Sónia Rocha

Ultrastructure and phylogeny of Ceratomyxa auratae n. sp. (Myxosporea: Ceratomyxidae), a parasite infecting the gilthead seabream Sparus aurata (Teleostei: Sparidae).

A new myxosporean parasite is described from the gall bladder of the gilthead seabream Sparus aurata in a Southern Portuguese fish farm, with basis on light and transmission electron microscopy, as well as in molecular procedures. In the bile, young and mature mono- to disporic plasmodia were elliptical and presented smooth surface membranes. Crescent-shaped myxospores measured 6.7±0.7 (5.3-7.6) μm in length and 27.0±3.0 (19.7-31.2) μm in thickness. The myxospore wall was constituted by two symmetrical valves united along a slightly curved suture line, each presenting a lateral projection with a rounded end. Two equal-sized subspherical polar capsules, measuring 3.6±0.2 (2.9-3.8) μm in length and 3.5±0.3 (2.9-3.8) μm in width, were located at the same level, each displaying a polar filament coiled in 5 turns. Molecular analysis of the SSU rRNA gene confirmed the parasite as a new member of the genus Ceratomyxa, making this the fourth report of Ceratomyxa from the gall bladder of S. aurata in the Iberian Peninsula. This reinforces the assumption that species richness of ceratomyxids in South European sparids is high, but the phylogenetic analysis performed disagrees with the existence of a common ancestor for Ceratomyxa species infecting sparid hosts, as well as their clustering according to geographical location. The main Ceratomyxa clade is not monophyletic due to the inclusion of Palliatus indecorus and Pseudoalatospora kovalevae; a situation that will probably be resolved by the taxonomic revision of these genera.

Morphology and phylogeny of Henneguya jocu n. sp. (Myxosporea, Myxobolidae), infecting the gills of the marine fish Lutjanus jocu

Henneguya jocu n. sp. (Myxosporea, Myxobolidae) is described from the gill lamellae of the marine teleost fish Lutjanus jocu, with a focus on ultrastructural and molecular features. This myxosporean forms subspherical cysts up to ∼260 μm × 130 μm long, and develops asynchronously. Mature myxospores ellipsoidal with a bifurcated caudal process. Myxospore length 10.9 ± 0.4 μm (n=50); width, 8.2 ± 0.3 μm (n=50); and thickness, 2.9 ± 0.5 μm (n=50). Two equal caudal processes, 34.1±1.0 μm long (n=50); and total myxospore length, 45.2 ± 1.0 μm (n=50). Two symmetric valves surround two ellipsoidal polar capsules, 5.0 ± 0.3 × 1.4 ± 0.2 μm (n=20), each containing an isofilar polar filament forming 4-5 coils along the inner wall of these structures, as well as a binucleated sporoplasm presenting a spherical vacuole and several globular sporoplasmosomes. Both the morphological data and molecular analysis of the SSU rDNA gene identify this parasite as a new species of the genus Henneguya. Maximum Likelihood and Maximum Parsimony analyses further indicate that the parasite clusters within others marine Myxobolidae species, forming a group alongside other Henneguya species described from marine hosts.

Description of Triangulamyxa psittaca sp. nov. (Myxozoa: Myxosporea), a New Parasite in the Urinary Bladder of Colomesus psittacus (Teleostei) from the Amazon River, with Emphasis on the Ultrastructure of Plasmodial Stages

A fish-infecting myxosporean was found in the urinary bladder of the teleostean Colomesus psittacus, collected from the Amazon River, Brazil. Specimens were sampled in three different periods: May and June, with water temperature ranging from 18-23oC; August, with water temperature ranging from 24-28oC; and November and December, with water temperature ranging from 29-32oC. Upon observation, several fish displayed abnormal behaviour, consisting of erratic movements, and mortality was recorded among them. Necropsy of all sampled fishes revealed hypertrophy of the urinary bladder only among specimens previously displaying the irregular behaviour. Microscopic analysis of this organ confirmed the parasitic infection, resulting in the observation of spores floating free in the urine, and numerous plasmodia attached to the epithelium of the urinary bladder. Light and ultrastructural studies allowed recognition of the spores and plasmodia morphological characteristics. Coelozoic plasmodia were polysporic with varying organizational structure, according to the sampling period. Spores were equilaterally triangular with rounded ends in valvar view, measuring 8.8 ± 0.4 μm (n = 30) in length and 8.4 ± 0.5 μm (n = 30) in width, and displaying a ridge surface pattern. Two polar capsules were observed in the anterior end of the spores, measuring 3.1-3.2 μm in diameter. The spores were morphologically identified as belonging to the recently described genus Triangulamyxa. Further observation and comparison to the morphological features described for Triangulamyxa amazonica, the only other species within this genus, allowed us to conclude our parasite as a new species, herein named Triangulamyxa psittaca sp. nov. from the Amazon River, Brazil. Also, three different stages were distinguished in the plasmodium evolution, based on the observed morphological features at the three sampling periods. Fish sampled during May and June displayed small plasmodia (up to ~ 15-20 μm long), containing early stages of sporogenic development. Fish sampled during November and December presented larger plasmodia (up to ~ 850 μm long), which appeared flattened against and lining the urinary bladder epithelial cells and contained the later stages of sporogenic development, including some mature spores. Fish sampled during August presented plasmodia displaying intermediate morphological features between those observed in infected fish from the other sampling periods. Several immature and mature spores were among the different developmental stages. The parasite-host interface evolution is described throughout the different observed stages, with emphasis on the formation of septate junctions. Considering several previous reports, as well as the different environmental conditions during the sampling periods, the plasmodium development here described appears to be influenced by environmental factors, namely water temperature.

Morphology and Phylogeny of Thelohanellus marginatus n. sp. (Myxozoa: Myxosporea), a Parasite Infecting the Gills of the Fish Hypophthalmus marginatus (Teleostei: Pimelodidae) in the Amazon River

Thelohanellus marginatus n. sp., a new myxosporean parasite infecting the primary gill filaments of the teleost fish Hypophthalmus marginatus (Pimelodidae) in the Amazon River, is described on the basis of microscopic and molecular procedures. The parasite forms whitish and ellipsoidal cysts up to 250 μm in diam. Myxospores ellipsoidal with a slightly more pointed anterior end, measuring 17.1 ± 0.6 μm in length, 6.9 ± 0.4 μm in width, and 5.1 ± 0.5 μm in thickness. A single pyriform polar capsule, 9.0 ± 0.3 μm long and 6.1 ± 0.4 μm wide, positioned slightly right to the medial plane in valvular view, contains a polar filament arranged in 4–5 coils. Molecular analysis of the SSU rRNA gene by Maximum Parsimony, Neighbor‐Joining, and Maximum Likelihood revealed the parasite clustering among other myxobolids, namely Henneguya and Myxobolus. Host affinity is supported as an important evolutionary signal for the phylogeny of myxobolids. The parasite here described represents the first record of the genus Thelohanellus Kudo, 1933 from the South American fauna.

Ortholinea auratae n. sp. (Myxozoa, Ortholineidae) infecting the urinary bladder of the gilthead seabream Sparus aurata (Teleostei, Sparidae), in a Portuguese fish farm

A new myxosporean species, Ortholinea auratae n. sp., is described from the gilthead seabream, Sparus aurata Linnaeus, 1758 (Teleostei, Sparidae) from a fish farm in Algarve, Portugal. Plasmodia and spores were found in the urinary bladder and, less frequently, in the posterior kidney. Plasmodia were polymorphic, presenting an irregular cellular membrane due to the presence of several peripheral projections, which in turn were covered by a glycocalyx-like sheet. Mature spores were subspherical in valvular view and ellipsoidal in sutural view, measuring 9.0 ± 0.3 (8.2–10.1) μm in length, 8.3 ± 0.4 (7.5–9.1) μm in width, and 7.2 ± 0.5 (6.3–8.4) μm in thickness. The two valves comprising the spores displayed an intricate pattern of surface ridges and were also enveloped by a glycocalyx-like sheet. Two subspherical polar capsules, 3.2 ± 0.1 (2.9–3.6) μm long and 2.7 ± 0.1 (2.4–2.9) μm wide, were located at the anterior pole and displayed divergent orientation. The polar filament coiled in three to four turns. The comprehensive analysis of the parasite’s ultrastructural observations and molecular data for the small subunit (SSU) ribosomal DNA (rDNA) gene identify O. auratae n. sp. as a new species, clustering together with other myxosporeans infecting the excretory system to form a subclade of the main freshwater clade.

Ultrastructure and phylogeny of Glugea nagelia sp. n. (Microsporidia: Glugeidae), infecting the intestinal wall of the yellowfin hind, Cephalopholis hemistiktos (Actinopterygii: Serranidae), from the Red Sea.

A new microsporidian species of the genus Glugea Thélohan, 1891 parasitising the marine teleost fish Cephalopholis hemistiktos Rüppell, collected from the Red Sea in Saudi Arabia, is described on the basis of microscopic and molecular procedures. Spherical and whitish xenoma were observed adhering to the intestinal wall. The numerous spores contained within these xenoma, were ovoid to pyriform and measured 4.3-6.0 µm (5.1 µm) in length and 1.8-2.9 µm (2.2 µm) in width. The spores wall was composed of two thick layers, which were thinner in the area contacting the anchoring disk. The latter appeared at the spores anterior pole, in an eccentric position to the longitudinal axis. A lamellar polaroplast surrounded the uncoiled portion of the polar filament projected to the basal region of the spore, giving rise to 26-29 turns with winding from the base to the anterior zone of the spore. The posterior vacuole, located at the spores posterior pole, and surrounded by the polar filament coils, was irregular and composed of light material. Molecular analysis of the rRNA genes, including the ITS region, was performed using maximum parsimony, neighbour-joining and maximum likelihood methods. The ultrastructural features observed, combined with the phylogenetic data analysed, suggest this parasite to be a new species of the genus Glugea. This is the first species of this genus to be reported from Saudi Arabia and is herein named Glugea nagelia sp. n.

Ultrastructural description of Ceratomyxa microlepis sp. nov. (Phylum Myxozoa): a parasite infecting the gall bladder of Hemiodus microlepis, a freshwater teleost from the Amazon River

A new ceratomyxid parasite was examined for taxonomic identification, upon being found infecting the gall bladder of Hemiodus microlepis (Teleostei: Hemiodontidae), a freshwater teleost collected from the Amazon River, Brazil. Light and transmission electron microscopy revealed elongated crescent-shaped spores constituted by two asymmetrical shell valves united along a straight sutural line, each possessing a lateral projection. The spores body measured 5.2 ± 0.4 µm (n = 25) in length and 35.5 ± 0.9 µm (n = 25) in total thickness. The lateral projections were asymmetric, one measuring 18.1 ± 0.5 µm (n = 25) in thickness and the other measuring 17.5 ± 0.5 µm (n = 25) in thickness. Two equal-sized subspherical polar capsules measuring 2.2 ± 0.3 µm in diameter were located at the same level, each possessing a polar filament with 5-6 coils. The sporoplasm was binucleate. Considering the morphometric data analyzed from the microscopic observations, as well as the host species and its geographical location, this paper describes a new myxosporean species, herein named Ceratomyxa microlepis sp. nov.; therefore representing the first description of a freshwater ceratomyxid from the South American region.

Morphological and Molecular Characterization of a New Myxozoan Species (Myxosporea) Infecting the Gall Bladder of Raja clavata (Chondrichthyes), from the Portuguese Atlantic Coast

Abstract: Microscopic and molecular procedures are used to describe a new myxosporean species, Chloromyxum clavatum n. sp., infecting the cartilaginous fish Raja clavata Linnaeus, 1758 (Chondrichthyes: Rajidae), collected from the northwest Atlantic coast of Portugal. Young plasmodia and mature spores were found floating free in the gall bladder of R. clavata. Spores were spherical to subspherical with a pointed anterior end, measuring14.4 ± 0.5 μm (n = 25) in length, 11.9 ± 0.5 μm (n = 25) in width, and 9.4 ± 0.5 μm (n = 15) in thickness. The spores wall was composed of 2 equally sized valves, each displaying 6–8 elevated surface ridges and a bundle of several tapering caudal filaments attached to the basal portion. Spores contained 4 pyriform equally sized polar capsules (5.5 ± 0.4 μm × 2.9 ± 0.5 μm) (n = 25), each possessing an obliquely arranged isofilar polar filament coiled in 7–8 coils. Morphological data, host specificity, tissue tropism, and molecular analysis of the SSU rDNA gene identify this parasite as a new species of Chloromyxum. Neighbor-joining and maximum likelihood further reveal the parasite clustering with other species of Chloromyxum infecting the gall bladder of marine cartilaginous fish to form a clade positioned at the base of the freshwater clade, therefore constituting an exception to the major division of the class Myxosporea into the freshwater and marine clades, while supporting the existence of a correlation between tissue tropism and myxosporean phylogeny.

Ultrastructure and phylogeny of the parasite Henneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil.

Microscopic and molecular procedures are used to describe a new myxosporean species, Henneguya carolina sp. nov., found infecting the intestine of the marine teleost fish Trachinotus carolinus on the southern Atlantic coast of Brazil. Spherical to ellipsoid cysts, measuring up to ~750 µm, display synchronous development. Mature myxospores are ellipsoidal with a bifurcated caudal process. Myxospore body length, width, and thickness are 12.7 ± 0.8 (12.0-13.4) µm, 8.8 ± 0.6 (7.5-9.6) µm, and 5.8 ± 0.4 (5.0-6.4) µm, respectively; 2 equal caudal processes are 16.8 ± 1.1 (15.9-18.0) µm long, and the total myxospore length is 29.4 ± 0.8 (28.4-30.4) µm. Two pyriform polar capsules measure 5.0 ± 0.5 (4.6-5.6) × 2.4 ± 0.4 (1.9-2.9) µm, and each contains a polar filament forming 3 to 4 coils. Sporoplasm is binucleated and presents a spherical vacuole surrounded by numerous globular sporoplasmosomes. Molecular analysis of the small subunit rRNA gene by maximum parsimony, neighbor joining, and maximum likelihood reveals the parasite clustering together with other myxobolids that are histozoic in marine fish of the order Perciformes, thereby strengthening the contention that the host phylogenetic relationships and aquatic environment are the strongest evolutionary signal for myxosporeans of the family Myxobolidae.

Ultrastructure and phylogeny of Glugea arabica n. sp. (Microsporidia), infecting the marine fish Epinephelus polyphekadion from the Red Sea.

A new microsporidian species, Glugea arabica n. sp., is reported infecting the intestinal wall of the marine teleost Epinephelus polyphekadion (=microdon) collected from the Red Sea coast off Saudi Arabia, and described on the basis of microscopic and molecular procedures. Spherical blackish xenomas formed parasitophorous vacuoles completely packed with several parasitic developmental stages, including spores. The nuclei were monokaryotic in all developmental stages. Spores were ellipsoidal to pyriform and measured 6.3 ± 0.3 (5.9-6.6) μm in length and 3.3 ± 0.4 (2.9-3.7) μm in width. A lamellar polaroplast surrounded the uncoiled portion of the polar filament, which extended into the spores posterior pole and formed 27-29 coils organized in three or four rows. The posterior vacuole, located at the spores posterior pole, appeared surrounded by the polar filament coils and displayed an irregular matrix composed of light material, in which was located the posterosome. Molecular analysis of the rRNA genes, including the ITS region, was performed using maximum parsimony, neighbor-joining and maximum likelihood methodologies. The ultrastructural features observed, in combination with the molecular data analysed, suggests the parasite to be a new species of the genus Glugea.