Cultivation and Lyophilization of Mastigina sp.

SYNOPSIS Mastigina sp. is an amoeboid flagellate isolated from pine frass collected in the Guadarrama Mountains in Spain. It feeds on bacteria and yeasts. It prefers yeasts that produce extracellular polysaccharides, and the 2 species that have been used predominantly for cultivation of the flagellate are Pachysolen tannophilus and Hansenula holstii. Mastigina sp. is easily isolated in axenic culture and grows abundantly therein. Its locomotive form, averaging 27 μ in length, resembles that of a limax amoeba, with a vesiculate nucleus at the anterior end. Cells are capable of simultaneous movement by pseudopodia and flagella. It develops rapidly on dead or living yeast cells in shaken cultures and the trophozoites may convert quantitatively to cysts. The cysts remain viable for long periods of time in refrigerated suspensions and in the lyophilized state. They are spherical or ovoid and smooth-walled cysts; the trophozoite emerges from them by breaking the wall.     

Two species of Entamoeba from white-tailed deer, Odocoileus virginianus, from Georgia

Large numbers of 2 species of Entamoeba, Entamoeba coli and Entamoeba bovis, were recovered from captive white-tailed deer from Georgia. The trophozoite of the first ameba was a large form with a well-defined vesicular nucleus bearing a prominent eccentric endosome, abundant peripheral chromatin, and numerous periendosomal granules. Cytoplasmic vacuoles containing bacteria and other substances were common. Mature cysts were octonucleate. Glycogen vacuoles and irregularly shaped chromatoids were frequently present. This ameba was identified as Entamoeba coli. The trophozoite of the second ameba was small, with a typical Entamoeba nucleus. Cysts were uninucleate, containing numerous chromatoids resembling bars, splinters of wood, and irregular masses. There was occasionally a glycogen vacuole. This ameba was identified as Entamoeba bovis.     

Light and Electron Microscopy of the Spore of Myxobolus heckelii n. sp. (Myxozoa), Parasite from the Brazilian Fish Centromochlus heckelii (Teleostei, Auchenipteridae)

ABSTRACT. A myxosporean parasitizing the gill filaments of the freshwater teleost fish Centromochlus heckelii collected in the Tocantins River (Lower Amazonian Region, Brazil) is described using light and electron microscopy. This parasite produces spherical to ellipsoidal cyst-like plasmodia up to 250 μm in diameter, with a thick wall strengthened by several stratified juxtaposed crossed collagen layers, whose thickness varies according to the number of the layers. Several compressed fibroblasts are observed among the collagen fibrils. Deposits of spherical dense material are scattered at the internal periphery of the cysts. Plasmodia and different developmental stages, including immature and mature spores, filled the central region of the cysts. The spore body is ellipsoidal in valvar view and biconvex in sutural view. It is formed by two equal-sized and symmetric valves measuring 12.7 μm long (12.2–13.1) ( n =50), 6.6 μm wide (6.3–6.9) ( n =25), and 4.0 μm (3.7–4.4) ( n =20) thick. A thin layer formed by fine and anastomosed microfibrils is observed at the spore surface. Two equal, elongated pyriform polar capsules measure 2.9 μm (2.7–3.3) × 1.7 μm (1.4–2.0) ( n =25), each containing four or five oblique polar filament coils. The binucleated sporoplasm contains numerous spherical sporoplasmosomes, glycogen particles, and a large vacuole with fine granular matrix. Based on the morphological and ultrastructural differences and specificity of the host, we describe this isolate as a new myxosporidian, Myxobolus heckelii n. sp. (Myxozoa, Myxosporea).     

Heat shock protein of HSP70 family revealed in some contemporary freshwater Amoebae and in Acanthamoeba sp. from cysts isolated from permafrost samples

A heat shock protein of HSP70 family was revealed for the first time in trophozoites of Acanthamoeba sp. (strain Am8) excysted from cysts previously isolated from samples of permafrost aging 30,000-35,000 years. The constitutive level of this HSP, shown by immunnoblotting in unstressed trophozoites of the ancient acanthamoebae, much surpassed that in unstressed cells of the three examined species of contemporary freshwater amebae of the genus Amoeba.     

Sphaerospora epinepheli N. Sp. (Myxosporea: Sphaerosporidae) Observed in Grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus , in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6–22.9 μm (length) × 8.4–21.6 μm (width). Spores were 7.8–10.0 μm (length) × 12.3–14.5 μm (thickness), and 7.0–9.5 μm (width) with two spherical polar capsules of equal size measuring 2.9–4.4 μm in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

The endogenous development of three new intranuclear species of Isospora (Apicomplexa: Eimeriidae) from agamid lizards

Three new species of Isospora Schneider, 1881 are described from agamid lizards, Isospora cannoni n. sp. in Diporiphora australis from northern Queensland, Australia, I. choochotei n. sp. in Calotes mystaceus from northern Thailand, and I. deserti n. sp. in Agama pallida from Israel. I. cannoni oöcysts are subspherical, 20–25 × 22.5–27.5 m with two ovoid sporocysts, 14–15.5 × 10–11.5 m. I. choochotei oöcysts are spherical to subspherical, 24–32 × 28–32.5 m with two ovoid sporocysts, 11 × 15.5–18 m. I. deserti oöcysts are spherical, 25–28 m in diameter with two ovoid sporocysts, 10–11 × 14–17.5 m. All species had sporocysts with Stieda bodies and underwent endogenous development in the nucleus of the host gut epithelial cells. At completion of merogony and gamogony, the host nucleus was reduced to a thin envelope. The significance of endogenous stage characteristics in Isospora taxonomy is discussed.     

Hexamastix dobelli n.sp., a New Trichomonad Parasitic in the Starred Tortoise

Hexamastix dobelli n. sp. (Trichomonadidae) is described from the large intestine of the starred tortoise, Testu-do elegans Schoepff. This is the first species of Hexamastix to be reported from a chelonian. The organism is pyriform, oval, or round in shape. There are five anterior flagella, of which two are long and three short and a single recurrent flagellum, which equals the short anterior flagella in length. The axostyle is very slender and projects for some distance outside the body. The nucleus contains three to six granules and has no endosome. The pelta is crescent-shaped and the parabasal body is rod-like. Perinuclear granules are present in the anterior half of the body. The parasite is 6.0 to 16.5 μ long, 2.0 to 10.3 μ wide and the diameter of nucleus is 2.5 μ.     

The Occurrence of Amphizoic Amebae in Domestic Animals

SYNOPSIS Random examination of domestic animals revealed the frequent presence of free-living amebae in their bodies. In diseased or dead cows, dogs, pigs, rabbits, pigeons, and turkeys 15 strains of amebae were found, belonging to the genera Acanthamoeba (A. polyphaga), Hartmannella (H. vermiformis) , and Vahlkampfia (V. avara, V. enterica, V. inornata). , They were usually accompanied by other infectious agents in different parts of the host bodies. Pathogenicity of 3 isolates could not be demonstrated by inoculation of laboratory animals.
Some features of the isolates differed from those previously known for members of these genera. These strains may be considered amphizoic amebae according to Page (1974).     

A new purple sulfur bacterium from stratified freshwater lakes,Amoebobacter purpureus sp. nov.

Six strains of a new purple sulfur bacterium were isolated from the chemocline of four different freshwater lakes. Single cells were spherical to oval, nonmotile and contained gas vacuoles in the central part of the cytoplasm. All strains contained bacteriochlorophyll a and okenone as the major carotenoid. The intracytoplasmic membrane system was of vesicular type. All strains resembled each other in growth conditions and utilization of simple organic carbon sources. The strains were able to grow microaerophilic in the dark, used hydrogen sulfide, elemental sulfur or thiosulfate as electron donor, and lacked assimilatory sulfate reduction. On the basis of all characteristics the new bacterium represents a new species of the genus Amoebobacter, A. purpureus sp. nov.     

The endogenous development of two new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from Thai geckoes

Two new species of Isospora are described from Thai geckoes. I. platyurusi n. sp. in Cosymbotus platyurus from Chiang Mai and Khon Kaen and I. uptoni n. sp. in Hemidactylus frenatus from Chiang Mai. Oöcysts of I. platyurusi are spherical to subspherical, 19–22.5 × 16.5–21.5 μm and have two ovoid sporocysts of 11.5–14 × 7.5–9 μm. Oöcysts of I. uptoni are spherical to subspherical, 24–31.5 × 20–27.5 μm and have two ovoid sporocysts of 12.5–15 × 9–11.5 μm. Both species have sporocysts with Stieda bodies and undergo endogenous development in the nucleus of the host gut epithelial cells. On the completion of merogony and gamogony, the host nucleus is reduced to a thin envelope.     

Autoradiographic Studies of H3-Thymidine Uptake in Entamoeba histolytica in CLG Medium*

SYNOPSIS. Entamoeba histolytica grown with H³-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome. The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H³-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H³-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.     

A Dermocystidium infection in Trichomycterus sp. (Osteichthyes, Trichomycteridae)

Two whitish elongate cysts in the left pectoral fin of Trichomycterus sp. (Osteichthyes, Trichomycteridae) were packed with Dermocystidium sp. spores. The spores were spherical and had a large PAS positive central refractile body, the cytoplasm being restricted to a narrow peripheral layer containing the nucleus. The cysts were surrounded by a thin homogeneous wall of parasite origin, and there was no encapsulation of the cysts by host tissue.     

Nuclearia thermophila sp. nov. (Nucleariidae), a new nucleariid species isolated from Yunoko Lake in Nikko (Japan)

A new species of unicellular opisthokont protist, Nuclearia thermophila sp. nov., was isolated from the warm spring water of Yunoko Lake, Japan, and has been described using light and electron microscopy. It exists as a spherical floating form and a flattened amoeboid form showing various shapes. The cells occasionally extended as branches or knobbed filopodia. The spherical form when suspended in medium measured 20–40 μm in diameter (excluding filopodia). The amoeboid form may exceed 65 μm along the longest axis. A nucleus with an obvious spherical nucleolus, dictyosomes, mitochondria with flat cristae, food vacuoles, and lipid droplet-like vacuoles with homogeneous contents were observed; no extracellular matrix or bacterial endosymbionts were present. The cells ingested flour particles. No cysts were seen. The molecular phylogenetic tree constructed on the basis of the small subunit ribosomal DNA revealed the novelty of N. thermophila and its relationships with previously known nucleariids.     

Isospora gallicolumbae sp. n. from Beccari's Ground Dove (Gallicolumba beccarii Salvadori) in Papua New Guinea

SYNOPSIS. Isopora gallicolumbae sp. n. is described from Beccari's ground dove, Gallicolumba beccarii Salvadori, in Papua New Guinea. The ellipsoidal oocysts average 16.0 × 20.0 μm. The oocyst wall is light green, single-layered (∼ 0.8 μm), and becomes distorted within 10 min in sugar solution. Micropyle and oocyst residuum are absent; one polar granule is present. Sporocysts are ovoid, 8.0 × 12.0 μm. with prominent conical Stieda body. Sporocyst residuum is a spherical mass (∼ 5.0 μm) of lighter and darker granules. This is the first species of Isospora reported from a host in the avian order Columbiformes.     

Amebic Parasites of Fishes*

SYNOPSIS. Entamoeba molae n. sp. was found in the hindgut of Mola mola collected from the coast of southern California. The trophic stages are actively motile and contain numerous food vacuoles. The nuclear peripheral chromatin is usually conspicuous. A subperipheral crescent of granules is the most distinctive feature of the nucleus. The endosome is eccentric and often surrounded by a periendosomal ring. Measurements: cell body 16.4 × 22.9 μ nucleus 4.8 μ in diameter. Cysts were not seen. We believe that few amebas have been described from fishes because few fishes have been examined for amebas.     

Widespread hermaphroditism in freshwater gastrotrichs

Abstract. Freshwater members of the phylum Gastrotricha were long thought to lack male gametes and to exist exclusively as parthenogenetic females. The surprising 1978 discovery of sperm in the common species Lepidodermella squamata raised the question of how many other freshwater gastrotrichs might likewise be hermaphroditic. In a comparative study of species from across both major families, sperm have been found in every species examined intensively. They were detected in 19 species of Chaetonotidae (from Aspidiophorus, Chaetonotus, Heterolepidoderma, Ichthydium, Lepidodermella , and Polymerurus ) and 3 species of Dasydytidae (from Haltidytes, Setopus , and Stylochaeta ), characteristically occurring ventrally in single unilateral or (more often) 2–12 bilateral packets. Their shape ranges from filiform (length in Chaetonotus bisacer , ∼40 μm) to rodlike, spindlelike, oval, and possibly spherical (some in Stylochaeta scirtetica measure only 1 μm). With light microscopy, a dense nucleus appears to fill the entire volume of these aflagellate cells. Spermatogenesis within cysts (maximally, 16 sperm/clone) is evidently characteristic of both families, each cyst generating one large residual body. Sperm-bearers display oocytes with sometimes distinctive cytoplasmic elements and a posterior X-organ whose organization can be complex. Evidence supports an unusual life cycle in which parthenogenesis is followed by simultaneous hermaphroditism. These findings may illuminate the reproductive characters as well as ancestry of marine and brackish-water taxa of Chaetonotida.     

Taxonomic Criteria for Limax Amoebae,with Descriptions of 3 New Species of Hartmannella and 3 of Vahlkampfia*

SYNOPSIS. Seven species of limax amoebae were isolated into clonal, monoxenic cultures with Aerobacter aerogenes from material collected from freshwater habitats. Studies were made of their trophic structure, nuclear division, cyst structure, some aspects of cytochemistry, and other characteristics. Six new species are described: Vahlkampfia inornata, V. avara, V. jugosa, Hartmannella limacoides, H. vermiformis, and H. exundans. The well-known species Naegleria gruberi (Schardinger, 1899) is re-described on the basis of 8 strains; its flagellated phase was found to be biflagellate, with rare exceptions. A correlation exists between the manner of locomotion and the pattern of nuclear division in the limax amoebae in the family Vahlkampfiidae and those in the genus Hartmannella. Trophic amoebae of all species had a PAS-positive surface layer, altho results with H. vermiformis and H. exundans were less definite than with other species. All species except H. limacoides formed cysts in culture. The cyst walls of all cyst-forming species were strongly PAS-positive, but results of the zinc chloroiodide test for cellulose were negative with the method used. The genus Hartmannella Alexeieff, 1912, is re-defined to include those species which assume a simple, monopodial limax-like form during locomotion and have nuclear division similar to that of metazoan cells and to distinguish it from the genus Acanthamoeba Volkonsky, 1931.     

New species of Eimeria Schneider, 1875 and Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the short-crested flycatcher Myiarchus ferox (Gmelin) (Passeriformes: Tyrannidae) in South America

In the current study, two new coccidian species (Protozoa: Apicomplexa: Eimeriidae) obtained from short-crested flycatcher Myiarchus ferox (Gmelin) are reported from Brazil. Isospora feroxis n. sp. has o?cysts which are spheroidal to sub-spheroidal, 18.7 × 18.0 μm, with a smooth and bi-layered wall, c. 1.2 μm. The micropyle and o?cyst residuum are absent, but two polar granules are present. Its sporocysts are broadly ellipsoidal and 11.7 × 8.5 μm. Stieda and substieda bodies are present. A sporocyst residuum is present and the sporozoites have a refractile body and nucleus. O?cysts of Eimeria sicki n. sp. are spheroidal to sub-spheroidal, 30.3 × 28.5 μm, with a smooth and bi-layered wall, c. 1.3 μm. The micropyle, o?cyst residuum and polar granule are absent. Its sporocysts are ellipsoidal, 18.4 × 10.0 μm. Stieda and substieda bodies are present. A sporocyst residuum is present and sporozoites have a refractile body and nucleus.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.