Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Further Consideration of the Phylogeny of Some "Traditional" Heterotrichs (Protista, Ciliophora) of Uncertain Affinities, Based on New Sequences of the Small Subunit rRNA Gene

ABSTRACT. The systematic relationships and taxonomic positions of the traditional heterotrich genera Condylostentor, Climacostomum, Fabrea , Folliculina, Peritromus , and Condylostoma , as well as the licnophorid genus Licnophora , were re-examined using new data from sequences of the gene coding for small subunit ribosomal RNA. Trees constructed using distance-matrix, Bayesian inference, and maximum-parsimony methods all showed the following relationships: (1) the "traditional" heterotrichs consist of several paraphyletic groups, including the current classes Heterotrichea, Armophorea and part of the Spirotrichea; (2) the class Heterotrichea was confirmed as a monophyletic assemblage based on our analyses of 31 taxa, and the genus Peritromus was demonstrated to be a peripheral group; (3) the genus Licnophora occupied an isolated branch on one side of the deepest divergence in the subphylum Intramacronucleata and was closely affiliated with spirotrichs, armophoreans, and clevelandellids; (4) Condylostentor , a recently defined genus with several truly unique morphological features, is more closely related to Condylostoma than to Stentor ; (5) Folliculina, Eufolliculina , and Maristentor always clustered together with high bootstrap support; and (6) Climacostomum occupied a paraphyletic position distant from Fabrea , showing a close relationship with Condylostomatidae and Chattonidiidae despite of modest support.     

Rearing of Fabrea salina Henneguy (Ciliophora, Heterotrichida) with three unicellular feeds

The growth rate of the ciliate Fabrea salina was studied in batch cultures in the presence of three feeds, tested separately from each other: the Prymnesiophyceae, Isochrysis galbana obtained from pure culture, the Chlorophyceae Dunaliella salina, and the commercially available yeast Saccharomyces cerevisiae. F. salina, and D. salina were harvested below the surface from the first evaporation pond and the crystallizer pond, respectively in multi-pond salterns (Sfax, Tunisia). The highest density of Fabrea was recorded with I. galbana (26 ind ml(-1)). However, the greatest length (243 microm) was recorded with Fabrea fed with D. salina. The lowest density, length and biovolume values were recorded with Fabrea fed with S. cerevisiae. The ANOVA test showed that density (F=18, d.f.=57), length (F=33, d.f.=57), and biovolume (F=19, d.f.=57) of Fabrea fed with yeast were significantly different (p<0.001) from those when Fabrea was fed with D. salina and I. galbana. The ciliate Fabrea encountered in the Sfax saltern (Tunisia) might be a valuable food source for Tunisian marine fish hatcheries.     

Simple synthesis of climacostol, a defensive secretion by the ciliate Climacostomum virens.

Climacostol [1,3-dihydroxy-5-[(Z)-2'-nonenyl]benzene, 1], a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43% overall yield in three steps by starting from methyl 1,3-bis(tert-butyldimethylsilyloxy) phenylacetate (3).     

A Rhodopsin-like Molecule on the Plasma Membrane of Fabrea salina

Immunofluorescence was used to reveal the presence of a rhodopsin-like molecule on the plasma membrane of the photoresponsive ciliate Fabrea salina. The ciliate, fixed on a glass support, showed a spontaneous red-orange fluorescence. Cells incubated with a fluoresceinated, anti-bovine opsin, sheep polyclonal antiserum showed instead a green fluorescence typical of fluorochrome excited at 436 nm, whereas cells incubated with a fluoresceinated, nonimmune sheep serum kept their original red fluorescence. Excess bovine opsin did not inhibit the antibody reaction, but detergent-coated purified bovine opsin was proven able to bind to the cell membrane. Since rhodopsin belongs to a gene-related protein family, which includes adrenergic or cholinergic receptors, the cell response to acute exposure to muscarinic and adrenergic agonist and antagonist drugs was investigated. Acetylcholine, atropine, hexamethonium, noradrenaline, or phenoxybenzamine in the culture medium (10^-4 M) did not influence motion behaviour nor phototaxis of Fabrea salina. These findings raise the possibility that a rhodopsin-like molecule is present on the surface of Fabrea salina. Nevertheless, they do not prove that this surface antigen is a rhodopsin, or that this molecule is responsible for photoresponsiveness in this organism.     

Simple Synthesis of Climacostol,a Defensive Secretion by the Ciliate Climacostomum virens

Climacostol {1,3-dihydroxy-5-[(Z)-2'-nonenyl]benzene,1}, a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43% overall yield in three steps by starting from methyl 1,3-bis(tert-butyldimethylsilyloxy) phenylacetate (3).     

Endosymbiotic bacteria and their relationship with chlorella in the ciliate Climacostomum virens

Structure of cytoplasmic bacterial symbionts of chlorella-free ciliate Climacostomum virens has been investigated. It is shown that ciliates are not able to support simultaneously growth and duplication of two different symbionts--bacteria and chlorella. Cells of C. virens lost bacterial symbionts after an artificial infection with chlorella by microinjection. Competitive relationships between two endopionts are discussed.     

青岛沿海7种异毛类纤毛虫形态学描述

借助活体观察及银染法等对采集于青岛沿海的7种异毛类纤毛虫(绿色爽口虫Climacostomum virens、蓝菌丽克虫Licnophora lyngbycola、盐蚕豆虫Fabrea salina、佛氏环须虫Peritromus faurei、简单瓶囊虫Folliculina simplex、延长类瓶囊虫Folliculinopsis producta、弯曲突口虫Condylostoma curva)进行了活体形态与纤毛图式水平的分类学研究, 其中绿色爽口虫为国内新记录。对迄今缺乏详细研究的简单瓶囊虫与延长类瓶囊虫补充了大量活体形态描述, 如皮层颗粒、色素颗粒的分布与颜色变化等, 同时运用现代银染技术首次报道了该两种游泳体的纤毛图式。       

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

The endosymbiotic unit ofClimacostomum virens andChlorella sp. I. Morphological and physiological studies on the algal partner and its localization in the host cell

Summary The ciliateClimacostomum virens forms an endosymbiotic association with coccoid chlorophycean algae which can be isolated and grown as sterile mass cultures with an inorganic medium. According to both morphological and physiological properties, the algae probably belong to the genusChlorella and have some features in common with symbiotic chlorellae isolated from the ciliatesParamecium bursaria andStentor polymorphus. These and other endosymbiotic chlorellae studied so far excrete maltose or glucose, but algae fromClimacostomum virens show a different excretion pattern by releasing glucose, fructose and xylose. Possible biosynthetic pathways are discussed. Algae inClimacostomum virens are either located individually in special perialgal vacuoles where they are probably protected against attack by host lytic enzymes or to a lesser extent in food vacuoles in different states of digestion. The endosymbiotic character of theClimacostomum virens-Chlorella sp.-association is discussed.Dedicated to Prof. H. A.von Stosch on the occasion of his 75th birthday.     

Defense function of pigment granules in the ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus (Rhizopodea) and Climacostomum virens (Ciliata)

The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.     

Insights into the Evolution of Nuclear Dualism in the Ciliates Revealed by Phylogenetic Analysis of rRNA Sequences

The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi , were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait.     

Climacostol inhibits <Emphasis Type="Italic">Tetrahymena</Emphasis> motility and mitochondrial respiration

Climacostol is a resorcinol derivative that is produced by the ciliate Climacostomum virens. Exposure to purified climacostol results in lethal damage to the predatory ciliate Dileptus margaritifer and several other ciliates. To elucidate the mechanism of climacostol toxic action, we have investigated the effects of this compound on the swimming behavior of Tetrahymena thermophila and the respiratory system of isolated rat liver mitochondria. When added to living T. thermophila cells, climacostol markedly increased the turning frequency that was accompanied by a decrease in swimming velocity and subsequently by cell death. Observations by DIC and fluorescence microscopy showed morphological alterations in climacostol treated T. thermophila, indicating that climacostol might exert cytotoxic action on this organism. In the experiment with isolated rat liver mitochondria, climacostol was found to inhibit the NAD-linked respiration, but had no apparent effect on succinate-linked respiration. This finding indicates that climacostol specifically inhibits respiratory chain complex I in mitochondria. The combination of results suggest that the inhibition of mitochondrial respiration may be the cytotoxic mechanism of climacostol’s defenses against predatory protozoa.     

Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, <Emphasis Type="Italic">Spirostomum ambiguum</Emphasis>

Several species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence.     

Lipid Composition of the Marine Ciliates Pleuronema sp. and Fabrea salina: Shifts in Response to Changes in Diet1

ABSTRACT. The composition and incorporation of lipids in two marine ciliates, Pleuronema sp. and Fabrea salina, was examined following growth on either an algal or bacterial diet. When allowed to feed on a natural bacterial community, Pleuronema sp. synthesized the triterpenoid alcohol gammaceran-3β-ol (tetrahymanol) and two hopanoids (hopan-3β-ol and one uncharacterized hopanoid). When fed the marine alga Isochrysis galbana, F. salina contained the major algal sterol 24-methylcholesta-5, 22-dien-3β-ol and several long chain ketones specific to the alga. In both ciliates, fatty acids composition showed a general correspondence to that of the diet. Using a series of antibiotic treatments to alter the bacterial prey community, and thus fatty acid composition of the ciliate's diet, promoted changes in the fatty acid composition of Pleuronema sp. to resemble that of the bacterial prey. The addition of a mixture of algal sterols to a bacterized culture of another scuticociliate, Parauronema acutum, inhibited tetrahymanol synthesis and resulted in the incorporation of sterols into the ciliate.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Seasonal change in a saline temporary lake (Fuente de Piedra,southern Spain)

Fuente de Piedra saline lake is located in an endorheic basin in the south of Spain. This lake is very shallow (0.5 m max. depth during 1987–88) and relatively large (± 1350 ha). It is a temporary playa lake, showing irregular cycles, with frequent seasonal drought and a high degree of unpredictability. The lake was sampled monthly during a relatively rainy year (1987–88, 10.5 months permanence). The result of combined analyses for environmental variables (salinity, temperature and soluble inorganic forms of nitrogen and phosphorus), variables related to biological activity (chlorophyll a, sediment organic matter and redox potential) and the direct analysis of the planktonic community, shows the existence of two periods of dominance by autotrophs. The first occurs during winter, exhibits a progressively higher surface to volume ratio for phytoplankton and is followed in the spring by high zooplankton densities (Moina salina, Fabrea salina) and very low phytoplankton densities, suggesting the existence of a period with a detritus-based food web. The summer period coincides with a community better adapted to high salinities that is dominated by Dunaliella salina, D. viridis, diatoms and the ciliate Fabrea salina, and associated with high ammonium concentrations. A new period of organic matter accumulation could be facilitated, in the last moments before the lake dries, by a progressive decrease in zooplankton abundance.     

Light and Electron Microscopic Observations on the Heterotrich Ciliate Climacostomum virens

SYNOPSIS. Alveolar membranes and an epiplasm exist under the cell membrane of the noncontractile heterotrich ciliate Climacostomum virens. Postciliary microtubular ribbons join at the right of each somatic kinety to form a Km fiber. Two transverse microtubular fibers occur per kinetosomal pair. A myonemal network interconnects the kinetosomal bases intrakinetally and interkinetally. Ultrastructural comparisons are made between the contractile and noncontractile heterotrichs.
The buccal cortex consists of an adoral zone of membranelles, a peristomal field, a buccal tube, the apical membranelles, and a haplokinety. The kineties of the peristomal field and buccal tube are rows of paired kinetosomes, with a postciliary ribbon of microtubules arising from the posterior kinetosome of each pair, and a transverse ribbon and an oblique ribbon from the anterior kinetosome. No Km fibers exist in this region. The haplokinety is a collar of paired kinetosomes surrounding the cytostome; a postciliary microtubular ribbon descends from each kinetosomal pair into the cytostomal region. Ultrastructural details of the buccal cortex of C. virens and other heterotrichs are compared. The nemadesmata which lie under the membranelles are implicated in the body bending of C. virens.
Algae endosymbiotic in the cytoplasm of C. virens are described.     

In vivo NMR metabolic profiling of Fabrea salina reveals sequential defense mechanisms against ultraviolet radiation

Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement.     

Suitability of Selected Marine Algae for Growing the Marine Heterotrich Ciliate Fabrea salina1

ABSTRACT. Forty-five axenically grown algal (sensu lato) species representing six divisions—that is. 13 Chlorophyceae, 14 Chrysophycophyta, five Dinophycophyta, seven Cryptophycophyta, two Rhodophycophyta, and four Cyanochloronta—were aseplicaily presented separately as potential food sources to the marine helerotrich ciliate Fabrea salina under standardized algal number, medium, lighting, and temperature. The algae can be placed into three groups based on their effect on the intrinsic growth rate of the ciliate. Nutritious: Rhodomonas lens, cryptomonad LIS₁, Dunaliella parva, Prasinodadus marinus, Chroomonas salina, D. tertiolecta, Chaeloceros galvestonensis, D. primolecta, Phaeodactylum tricornutum, D. salina, Isochrysis galbana, Cylindrothecaclosterium, cryptomonad strains M₂, WH₂ & FSA, Chroomonas sp., P. lubricus, and Peridinium trochoideum. Maintamers: Cyanobacterium strain Tigriopus blue green, P. triquetum. Monochrysis lutheri, Exuviella gracilis, Platymonas tetrathele. Cyclotella caspa, Crypthecodinium cohnii, Prasinocladus C₅ strain, D. viridis, Nannochloris occulata, Tetraselmis gracilis, Anacystis marinum, Rhodosorus marinum, and Thalassiosira pseudonana. Nonnutritious: Stichococcus immobilis, Hymenomonas sp. strain 150, Syracosphaera sp. strain 181, Tetraselmis verrucosa, Thalassiosira fluviatilis, Microcoleus chthonoplastes, Synechococcus sp., Pavlova gyrans, Prymnesium parvum, Coccolithus huxleyi, Olisthodiscus luteus, Amphidinium carterii, and Porphyridium aerugineum. There was no apparent relationship between a given taxon and the nutritional value of the group, with the possible exception of the Cryptophycophyta.