A new species of Myxozoa, Henneguya rondoni n. sp. (Myxozoa), from the peripheral nervous system of the Amazonian fish, Gymnorhamphichthys rondoni (Teleostei)

Henneguya rondoni n. sp. found in the peripheral lateral nerves located below the two lateral lines of the fish Gymnorhamphichthys rondoni (Teleostei, Rhamphichthyidae) from the Amazon river is described using light and electron microscopy. Spherical to ellipsoid cysts measuring up to 110 microm in length contained only immature and mature spores located in close contact with the myelin sheaths of the nervous fibres. Ellipsoidal spores measured 17.7 (16.9-18.1)-microm long, 3.6 (3.0-3.9)-microm wide, and 2.5 (2.2-2.8)-microm (n=25) thick. The spore body measuring 7.0 (6.8-7.3)-microm long was formed by two equal symmetric valves, each with an equal tapering tail 10.7 (10.3-11.0) microm in length. The tails were composed of an internal dense material surrounded by an external homogeneous sheath of hyaline substance. The valves surrounded two equal pyriform polar capsules measuring 2.5 (2.2-2.8)-microm long and 0.85 (0.79-0.88)-microm (n=25) wide and a binucleated sporoplasm cell containing globular sporoplasmosomes 0.38 (0.33-0.42) microm (n=25) in diam. with an internal eccentric dense structure with half-crescent section. Each polar capsule contains an anisofilar polar filament with 6-7 turns obliquely to the long axis. The matrix of the polar capsule was dense and the wall filled with a hyaline substance. The spores differed from those of previously described species. Based on the ultrastructural morphology of the spore and specificity to the host species, we propose a new species name H. rondoni n. sp.     

A new myxozoan parasite from the Amazonian fish Metynnis argenteus (Teleostei, Characidae): light and electron microscope observations

Myxobolus metynnis n. sp. (Phylum Myxozoa) is described in the connective subcutaneous tissues of the orbicular region of the fish, Metynnis argenteus (Characidae), collected in the lower Amazon River, near the city of Peixe Boi, Pará State, Brazil. Polysporic, histozoic plasmodia were delimited by a double membrane with numerous microvilli on the peripheral cytoplasm. Several life-cycle stages, including mature spores, were observed. An envelope formed by numerous fine and anastomosed microfibrils was observed at the spore surface. The spore body presented an ellipsoidal shape and was about 13.1 microm long, 7.8 microm wide, and 3.9 microm thick. Elongated-pyriform polar capsules were of equal size, measuring 5.2 microm in length, 3.2 microm in width, and possessing a polar filament with 8-9 turns around the longitudinal axis. The binucleated sporoplasm contained a vacuole and numerous sporoplasmosomes. These were circular in cross-section, showing an adherent eccentric, dense structure, with a half-crescent section. Based on the morphological differences and host specificity, we propose that the parasite is a new species named Myxobolus metynnis n. sp.     

Ultrastructural data on the spore of Myxobolus maculatus n. sp. (phylum Myxozoa), parasite from the Amazonian fish Metynnis maculatus (Teleostei)

Light and electron microscopy studies of a myxosporean, parasitic in the intertubular interstitial tissue of the kidney of the freshwater teleost fish Metynnis maculatus Kner, 1860 (Characidae) from the lower Amazon River (Brazil), are described. We observed polysporic histozoic plasmodia delimited by a double membrane and with several pinocytic channels and containing several life cycle stages, including mature spores. The spore body was of pyriform shape and was 21.0 microm long, 8.9 microm wide and 7.5 microm thick. Elongated-pyriform polar capsules were of equal size (12.7 x 3.2 microm) and contained a polar filament with 14 or 15 coils. The spore features fit those of the genus Myxobolus. Densification of the capsular primordium matrix, which increased in density from the inner core outwards, differentiating at the periphery into small microfilaments measuring 45 nm each, and tubuli arranged in aggregates and dispersed within the capsular matrix of the mature spores, are described. Based on the morphological differences and specificity of the host, we propose the creation of a new species named Myxobolus maculatus n. sp.     

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.     

Ultrastructural studies of Henneguya rhamdia n. sp. (Myxozoa) a parasite from the Amazon teleost fish, Rhamdia quelen (Pimelodidae)

Henneguya rhamdia n. sp. is described in the gill filaments of the teleost fish Rhamdia quelen, collected from the Peixe Boi River, State of Pará, Brazil. This myxosporean produced spherical to ellipsoidal plasmodia, up to 300 microm in diameter, which contained developmental stages, including spores. Several dense bodies up to 2 microm in diameter were observed among the spores. The spore body was ellipsoidal (13.1 microm in length, 5.2 microm in width, and 2.5 microm in thickness) and each of the two valves presented a tapering tail (36.9 microm in length). These valves surrounded the binucleated sporoplasm cell and two equal ellipsoidal polar capsules (4.7 x 1.1 microm), which contained 10-11 (rarely 12) polar filament coils. The sporoplasm contained sporoplasmosomes with a laterally eccentric dense structure with a half-crescent section. Based on the data obtained by electron microscopy and on the host specificity, the spores differed from previously described Henneguya species, mainly in their shape and size, number and arrangement of the polar filament coils, and sporoplasmosome morphology.     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

Henneguya schizodon n. sp. (Myxozoa, Myxobolidae), a parasite of the Amazonian teleost fish Schizodon fasciatus (Characiformes, Anostomidae)

A new histozoic species of myxosporean (Henneguya schizodon n. sp.) is described from the Amazon River teleost fish Schizodon fasciatus Spix & Agassiz, 1892 (Characiformes, Anostomidae). The plasmodia, which showed asynchronous development, were located in the kidney of the host. The spore body was ellipsoidal and was 13.1 (12-14) micron long by 3.3 (3-4) micron wide. The total length of the spore was 28.9 (27-30) micron, and each value had a caudal process measuring 16.3 (15-17) micron. The polar capsules were 5.4 (5-6) micron long by 1.3 (1-1.5) micron wide, and each had a polar filament with 8-10 coils. The characteristics of the species were compared with nearly all the species described so far, including all the species reported from South American fishes. This comparison allows to consider the materials as a new species, and the name Henneguya schizodon n. sp. is proposed.     

Myxobolus insignis sp. n. (Myxozoa, Myxosporea, Myxobolidae), a parasite of the Amazonian teleost fish Semaprochilodus insignis (Osteichthyes, Prochilodontidae)

A new myxosporean species is described from the fish Semaprochilodus insignis captured from the Amazon River, near Manaus. Myxobolus insignis sp. n. was located in the gills of the host forming plasmodia inside the secondary gill lamellae. The spores had a thick wall (1.5-2 microm) all around their body, and the valves were symmetrical and smooth. The spores were a little longer than wide, with rounded extremities, in frontal view, and oval in lateral view. They were 14.5 (14-15) microm long by 11.3 (11-12) microm wide and 7.8 (7-8) microm thick. Some spores showed the presence of a triangular thickening of the internal face of the wall near the posterior end of the polar capsules. This thickening could occur in one of the sides of the spore or in both sides. The polar capsules were large and equal in size surpassing the midlength of the spore. They were oval with the posterior extremity rounded, and converging anteriorly with tapered ends. They were 7.6 (7-8) microm long by 4.2 (3-5) microm wide, and the polar filament formed 6 coils slightly obliquely to the axis of the polar capsule. An intercapsular appendix was present. There was no mucous envelope or distinct iodinophilous vacuole.     

Production and characterization of collagenase from a new Amazonian Bacillus cereus strain

Abstract

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39?±?5.15?U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45?°C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2–11.0 and 25–50?°C, respectively) and was strongly inhibited by 10?mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76?kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.     

A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the Teleostean fish, Potamorhaphis guianensis from the River Amazon. Morphological, ultrastructural and molecular characterization

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Light and ultrastructural data on Henneguya testicularis n. sp. (Myxozoa,Myxobolidae), a parasite from the testis of the Amazonian fish Moenkhausia oligolepis

Light and electron microscopical data on a myxosporidian found in the testis of Moenkhausia oligolepis Gunther (Teleostei, Characidae, Tetragonopterinae) from the lower Amazon River near Belém, Brazil, are described. Based on spore morphology, we conclude that this species belongs to the family Myxobolidae and genus Henneguya. Mature spores (total length 27.5 (27.0–28.5) µm) were observed at the periphery of the testis. The ellipsoidal spore body consists of two unequal shell valves adhering together along the suture lines. The spore body is ellipsoidal, 14.0 µm long and 6.5 µm wide. Each valve tapers to a single caudal projection, forming a 13.5 µm long tail (13.0–14.5 µm). The spore is surrounded by a homogenous dense sheath. The polar capsules, measuring 9.0 × 2.0 µm, contain 12–13 coils of the polar filaments. Morphological differences between this material and other species of the genus Henneguya indicated the erection of a new species, which was named H. testicularis. The taxonomic affinities of this parasite are discussed.     

Morphological and molecular characterization of a new microsporidian species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae)

A new microsporidian species is described from the predatory mite Metaseiulus (formerly Typhlodromus or Galendromus) occidentalis (Nesbitt) (Acari, Phytoseiidae). The ultrastructure of this new species is presented together with the first molecular characterization for a microsporidium of mites. All stages of this new microsporidium are haplokaryotic and develop in direct contact with the host-cell cytoplasm. Sporogony is disporoblastic and spores are formed in eggs, immature stages, and adults of M. occidentalis. There are two morphological classes of spores, one with a short polar filament (3-5 coils) that measured 2.53 x 1.68 microm and one with a longer polar filament (8-9 coils) that measured 3.14 x 1.77 microm. Horizontal transmission of this new species occurs by cannibalism of eggs and other stages and perhaps involves the spores with the long polar filament. Spores with the short polar filament may play a role in autoinfection and vertical (transovarial) transmission that is highly efficient in transferring the microsporidium from adults to progeny. Analysis of the small subunit ribosomal DNA indicated that this species from M. occidentalis is most closely related to the Nosema/Vairimorpha clade of microsporidia. A conflict between the morphological and molecular data is discussed. The species is compared to previously described microsporidia of arachnids resulting in creation of Oligosporidium occidentalis n. sp. in the family Unikaryonidae.     

A new Culicoides from the Amazonian region, Brazil (Diptera: Ceratopogonidae)

A new Neotropical species of biting midge Culicoides (Haematomyidium), C. kampa Felippe-Bauer, Veras & Castellon, is described and illustrated based on female specimens from the Amazonian Region.     

Ultrastructural aspects of the myxosporean Henneguya astyanax n. sp. (Myxozoa: Myxobolidae), a parasite of the Amazonian teleost Astyanax keithi (Characidae)

This study reports light and electron microscopical aspects of a myxosporean found in the gills of the freshwater teleost Astyanax keithi Géry, Planquete & Le Bail, 1996 (family Characidae), collected from the estuarine region of the Amazon River, near Belém, Brazil. The prevalence of infection was 23%. In interlamellar spaces of the gills, ellipsoidal whitish cyst-like plasmodia structures were present, which contained spores. The spores had a spermatozoa-like appearance (47.8 +/- 0.71 microm in total length) with a fusiform body (15.2 +/- 0.77 pm in length, 5.7 +/- 0.71 microm in width and 4.2 +/- 0.31 microm in thickness), and each of the 2 valves presented a tapering tail (32.6 +/- 1.11 microm in length). The valves surrounded a binucleate sporoplasm cell and 2 polar capsules (5.0 +/- 0.13 microm in length, 1.5 +/- 0.07 microm in width) that contained 8 to 9 coils of the polar filament. In the sporoplasm, several unique sporoplasmosomes were visible. A synoptic table of spore measurements of known Brazilian Henneguya species is presented. The spores differed from those of previously described species. Based on spore morphology, it is concluded that this species belongs to the family Myxobolidae, genus Henneguya, and that it constitutes a new species: H. astyanax n. sp.     

Ultrastructural and molecular identification of a new Rickettsia endosymbiont in the springtail Onychiurus sinensis (Hexapoda, Collembola)

In this paper we provide microscopic and molecular evidence for the presence of an endosymbiontic bacterium in male and female gonads of the soil arthropod Onychiurus sinensis. The sequence of the gene encoding for the 16S rRNA shows that the bacterium is a member of the genus Rickettsia, and some anomalies presumably associated with the presence of these microorganisms have been detected. Although the Rickettsia found in O. sinensis has the smallest genetic divergence with Rickettsia bellii, the phylogenetic analysis fails to find support for a sister-group relationship between these two species, rather suggesting that most Rickettsia species/strains isolated in various arthropods have rapidly evolved and diversified in what appears to be a sudden burst of evolution.     

Leishmania (Viannia) utingensis n. sp., a parasite from the sandfly Lutzomyia (Viannamyia) tuberculata in Amazonian Brazil

A leishmanial parasite isolated in 1977 from a specimen of the sandfly Lutzomyia tuberculata from Pará State, Amazonian Brazil, has been characterized following its comparison with other species of Leishmania from the same region, using isoenzyme profiles, monoclonal antibodies and characterization of the miniexon gene repeat, using the polymerase chain reaction technique (PCR). It is described here under the name of Leishmania (Viannia) utingensis n. sp.     

Chlorophycean parasite on a marine fish,Sillago japonica (Japanese sillago)

A green spotted Japanese sillago (Sillago japonica) was caught by a fisherman and brought to the laboratory for pathological inspection. The green spots were abundant on the lateral line and more extensively so within the mouth cavity. In both sites, green spots were embedded within the fish flesh and formed 2–3 mm dome-shaped colonies. SEM revealed these colonies to harbor numerous unknown cells with small, surface warts (ornamentations). Molecular analysis showed the cells were Desmodesmus (D. komarekii), a common freshwater coccoid green alga found in ponds and rivers worldwide. It is uncertain how the host fish came to be infected with the alga which was not merely attached externally but embedded within the flesh and inside the mouth cavity. This is the first case of parasitic form of coccoid green algae in marine fish and provides new insights into the variable nature of green algae.