Environmental factors and chemical agents affecting the growth of the pathogenic marine ciliate Uronema nigricans.

The scuticociliate Uronema nigricans is an opportunistically parasitic marine ciliate known to cause disease in some aquacultural environments with epizootics documented from marine larval rearing systems, marine aquaria and in southern bluefin tuna Thunnus macoyii growout enclosures. This study examined growth responses of laboratory cultures of the ciliate and prey bacteria to variations in temperature and salinity, and the efficacy of potential chemotherapeutants for control of U. nigricans infections. Differences in ciliate growth responses were marginal at temperatures of 10 to 25 degrees C and at salinities between 15 and 35 ppt, though 3.5 ppt or less was lethal. Ciliates were found to be sensitive to fluctuations in bacterial densities, which may be a factor in the seasonal occurrence of the ciliate-related disease in tuna. Commonly used chemotherapeutants such as formalin, malachite green and hydrogen peroxide were all effective against the ciliate during in vitro trials.     

Importance of passive diffusion in the uptake of polychlorinated biphenyls by phagotrophic protozoa

Unicellular protozoan grazers represent a size class of organisms where a transition in the mechanism of chlorobiphenyl (CB) introduction, from diffusion through surface membranes to ingestion of contaminated prey, could occur. This study compares the relative importance of these two processes in the overall uptake of polychlorinated biphenyls by protists. Uptake rates and steady-state concentrations were compared in laboratory cultures of grazing and nongrazing protozoa. These experiments were conducted with a 10-microm marine scuticociliate (Uronema sp.), bacterial prey (Halomonas halodurans), and a suite of 21 CB congeners spanning a range of aqueous solubilities. The dominant pathway of CB uptake by both grazing and nongrazing protozoa was diffusion. Organic-carbon-normalized CB concentrations (in the protozoan cell) were equivalent in grazing and nongrazing protozoa for all congeners studied. Rate constants for uptake into and loss from the protozoan cell were independently determined by using [3,3',4, 4'-(14)C]tetrachlorobiphenyl (IUPAC no. 77), 0.38 +/- 0.03 min(-1) and (1.1 +/- 0.1) x 10(-5) (g of organic carbon)(-1) min(-1), respectively. Magnitudes of the uptake and loss processes were calculated and compared by using a numerical model. The model result was consistent with data from the bioaccumulation experiment and supported the hypothesis that diffusive uptake is faster than ingestive uptake in phagotrophic unicellular protozoa.     

Balanion masanensis n. sp. (ciliophora: prostomatea) from the coastal waters of Korea: morphology and small subunit ribosomal RNA gene sequence

The planktonic ciliate Balanion masanensis n. sp. is described from living cells, from cells prepared by quantitative protargol staining (QPS), scanning electron microscopy (SEM), and transmitted electron microscopy (TEM) preparations, and the sequence of its nuclear small subunit rDNA (SSU rDNA) is reported. This species is almost ovoid with a flattened anterior oral region when the cells are alive and stained. The flattened anterior region of a living cell often forms a dome with the perimeter receded in a groove, and this region is easily inflated or depressed. In SEM photos, a brosse of six to nine monokinetids (or possibly three to five dikinetids) was observed inside the circumoral dikinetids. In TEM photos, circumoral microtubular ribbons were observed below the oral cilia, which along with the oral flaps were 8-16 microm in length. The cytostome is a slight funnel-like central depression on the flattened anterior end. The morphological characteristics of this ciliate are identical to those of the genus Balanion (Order Prorodontida). The ranges (and mean+/-standard deviation) of cell length, cell width, and oral diameter of living cells (n=23-26) were 27-43 microm (35.2+/-4.6), 25-32 microm (28.6+/-2.3), and 25-30 microm (27.6+/-1.3), respectively, while those of the QPS-stained specimens (n=70) were 23-37 microm (30.6+/-3.5), 26-35 microm (30.7+/-2.2), and 26-33 microm (29.5+/-1.5), respectively. Forty-six to 55 somatic kineties (SKs) were equally spaced around the cell body and extended from the oral to near the posterior regions with 24-50 monokinetids per kinety. Each kinetid bore a cilium 2.8-7.2 microm long. A caudal cilium (ca 14 microm long) arose on the posterior end. The single ellipsoid macronucleus is 6.8-13.4 x 6.8-10.5 microm, accompanied by a single micronucleus (2.0-2.8 x 1.5-2.5 microm) visible only in QPS specimens. Because, the cell size, the number of SKs, and the number of kinetosomes per SK of this ciliate were much greater than those of Balanion comatum and Balanion planctonicum, the only two Balanion species so far reported, we have established B. masanensis n. sp. When properly aligned, the sequence of the SSU rDNA of B. masanensis n. sp. (GenBank Accession No. AM412525) was approximately 9% different from that of Coleps hirtus (Colepidae, Prorodontida) and 12% different from that of Prorodon teres (Prorodontidae, Prorodontida).     

An ultrastructural and histopathological study of Henneguya pellucida n. sp. (Myxosporea: Myxobolidae) infecting Piaractus mesopotamicus (Characidae) cultivated in Brazil

During a study of myxosporean parasites of cultivated freshwater fish, a new myxosporean species, Henneguya pellucida n. sp., was discovered. Of the 120 Piaractus mesopotamicus sampled, only 10 specimens (8.3%) were infected. Yellow, round plasmodia measuring 0.5-3 mm were found in the serous membrane of the visceral cavity and in the tunica externa of the swim bladder. Sporogenesis was asynchronous, with the earliest developmental stages aligned prevailingly along the endoplasmic periphery and mature spores in the central zone. The mature spores were pear shaped (total length: 33.3 +/- 1.5 microm, mean +/- SD; width: 4.1 +/- 0.4 microm; body length: 11.4 +/- 0.3 microm; caudal process length: 24.1 +/- 1.5 microm). The polar capsules were elongated (length: 4.0 +/- 0.4 microm; width: 1.6 +/- 0.2 microm). The development of the parasite in the swim bladder produced thickening of the tunica externa and a granulomatous reaction. There was no correlation between the prevalence of the parasite and the chemical and physical characteristics of the water. Infection was recorded only in juvenile specimens ranging in size from 9.5 to 20 cm.     

Histophatology and ultrastructure of Henneguya caudalongula sp. n. infecting Prochilodus lineatus (Pisces: Prochilodontidae) cultivated in the state of São Paulo, Brazil

The histological and ultrastructural characteristics of a new species of Henneguya and the host reactions to infection by this species are reported. Henneguya caudalongula sp. n. was found in the inter and intralamellar regions of the gills of Prochilodus lineatus (Valenciennes, 1836) cultivated at Center for the Research and Management of Continental Fishing Resources located in the municipality of Pirassununga, state of S?o Paulo, Brazil. The plasmodia were white and round or ellipsoidal and measured 0.2 to 1 mm in length. The development of the parasite was asynchronous and the mature spores were fusiform, with a total length 71 +/- 1.4 microm, body length of 16.6 +/- 0.54 microm and width 4.6 +/- 0.2 microm. The caudal process was 52.6 +/- 1.5 microm long. The polar capsules were elongate (length 6.1 +/- 0.19 microm, width 1.6 +/- 0.15 microm) and of equal size. The polar filament was coiled in 10-11 turns. The prevalence of the parasite was 48.3% and did not vary significantly with the season or host size.     

Cultural characteristics of the marine ciliated protozoan, Uronema marinum Dujardin

The marine hymenostome ciliate Uronema marinum Dujardin was isolated from sediment from Robin Hood's Bay, Yorkshire. Experiments on the growth of U. marinum were carried out in batch cultures in which the ciliate was maintained on a diet of Vibrio sp. The duration of the lag phase varied directly with the age of the inoculum. Factorially designed experiments were used to investigate the effects of salinity and temperature on the rate of logarithmic growth of the ciliate. A response surface analysis of the data indicated that there was interaction between salinity and temperature. Regression equations predicted a minimum doubling time of 2.5 h at 32‰ and 25 °C. The cell volume of U. marinum varied during growth; it reached a maximum at the end of the lag phase and decreased during the logarithmic phase to become minimal in the stationary phase.     

Metacystis borrori n. sp. (Ciliophora: Metacystidae) on the seagrass Thalassia testudinum

A new epibiontic ciliate of the genus Metacystis is described on the seagrass Thalassia testudinum of the coral reef lagoons of Veracruz, Mexico. The ciliate was studied in living and stained specimens and under the scanning electron microscope. The cell body (10-35 x 10-18 microm in vivo) is transversely annulated (4-6 rings). The somatic ciliature consists of 22-30 longitudinal kineties, and patterned as 5-7 transverse kineties. The circumoral kinety is composed of kinetosomes closely spaced. The macronucleus diameter measures about 3-7 microm. The lorica (18-61 x 11-26 microm) has the posterior end round to conical or irregular with mucoid filaments. This prostomatid colonizes both natural and artificial substrates placed in an aquarium. Metacystis borrori n. sp. is a species that forms part of the ciliate community on Thalassia testudinum with a temperature range of 21-26 degrees C and a salinity of 32-40 per thousand.     

Strombidinopsis jeokjo n. sp. (ciliophora: choreotrichida) from the coastal waters off western Korea: morphology and small subunit ribosomal DNA gene sequence

The planktonic ciliate Strombidinopsis jeokjo n. sp. is described from Quantitative Protargol-Stained (QPS) preparations, and the sequence of the small subunit rDNA (SSU rDNA) from cultured cells is reported. This species is ovoid and bluntly tapered towards the posterior. The ranges (and mean +/- standard deviation, n = 31) of cell length, cell width, and oral diameter of the QPS-stained specimens were 100-190 microm (149 +/- 25), 60-105 microm (79 +/- 13), and 55-80 microm (64 +/- 5), respectively. Fifteen to seventeen external oral polykinetids had oral membranelle cilia 20-35 microm long. Twenty-six to twenty-eight somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 23-44 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-7 microm long. Strombidinopsis jeokjo had two ovoid macronuclei of 25-38 microm x 12-15 microm. When properly aligned, the sequence of the SSU rDNA of S. jeokjo (GenBank Accession No. AJ628250) was approximately 2% different from that of an unidentified Strombidinopsis species (GenBank Accession No. AF399132-AF399135), the closest species in the SSU rDNA sequence.     

Parastrombidinopsis shimi n. gen., n. sp. (Ciliophora: Choreotrichia) from the coastal waters of Korea: morphology and small subunit ribosomal DNA sequence

The planktonic ciliate Parastrombidinopsis shimi n. gen., n. sp. is described from both living cells and quantitative protargol-stained (QPS) preparations and the sequence of the small subunit rDNA (SSU rDNA) is reported. This species is almost oval when the cells are alive; when stained, it is cylindrical for the upper two-fifths, half-bowl shaped for the middle two-fifths, and narrow rodshaped for the lower one-fifth. The ranges (and mean +/- standard deviation, n = 20) of cell length, cell width, and oral diameter of living cells were 112-221 microm (168 +/- 39), 88-176 microm (121 +/- 30), and 53-110 microm (80 +/- 14), respectively, while those of the QPS-stained specimens (n = 54) were 88-225 microm (162 +/- 29), 55-163 microm (102 +/- 19), and 53-98 microm (69 +/- 9), respectively. Thirty-six to 48 external oral polykinetids had cilia 25-40 microm long. However, unlike Strombidinopsis species sensu stricto, P. shimi has an external oral polykinetid zone that is an open circle. This species has two shorter polykinetids associated with the end of the oral polykinetid zone, deep in the oral cavity. Like Strombidinopsis species in the subclass Choreotrichia, 36-50 somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 68-105 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-10 microm long. Parastrombidinopsis shimi had 2 (1-4) ovoid macronuclei of 20-82 x 15-32 microm. When properly aligned, the sequence of the SSU rDNA of P. shimi (GenBank Accession No. AJ786648) was approximately 5% different from that of Strobilidium caudatum and 6% different from that of two Strombidinopsis species. Based both on morphology and gene sequence divergence, we establish this is as a new species in a new genus belonging to the family Strombidinopsidae.     

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.     

Testicular myxosporidiasis in anurans,with a description of Myxobolus fallax n. sp.

During studies of amphibian sperm cryopreservation, a new species of myxosporidean parasite (Myxozoa, Myxosporae) was observed in the testes of the Australian dwarf green tree frog Litoria fallax (Peters). Myxosporidiasis was found to have no affect on L. fallax body condition or sperm numbers. Myxobolus spores from L. fallax are morphologically distinct from Myxobolus hylae spores (infecting the sympatric Litoria aurea Lesson) and the three previously named (exotic to Australia) Myxobolus species found in anurans. Myxobolus fallax n. sp. is characterised by: pseudocyst white, spherical to ovoid, 141 x 74 to 438 x 337 microm in diameter (mature); plasmodium with spores loosely arranged within interior. Spores ovoid 13.4 +/- 0.5 (12.6-14.6) microm length, 9.5 +/- 0.4 (8.3-10.6) microm width, 6.8 +/- 0.4 (6.5-7.6) microm depth, 1.4 +/- 0.1 (1.3-1.6) length/width; polar capsules broadly pyriform and equal in size 4.2 +/- 0.3 (3.3-4.7) microm length, 2.4 +/- 0.2 (2.1-2.8) microm width; filament coils 7-8, wound tightly and perpendicular to the longitudinal axis of the capsule; polar filament 34 +/- 7.0 (18-50) microm length; intercapsular appendix and sutural ridge folds absent; and iodinophilous vacuole and mucous envelope lacking. In addition to this new species, data from archival samples of M. hylae are provided which show two morphologically distinct spore types. Both appeared rarely in the same pseudocysts and we cautiously retain the single species.     

Replacement of the preoccupied name Davisia Laird 1953 and description of a new myxozoan species (Myxosporea: Sinuolineidae) from Sebastiscus marmoratus (Cuvier, 1829) in the East China Sea

The myxozoan genus Davisia Laird 1953 is preoccupied by Davisia Del Guercio 1909 (Insecta: Hemiptera). Here, Myxodavisia nomen novum is proposed to replace the preoccupied name, and a new species is described. Myxodavisia sebastisca n. sp. was found in the urinary bladder of Sebastiscus marmoratus, collected from coastal waters off Xiamen in the East China Sea. The parasite is characterized by a disporous trophozoite; spherical to subspherical spore, 13.1 +/- 0.3 (12.7-13.6) by 12.3 +/- 0.9 (10.9-13.5) microm in size; curved sutural line; 2 shell valves each with a long lateral appendage 119.4-335.2 microm in length; and 2 spherical or subspherical polar capsules, equal in size, 4.6 +/- 0.6 (3.2-4.6) microm in diameter. Traditionally, Myxodavisia is distinguished from Ceratomyxa, Sinuolinea, and Sphaerospora spp. by having spores that possess a distinct central chamber and lateral appendages. A review of the literature reveals that the presence or absence of a clear septum between these spore components is open to interpretation. Indeed, in immature spores of M. sebastisca n. sp., there was an indication of a demarcated appendage, but in some mature spores, no clear separation was apparent. Our findings suggest that future revision of this genus is warranted, particularly once DNA sequence data become available.     

Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure

The characteristics of Myxobolus cuneus n. sp. and its relationship to the host Piaractus mesopotamicus are described based on light and electron microscopy and histological observations. Polysporic plasmodia measuring 20 microm to 2.1 mm in size were found in 63.3 % of the P. mesopotamicus examined. The parasite was found in the gall bladder, urinary bladder, gills, spleen, fins, head surface, liver and heart. Generative cells and disporoblastic pansporoblasts occurred along the periphery of the plasmodia, and mature spores were found in the internal region. The mature spores had a pear shaped body in frontal view, with a total length of 10.0 +/- 0.6 microm and a width of 5.1 +/- 0.3 microm (mean +/- SD). The spore wall was smooth with sutural folds. The polar capsules were elongated, were pear shaped, and equal in size (length 5.7 +/- 03 microm; width 1.7 +/- 0.2 microm), with the anterior ends close to each other. The polar filaments were tightly coiled in 8-9 turns perpendicular to the axis of the capsule. The plasmodia were always found in connective tissue (wall of the arterioles of the gill filaments, serous capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). The parasite caused important damage in the gills, where development occurred in the wall of gill filament arterioles; a mild macrophage infiltrate was also observed. In advanced developmental stages, the plasmodia caused deformation of the arteriole structure, with a reduction and, in some cases, obstruction of the lumen. The parasite was found throughout the period studied and its prevalence was unaffected by host size, season or water properties.     

Description of a marine peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.     

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.     

Isolation and culturing of a most common anaerobic ciliate, Metopus sp

The study includes isolation of anaerobic ciliate, Metopus sp. from an anaerobic reactor and development of its monoculture under laboratory conditions. Separation by centrifugation followed by micromanipulatory isolation resulted in obtaining pure Metopus culture with less bacterial contamination. The isolated Metopus sp. had the mean dimensions of 32 x 123 microm with the generation time of 53 h. Among the different basal media tried, the ciliate mineral medium (CMV) with 1% wheat powder suspension was the most suitable one for Metopus growth. The temperature and pH ranges, for the best growth of Metopus, were 30-35 degrees C and 6-7, respectively. Higher concentrations of volatile fatty acids (VFA) such as acetate, butyrate and propionate had adverse effect on Metopus growth and prevented its growth beyond 0.05 M concentration. Maximum COD removal was in CMV medium by the growth of anaerobic Metopus sp.     

The life cycle of Gregarina ronderosi n. sp. (Apicomplexa: Gregarinidae) in the Argentine grasshopper Dichroplus elongatus (Orthoptera: Acrididae)

Gregarina ronderosi n. sp. is described based on life cycle observations conducted on nymphs and adults of its natural host, the grasshopper Dichroplus elongatus. Following ingestion of oocysts by the host, parasite development occurs between the epithelium and the food mass in the midgut and gastric caeca. Gametocysts are liberated in the faeces. Natural prevalence in the type locality, Girondo, northwestern Buenos Aires Province, was 39.7% (n=131). The earliest trophozoites seen were small (< or = 10 microm), somewhat ovoid, unsegmented bodies. Fully developed trophozoites (the body is divided into epimerite, protomerite, and deutomerite) were slender, with conical or globular epimerites in attached or unattached forms, respectively. Trophozoites varied greatly in size [total length: 10.4-275.1 microm; mean (+/-S.E.): 126.3+/-78.9]. Gamonts, which were the most common stages observed and filled the midgut and gastric caeca in grasshoppers kept in rearing rooms, had a stocky appearance and also varied greatly in size (total length: 80-348 microm; 205+/-13). Association of gamonts was precocious, biassociative, and caudofrontal. Gametocysts were spherical and highly variable in size (96-376 microm in diameter; 202.8+/-52.5), and normally have 14 sporoduct basal discs. Everted sporoducts were up to 60 microm long. Oocysts were uniformly doliform in shape, measured (5+/-0.08 by 3.2+/-0.06 microm) and contained eight sporozoites. Wall reinforcements (carinae) were present. No infection resulted in experimentally inoculated Locusta migratoria, which is a host of Gregarina acridiorum. G. ronderosi is strikingly similar to G. acridiorum, but has larger oocysts.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum

A cDNA encoding cathepsin B was cloned from the scuticociliate, Uronema marinum, which invades the olive flounder, Paralichthys olivaceus, leading to high mortalities in culturing fish. The full-length scuticociliate cathepsin B (ScCtB) gene contains an open reading frame of 1053 base pairs encoding 350 amino acids. A homology search revealed that ScCtB shares sequence identity with several piscine cathepsin Bs (48%-45%). The protein of ScCtB from U. marinum extracts was purified 12.8-fold by a one step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. It had a molecular mass of 30 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which was consistent with predicting molecular mass of mature protein (29.2 kDa) of ScCtB. The protease activity of the ScCtB enzyme was demonstrated by electrophoresis in a gelatin-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate, Z-arginyl-arginyl-7-amido-4-methylcoumarin (Z-Arg-Arg-AMC). The optimum pH for the protease activity was 5.5. Typical of cysteine proteases, the enzyme was inhibited by trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64) and leupeptin.     

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.