Ultrastructure of Mitosis and Cytokinesis In the Colorless Flagellate Katablepharis Ovalis Skuja

ABSTRACT. Mitosis and cytokinesis in Katablepharis ovalis , a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc-shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron-translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.     

Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.     

Subgenus Systematics of Acanthamoeba: Four Nuclear 18S rDNA Sequence Types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes ( Rns ) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes , or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.     

Isospora atrata N. Sp. (Apicomplexa, Eimeriidae): a New Coccidium Isolated from Carduelis atrata (Passeriformes, Fringillidae)

ABSTRACT Large numbers of coccidian oocysts belonging to the genus Isospora were obtained from the intestinal contents of 98 Carduelis atrata imported into Italy from South America during the months of August through December 1994. The oocysts are sub-spherical and average 21 × 20.3 μm (19.4–23.5 μm × 18.5–22 μm), have a bilayered wall, and an oval polar granule (rarely two). The sporocysts are elliptical and measure 18.8 μm × 10.3 μm (17.5–18.94 μm × 9.5–11.0 μm). The Stieda body protrudes slightly from the end of the sporocyst. A large sporocyst residuum is present, consisting of many granules that may be in a compact mass or scattered. Since this Isospora sp.does not resemble any other species of Isospora previously described from birds of the genus Carduelis , it has been named Isospora atrata n. sp. after the host. Disseminated asexual stages were found in mononuclear cells derived from formalin-fixed post mortem material, suggesting this coccidian may represent an Atoxoplasma -like parasite. Four coccidia-free Serinus canarius L. cohabitated for a long period (4 mo) with infected C. atrata but oocysts were never found in the stool of these birds.     

Novel Structure In the Pellicular Complex of Plasmodium Falciparum Gametocytes1

ABSTRACT. Using transmission electron microscopy, transverse dense bands were found to be associated with subpellicular microtubules and inner membraner in Plasmodium falciparum gametocytes. These dense bands may act as supportive structures to maintain the parallel arrangement of the microtubules, and/or to connect them to the inner membranes.     

Effects of Mesoionic Xanthine Analogs On Trypanosoma Musculi Development In Mice

ABSTRACT. Two derivatives of the mesoionic thiazolo[3,2-a]pyrimidine-5,7-diones 1 were prepared and examined for in vivo antiprotozoan activity to study structure-activity relationships that might lead us to more active derivatives. Mesoionic compounds 1A and 1B were inoculated into Swiss Webster male mice with Trypanosoma musculi infection. the effects were measured by studying parasite populations during the course of patent period (days 9 through 15 post-infection).
The injection of 200 μg of compound 1A along with 5 times 10⁴ trypanosomes affected the level of parasitemia at both the peak and during days 9 to 13 post infection. Experimental animals that received drug 1A prior to and after infection with T. musculi developed significantly lower parasitemia as compared to the control animals at the height of parasite populations (day 11 of observation). the group that received the drug simultaneously with trypanosome infection had significantly lower parasitemias on day 11 and 13 of infection compared to the controls.     

Ultrastructure and formation of the physogastric termite queen cuticle

The physogastric termite queen is the most striking example in insects of growth in size without cuticular moulting. This phenomenon has been studied with electron microscopy and histochemical tests in two species of higher termites, Cubitermes fungifaber and Macrotermes bellicosus. The abdominal hypertrophy (physogastry) is allowed by growth of the arthrodial membranes of the swarming imago. The growth is slow (over several years) but important: the cuticular dry weight is multiplied by 20 in C. fungifaber, by 100-150 in M. bellicosus. The termite queen cuticle arises from the transformation of the cuticle of the swarming imago or imaginal cuticle (unfolding and growing of the epicuticle, stretching of the endocuticle, resorption of the subcuticle) and from the secretion of a new endocuticle or royal endocuticle. The termite queen is the first example known in insects of epicuticular growth. In the physogastric queen, three cuticular types are observed: the rigid cuticle of the sclerites, the soft cuticle of the arthrodial membranes and the partially rigid cuticle of special structures, the neosclerites, which show both rigidity and growth. The fibrillar architecture varies according to the abdominal zones and the position within the cuticle. It appears to be determined by the forces arising from the musculature and the anisometric abdominal growth. The king does not become physogastric, although its cuticle is also modified.     

In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus , a pathogen of eastern oysters ( Crassostrea virginica ), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus -1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue-black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus -1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus -1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell-free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.     

Oral Morphogenesis of the Microstome to Macrostome Transformation in Miamiensis avidus Strain Ma/21

ABSTRACT. Miamiensis avidus, a marine scuticociliate, undergoes microstome to macrostome transformation. This process is induced by prey ciliates. the morphogenesis of this process was examined using scanning electron microscopy and light microscopy of quantitative protargol-stained specimens. Several stages are identified and described.     

Antagonistic Action of the Bacterium Bacillus Licheniformis M-4 Toward the Amoeba Naegleria Fowleri

ABSTRACT. Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

Biological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 10⁴ CFU mL^‐1) and Beijerinckia (13.2 x 10⁴ CFU mL^‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 10⁴ CFU mL^‐1) and Clostridium pasteurianum 39.1 x 10⁴ CFU mL^‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.     

Transformation of Paramecium tetraurelia by electroporation or particle bombardment

Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2+ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.     

Khawkinea quartana, a Colorless Euglenoid Flagellate. I. Ultrastructure

Khawkinea quartana, a naturally occurring colorless homologue of Euglena, was examined with the electron microscope. The organism is biflagellate though only one of the 2 flagella emerges from the anterior reservoir. The pellicular strips covering the body of the organism are supported by microtubules which are continuous in part with microtubules bordering the reservoir. Additional rows of microtubules are found associated with the kinetosomes. An eyespot is located in the wall of the reservoir and, adjacent to it, the contractile vacuole. The nucleus, mitochondria, and Golgi complexes are similar to those described in other euglenoid flagellates. The food reserve is paramylon. The study supports the phylogenetic origin of Khawkinea from pigmented Euglena through the loss of chloroplasts.     

替吉奥对BALB/c小鼠肠道菌群的影响

目的应用PCR-DGGE方法研究抗癌剂替吉奥对肠道菌群的影响。方法取BALB／c小鼠10只,灌服替吉奥（441mg／kg）7d。应用PCR—DGGE方法获得肠道菌群分子指纹图谱,进行相似性、多样性分析及优势条带的序列分析。结果实验0d与实验7d的小鼠肠道菌群结构差异存在统计学意义（P〈0．01）。结论替吉奥能够杀灭肠道中的有益菌,促使致病菌过度生长,导致肠道菌群严重失调。     

Growth and Grazing Rates of the Marine Planktonic Ciliate Strombidinopsis sp. on Red-Tide and Toxic Dinoflagellates

ABSTRACT We investigated growth and grazing rates of Strombidinopsis sp. when feeding on several species of red-tide and/or toxic dinoflagellates. Strombidinopsis sp. one of the largest aloricate choreotrichs so far reported, grew well on Lingulodinium polyedrum, Gymnodinium sanguineum, Scrippsiella trochoidea, Cochlodinium polykrikoides , and Prorocentrum minimum , but failed to grow on Amphidinium carterae. Specific growth rates of Strombidinopsis sp. increased rapidly with increasing prey density up to ca. 100 ng C ml^-1, but were saturated or increased slightly at higher concentrations. Maximum specific growth rates of Strombidinopsis sp. on various prey species were 1.38 day^-1 for C. polykrikoides , 1.27 for G. sanguineum , 1.06 for P. minimum , 0.83 for L. polyedrum , and 0.67 for S. trochoidea. Threshold prey concentrations (where net growth = 0) were 12–38 ng C ml^-1. Maximum ingestion and clearance rates of Strombidinopsis sp. were 353 ng C grazer^-1 day^-1 and 110 μ, l grazer^-1 h^-1, respectively. Strombidinopsis sp. exhibited higher maximum growth, ingestion, and clearance rates than the mixotrophic dinoflagellate Fragilidium cf. mexicanum or the heterotrophic dinoflagellates Protoperidinium cf. divergens and P. crassipes , when grown on the same prey species. In addition, the sequence of prey species arranged according to growth response of Strombidinopsis sp. differed considerably from those of Fragilidium cf. mexicanum, Protoperidinium cf. divergens , and P. crassipes.     

Ultrastructure and Organization of the Cytoskeleton in Oxymonas, an Intestinal Flagellate of Termites

ABSTRACT. Oxymonas has the characteristic structures and organization of other oxymonads including two separated pairs of basal bodies/flagella, a preaxostylar lamina, a paracrystalline axostyle, and an absence of mitochondria and Golgi. Like other Oxymonadinae genera it possesses a long proboscis, the rostellum which is terminated by the holdfast. Like the genera Pyrsonympha and Streblomastix, Oxymonas possesses a holdfast which permits it to attach to the cuticle of the termite hind-gut. This holdfast is subdivided into rhizoids and is filled with microfilaments. The rostellum is variable in length and contains two distinct microtubular bundles. One bundle is composed of convoluted microtubular ribbons which originate at the base of the holdfast and extend posteriorly along the rostellum and before penetrating into the cell body. The second bundle is composed of flexuous free microtubules which originate at different levels of the rostellum, increasing in number from top to base. They occupy the axial part of the rostellum and incorporate into the axostylar rows at the basal body/flagellar level. Microtubules of the paracrystalline axostyle are cross-linked by bridges forming parallel rows like in the contractile axostyles of other oxymonads such as Pyrsonympha and Saccinobaculus . Most of the microtubules of the axostyle originate at the flagellar/preaxostylar level but some originate from the axial flexous free microtubules of the rostellum, as indicated above. The possibility of an extension/retraction of the rostellum, suggested by other authors, is discussed.     

UV-B辐射对几种木本植物幼苗生长和叶绿体超微结构的影响

在实验室条件下研究了UV－B辐射对广东省南亚热带森林中5种木一植物九节,鸭脚木,猴耳环,半枫荷,山乌桕和绿化树种大叶合欢的幼苗形态和细胞超微结构。特别是叶绿体超微结构的影响。结果表明,UV－B辐射抑制幼苗主根伸长和减少侧根数,抑制茎伸长,减少叶数和叶面积。UV－B辐射破坏叶片细胞膜细胞,特别是叶绿体膜结构,但不同种受影响的程度不同,所表现的症状也不完全相同,鸭脚木的质膜收缩,导致质壁分离;鸭脚木,半枫荷和猴耳环的叶绿体膨胀,前二者的甚至破裂,鸭脚木的质膜收缩,导致质壁分离;鸭脚木,半枫荷和猴耳环的叶绿体膨胀,前二者的甚至破裂;鸭脚木和半枫荷的类囊体膨胀粘连;猴耳环的线粒体出现空泡化,半枫荷和猴耳环的叶绿体内大的淀粉粒增多;鸭脚木和山乌桕的叶绿体内或膜上黑色颗粒增多,鸭脚木的线粒体膜上也出现黑色颗粒。     

Phylogeny of Trichomonads Based On Partial Sequences of Large Subunit Rrna and On Cladistic Analysis of Morphological Data

ABSTRACT. Several domains of large subunit rRNA from nine trichomonad species have been sequenced. Molecular phylogenies obtained with parsimony and distance methods demonstrate the trichomonads are a monophyletic group which branches very early in the eukaryotic tree. the topology of the trees is in general agreement with traditional views on evolutionary and systematic relationships of trichomonads. A clear dichotomy is noted between the subfamily Trichomonadinae and the subfamily Tritrichomonadinae. In the latter subfamily, a second division separates the " Tritrichomonas muris -type" species from the " Tritrichomonas augusta -type" ones. Previous evolutionary schemes in which the Monocercomonadidae were regarded as the most "primitive" and the Trichomonadidae as more "evolved" are not in agreement with our molecular data. the emergence of Monocercomonas and Hypotrichomonas at the base of the Tritrichomonas lineage suggests a secondary loss of some cytoskeletal structures, the costa and undulating membrane in these genera. This is corroborated by the early branching position of Trichomitus. which possesses a costa and an undulating membrane and has usually been placed among the Trichomonadidae on the basis of cytological characters. A cladistic analysis was applied to the available morphological characters in order to produce a hierarchical grouping of the taxa reflecting their morphological diversity. Supplementary key words. Evolution, molecular phylogeny, morphological cladistic analysis.