariensis n. sp. and 16% had infection of Myxobolus arapiuns n. sp. in the pyloric cecum. The morphological analysis revealed H. tapariensis n. sp. myxospores with an ellipsoid shape and caudal process larger than the length of the body. The polar capsules of same size were elongated and occupied less than half the body. Sequencing of the SSU rDNA generated a partial sequence of 1946 bp. In M. arapiuns n. sp. the myxospores had oval-shaped body and polar capsules of the same size, occupying less than half the body. Sequencing of the SSU rDNA generated a partial sequence of 1950 bp. Phylogenetic analysis revealed a cluster according to the order/family of the host, where H. tapariensis n. sp. was grouped in a subclade with Henneguya brachypomus and Henneguya piaractus and M. arapiuns grouped in a subclade with Myxobolus colossomatis, Myxobolus matosi and Myxobolus pirapitingae.

Novel myxosporean species parasitizing an economically important fish from the Amazon basin.

This paper provides morphological and phylogenetic analyses of two new myxobolid species found infecting Piaractus brachypomus from the Amazon basin. The fish were caught in the Tapajós River, in the municipality of Santarém, in the state of Pará, Brazil. The plasmodial development of Henneguya brachypomus n. sp. occurred in the gill lamellae while Myxobolus pirapitingae n. sp. developed in the pyloric cecum. Morphological analyses did not identify inflammatory infiltrate for either species, but H. brachypomus n. sp. induced stretching of the epithelium, compression of the adjacent tissues, and displacement and deformation of the neighboring lamellae. The mature myxospores of H. brachypomus n. sp. were ellipsoid, with a length of 11.7-13.8 µm, a width of 4.0-4.6 µm, and a thickness of 3.5-4.3 µm. The polar capsules were elongated, with a length of 5.6-7.3 µm and a width of 1.3-2.0 µm, and each contained a polar filament with 8-9 coils. The caudal process was 40.5-48.1 µm long and the total length of the myxospore was 52.4-61.6 µm. Myxobolus pirapitingae n. sp. exhibited rounded mature myxospores measuring 10.0-11.1 µm in length, 7.0-7.6 µm in width, and 5.4-6.3 µm in thickness. The polar capsules were of equal size and occupied less than half the myxospore, measuring 3.5-4.0 µm in length and 2.0-2.6 µm in width, with each containing a polar filament with 6-7 coils. Phylogenetic analysis based on partial small subunit ribosomal DNA (ssrDNA) sequences showed that H. brachypomus n. sp. clustered as a sister species of Henneguya piaractus, while M. pirapitingae n. sp. was grouped in a sub-clade together with Myxobolus matosi and Myxobolus colossomatis.

Taxonomy, phylogeny and host-parasite interaction of two novel Myxobolus species infecting Brycon orthotaenia from the São Francisco River, Brazil.

Two new Myxobolus species were described infecting Brycon orthotaenia from the São Francisco River, in the state of Minas Gerais, Brazil. From a total of 39 B. orthotaenia collected, two specimens (5.1%) exhibited infection of the ovary and 12 specimens (30.8%) displayed infection of the liver. The plasmodia of both Myxobolus species were white and spherical measuring around 1 mm in length. The plasmodium found in the ovary showed mature myxospores, which were oval shaped from the frontal view and measured 9.2-11.0 (9.8 ± 0.4) µm in length, 5.9-6.9 (6.5 ± 0.3) µm in width and 4.6-5 (4.9 ± 0.1) µm in diameter. The two polar capsules were the same size and measured 3.9-6.2 (4.7 ± 0.5) µm in length and 1.8-2.4 (2.1 ± 0.2) µm in width. The polar tubules had 9 coils. The plasmodium found in the liver showed mature myxospores which were ellipsoidal in shape from the frontal view and measured 10.0-11.4 (10.7 ± 0.5) µm in length, 7.3-8.6 (8.1 ± 0.4) µm in width and 5.3-7.0 (6.8 ± 0.4) µm in diameter. The two polar capsules were the same size and measured 4.2-5.4 (4.9 ± 0.3) µm in length and 1.9-2.9 (2.7 ± 0.3) µm in width. The polar tubules had 8 coils. Ultrastructural analysis revealed an asynchronous sporogenesis process, with young developmental myxospore stages more often found in the periphery of the plasmodium and mature myxospores in the centre of the plasmodium. The plasmodial wall was formed by a single membrane which was not surrounded by a layer of host tissue. A thick layer of fibrous material was found in the peripheral ectoplasm close to the plasmodial wall of the plasmodium found in the ovary. Phylogenetic analysis based on the small-subunit ribosomal DNA - ssrDNA sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data and Henneguya/Myxobolus/Thelohanellus species parasitizing fish from South American, revealed that the new species are grouped in a subclade together with other Myxobolus species parasitizing bryconid hosts.

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.

The resolution of the taxonomic dilemma of Myxobolus colossomatis and description of two novel myxosporeans species of Colossoma macropomum from Amazon basin.

This study presents morphologic, molecular and phylogenetic data about two new species of the genus Myxobolus and of the previously described Myxobolus colossomatis, all which are found infecting the Colossoma macropomum, a fish whose natural habitat is the Amazon Basin of Brazil, from where the specimens for this study were caught. A total of 51 C. macropomum specimens were examined between October of 2014 and January of 2016. Plasmodia of the myxosporeans were found infecting several organs: Myxobolus matosi n. sp. and Myxobolus longissimus n. sp. were respectively found in the inner face of the operculum and in the wall external surface of the stomach and gill arch. M. matosi n. sp. were 9.6 ± 0.4 µm in length, 7.0 ± 0.3 µm in width and 5.0 ± 0.3 µm in thickness of the myxospore. M. longissimus n. sp. measured 19.1 ± 0.4 µm in length, 9.4 ± 0.3 µm in width and 8.3 ± 0.4 µm in thickness. The polar capsules, which were elongated, showed 4.3 ± 0.4 µm in length and 1.9 ± 0.1 µm in width for M. matosi n. sp. and 10.5 ± 0.2 µm in length and 2.5 ± 0.1 µm in width for M. longissimus n. sp. The Myxobolus colossomatis had two myxospore morphotypes: 1) Ellipsoidal myxospores measuring 11.6 ± 0.4 µm in length and 7.6 ± 0.2 µm in width. Their elongated polar capsules measured 5.6 ± 0.2 µm in length and 2.5 ± 0.2 µm in width; 2) Oval myxospores measuring 10.4 ± 0.5 µm in length and 7.7 ± 0.3 µm in width. Their polar capsules were 5.4 ± 0.2 µm in length and 2.4 ± 0.0 µm in width. The number of turns of the polar filament was 7-8 coils. The molecular comparison of the small subunit ribosomal DNA (ssrDNA) showed a genetic divergence of 10.3% between M. matosi n. sp. and M. colossomatis, 22.4% between M. matosi n. sp. and M. longissimus n. sp., and 23.2% between M. longissimus n. sp. and M. colossomatis. Myxobolus cf. colossomatis, a parasite of Piaractus mesopotamicus, showed 11.1% of genetic divergence to M. colossomatis, demonstrating them to be distinct species. Phylogenetic analysis, based on sequences of the ssrDNA, showed the M. matosi n. sp. to be a sister species of M. colossomatis, and it also showed M. longissimus n. sp. to be a sister branch in the lineage composed by Myxobolus cf. cuneus and Henneguya pellucida.

Morphology and 18S rDNA sequencing of Henneguya peruviensis n. sp. (Cnidaria: Myxosporea), a parasite of the Amazonian ornamental fish Hyphessobrycon loretoensis from Peru: A myxosporean dispersal approach.

Myxosporean are endoparasitic cnidarians of wide distribution and responsible for important economic losses in fisheries and aquaculture. A new myxosporean species, Henneguya peruviensis n. sp., is herein described as obtained from the gill filaments of Hyphessobrycon loretoensis caught in the Nanay River, Department of Loreto, Peru. The parasite was found in 37 of 45 (82.2%) examined H. loretoensis. The new species was characterized based on morphological features and 18S rDNA gene sequence data. The sequencing of the 18S rDNA gene from the spores of H. peruviensis n. sp. resulted in 1632 nucleotides and this sequence did not match any of the myxozoan available in the GenBank. Phylogenetic analysis showed that H. peruviensis n. sp. closed together with H. leporinicola. Nonetheless, the 18S rDNA sequences of H. peruviensis n. sp. and H. leporinicola have only 82% similarity. This is the first description and molecular study of a Myxozoa parasitizing fish of the genus Hyphessobrycon in the Amazon basin. Given the importance of the ornamental fish industry in translocation of aquatic organisms worldwide, the international movement of myxosporeans in infected fish is discussed in terms of disease outbreaks and the need for preventative action.

Molecular phylogeny of the gill parasite Henneguya (Myxosporea: Myxobolidae) infecting Astyanax lacustris (Teleostei: Characidae) from fish farm in Brazil.

Molecular data of Henneguya chydadea Barassa, Cordeiro and Arana, 2003, found in the gill filaments of Astyanax lacustris bred in fish farm in the State of Mato Grosso do Sul, Brazil was obtained in order to estimate their phylogenetic position among other platysporines myxosporean. The prevalence of the parasite was 28.1% and the range intensity was 1-3 plasmodia per fish. The shape and measurements of mature myxospores were consistent with the characteristics previously defined to H. chydadea. The SSU rDNA sequence of the myxospores of H. chydadea resulted in a total of 1405 nucleotides, and this sequence did not match any of the myxozoan available in the GenBank. Phylogenetic analysis showed H. chydadea within the clade of histozoic myxosporeans and closed together with Henneguya rotunda and Myxobolus pantanalis reported in the gill arch and fins and gill filaments of Salminus brasiliensis respectively. Nonetheless, the SSU rDNA sequences of H. chydadea, H. rotunda and M. pantanalis have only 85.2% and 84.4% similarity, respectively. This is the first molecular study of a Henneguya species that parasitizes a fish belonging to the genus Astyanax in South America. The importance of myxosporeans introduction to new locations along with infected cultured host is emphasized.

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.

Occurrence of two novel actinospore types (Cnidaria: Myxozoa) in fish farms in Mato Grosso do Sul state, Brazil.

We investigated the involvement of oligochaetes in the life cycles of fresh water myxozoan parasites in Brazil. In a fish farm in the State of Mato Grosso do Sul, we examined 192 oligochaetes and found that two (1%) released Aurantiactinomyxon type actinospores. We identified infected oligochaetes by morphology: both were Pristina synclites, from family Naididae. This is the first report of the involvement of this species in the life cycle of myxozoans. Small-subunit ribosomal DNA sequences of Aurantiactinomyxon type 1 (1882 nt) and Aurantiactinomyxon type 2 (1900 nt) did not match any previously sequenced myxozoan in the NCBI database, with the highest BLAST search similarities of 83% with Myxobolus batalhensis MF361090 and 93% with Henneguya maculosus KF296344, respectively, and the two aurantiactinomyxons were only 75% similar to each other (over ~ 1900 bases). Phylogenetic analyses showed that Aurantiactinomyxon type 1 had closest affinities with myxozoans from fish hosts in Order Characiformes, and Aurantiactinomyxon type 2 had affinities with myxozoans from fish of Order Siluriformes.

New myxosporeans parasitizing Phractocephalus hemioliopterus from Brazil: morphology, ultrastructure and SSU-rDNA sequencing.

Myxozoans are a diverse group of parasitic cnidarians, with some species recognized as serious pathogens to their hosts. The present study describes 2 new myxobolid species (Myxobolus figueirae sp. nov. and Henneguya santarenensis sp. nov.) infecting skin and gill filaments of the Amazonian pimelodid fish Phractocephalus hemioliopterus, based on ultrastructural, histology and phylogenetic analysis. The fish were caught in the Amazon River, Pará, Brazil. The plasmodial development of M. figueirae sp. nov. was in the dermis and those of H. santarenensis sp. nov. were of the intralamellar type. For both species, the plasmodia were surrounded by a connective tissue layer, but there was no inflammatory infiltrate. For M. figueirae sp. nov., mature spores were ovoid measuring 9.1 to 10 (9.5 ± 0.3) µm in length, 5.8 to 6.9 (6.4 ± 0.3) µm in width and 4.4 to 4.5 (4.5 ± 0.1) µm in thickness. Two polar capsules were elongated and of unequal size. For H. santarenensis sp. nov., mature spores were ellipsoidal in the frontal view, measuring 26.3 to 36.1 (31.9 ± 3) µm in total length, 9.6 to 11.9 (10.8 ± 0.5) µm in body length, 3.7 to 4.9 (4.3 ± 0.3) µm in width and 16.6 to 25.6 (21 ± 3.1) µm in caudal process. The polar capsules were elongated and of equal size. Phylogenetic analysis, based on partial small subunit ribosomal DNA (SSU rDNA) sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data, showed M. figueirae sp. nov. and H. santarenensis sp. nov. clustering in distinct lineages. While H. santarenensis sp. nov. clustered in a well-supported subclade composed of Henneguya species that infect gills of South American pimelodid hosts, M. figueirae sp. nov. clustered in a weakly supported subclade containing parasite species of bryconid hosts.

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.

Novel Henneguya spp. (Cnidaria: Myxozoa) from cichlid fish in the Amazon basin cluster by geographic origin.

We describe three new Henneguya spp. (Myxobolidae) found parasitizing two species of cichlid fish from the Amazon basin, Brazil: H. tucunarei n. sp. from gill filaments of Cichla monoculus and H. tapajoensis n. sp. from gill filaments of Cichla pinima, both from the Tapajós River, Pará State and H. jariensis n. sp. in the fins of Cichla monoculus from the Jari River, Amapá State. We based descriptions on myxospore morphology and small subunit ribosomal DNA sequences, and used a phylogenetic analysis to compare the new Henneguya species with known relatives. Spores of the three species had similar morphology and morphometrics, but differed molecularly 5-7.5%, and were no more than 94% similar to any other sequence in GenBank. Together with having different hosts, these data supported the diagnosis of the parasites as distinct, novel species. Maximum likelihood and Bayesian analyses showed that H. tucunarei n. sp., H. tapajoensis n. sp., and H. jariensis n. sp. plus Henneguya paraensis (which parasitizes Cichla temensis) formed a well-supported sub-clade of Henneguya parasites of cichlids from the Amazon basin, in a lineage sister to those in characiforms hosts. Our analysis was consistent with previous studies that suggest that aquatic environment and vertebrate host group are the strongest correlates with phylogenetic signals in the Myxobolidae.

Characterization and gene expression analysis of pacu (Piaractus mesopotamicus) inducible nitric oxide synthase (iNOS) following Aeromonas dhakensis infection.

Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red-bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48 h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu.