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Characterization of proteins in flagellates and growing amebae of Naegleria fowleri.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.

Amoeba↗

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini.

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.

Animals↗

Phylogeny of the colonial green flagellates: a study of 18S and 26S rRNA sequence data.

A study of phylogenetic relationships of the colonial green algal flagellates based on nuclear 18S and 26S rRNA sequence data suggests that the colonial habit has had at least two independent origins. All colonial taxa included in the analysis, except Stephanosphaera, are allied in a clade with Chlamydomonas reinhardtii and other Chlamydomonas taxa ascribed to the Euchlamydomonas group by Ettl. In contrast, Stephanosphaera is allied with other unicellular flagellates including Haematococcus. Comparison of the 18S and 26S data shows that the two sets of data yield different results following cladistic analysis. The 18S data provide the principal signal that supports the more basal divergences, but the data do not unambiguously address relationships among taxa in the clade that includes most colonial flagellates and Chlamydomonas taxa representative of the Euchlamydomonas group (sensu Ettl). In contrast, the 26S data have fewer informative sites that support basal divergences than the 18S data, but provide much of the signal that supports resolution of taxa in the colonial flagellate clade in an analysis of the combined 18S and 26S rRNA sequence data. Additional sequence data from the 26S molecule and additional taxa may reduce the topological ambiguity inferred from the sequence data for the colonial flagellates. Alternatively, an ancient and rapid radiation of taxa in the colonial lineage could account for the topological ambiguity. Despite some unresolved questions of relationships, cladistic analysis of the combined data sets provides some robustly supported concepts of evolution in these flagellates.

Animals↗

Heavy metal toxicity and bioavailability of dissolved nutrients to a bacterivorous flagellate are linked to suspended particle physical properties.

Many dissolved substances attach easily to sediment particles. In the presence of suspended sediments bioavailability of dissolved substances is therefore, usually reduced and clays are even applied to "wash" natural waters upon pollution. In organisms which feed on food organisms in the size range of these suspended sediment particles, however, bioavailability of such substances may even increase. For microorganisms the interaction with dissolved substances and suspended sediment particles so far has hardly been investigated. We specifically tested: (1) the importance of suspended particles as an uptake route for dissolved substances; and (2) the significance of particle surface properties, i.e. surface load and mineralogy. As a model system we used an axenically cultured strain of a widespread and often abundant flagellate ("Spumella-like" flagellate strain JBM10). We tested the toxicity of cadmium (II) and mercury (II) as well as availability of dissolved organic matter (DOM) in the absence as well as in the presence of different natural clays, i.e. a kaolinite, a montmorillonite, and a mixed clay, and of artificial silicate particles of different surface charge. When applied separately the presence of the heavy metals cadmium and mercury as well as of suspended particles negatively affected the investigated flagellate but nutritive organics supported growth of the investigated flagellate. Toxic stress response comprises behavioral changes including enhanced swimming activity and stress egestion of ingested particles and was generally similar for a variety of different flagellate species. In combination with suspended particles, the respective effect of trace metals and nutritive substances decreased. Regarding the particle quality, cadmium toxicity increased with increasingly negative surface charge, i.e. increasing surface density of silanol groups (Pearson's product moment, P = 0.005). For mercury particle mineralogy still had a significant effect (P < 0.001) but surface load seems to play a minor role and for nutritive organics no significant effect of the investigated particle properties was found. We conclude that: (i) flagellates are as sensitive as higher animals to heavy metal pollution; (ii) suspended particles decrease bioavailability of dissolved substances and ingestion of suspended particles probably play a minor role as uptake route for dissolved substances; and (iii) suspended sediment particle properties, i.e. surface charge and mineralogy, are key factors for the interaction between microorganisms and dissolved substances in the presence of suspended sediments.

Aluminum Silicates↗

Species diversity of heterotrophic flagellates in White Sea littoral sites.

The species diversity and distribution of benthic heterotrophic flagellates in sediment samples from along the salinity gradient in the Chernaya River Estuary and from Velikaya Salma Strait (Kandalaksha Bay, the White Sea) were investigated during August 2004. One hundred and six taxa have been identified by means of phase and interference contrast light microscopy and transmission electron microscopy. The majority of observed flagellates were bacterivores. The species diversity of the following groups: choanoflagellates, euglenids, kinetoplastids, bicosoecids, chrysomonads, thaumatomonads and flagellates Incertae sedis was the highest. Ancyromonas sigmoides and Petalomonas pusilla were the most common species. The species richness was lowest in the brackish water estuarine part with salinity levels between 5 per thousand and 8 per thousand. The distribution of heterotrophic flagellates conforms to the so-called "rule of critical salinity", possessing, apparently, the same universal character for organisms of different size levels. Heterotrophic flagellate communities in these littoral sites were highly heterogeneous. The curve of "cumulative species number vs. sampling effort" is well fitted by equation S=21.17N(0.50) and unsaturated, which indicates that more intensive investigations of the heterotrophic flagellates in the White Sea should be expected to reveal more species.

Animals↗

Quantitative estimation of flagellate community structure and diversity in soil samples.

Heterotrophic flagellates occur in nearly all soils and, in most cases, many different species are present. Nevertheless, quantitative data on their community structure and diversity are sparse, possibly due to a lack of suitable techniques. Previous studies have tended to focus on either total flagellate numbers and biomass, or the identification and description of flagellate species present. With the increased awareness of the role of biodiversity and of food web interactions, the quantification of species within the community and their response to environmental change is likely to become more important. The present paper describes a modification of the most probable number method that allows such a quantification of individual flagellate morphotypes in soil samples. Observations were also made on the biomass of flagellate morphotypes in soil. 20 to 25 morphotypes of heterotrophic flagellates were detectable per gram of two different arable soils, which were treated experimentally to test the technique. One of the soils was fumigated with chloroform vapour for different lengths of time (0, 0.5, 2 or 24 hours); this led to a reduction in the number of morphotypes, in the Shannon diversity index and in the evenness. The other soil was planted with wheat, and while rhizosphere soils contained the same morphotypes as bulk soil, the abundance of individual morphotypes was significantly different and the Shannon diversity index in rhizosphere soils was significantly higher. Soil influenced by an elevated CO2 level likewise differed significantly in morphotype abundance when compared to soil exposed to ambient levels of CO2. The technique recovered more than 80% of the discernible morphotypes and could also be used to quantify amoebal and ciliate communities in a similar way.

Animals↗

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.

Amoeba↗

Studies on the rumen flagellate Neocallimastix frontalis.

The vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N. frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within I h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those occurring in the rumen: pH 6-5, 39 degrees C, absence of O2, presence of CO2. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.

Animals↗

High diversity of the 'Spumella-like' flagellates: an investigation based on the SSU rRNA gene sequences of isolates from habitats located in six different geographic regions.

We isolated 28 strains of 'Spumella-like' flagellates from different freshwater and soil habitats in Austria, People's Republic of China, Nepal, New Zealand, Uganda, Kenya, Tanzania and Hawaii by use of a modified filtration-acclimatization method. 'Spumella-like' flagellates were found in all of the samples and were often among the dominant bacterivorous flagellates in the respective environments. The small subunit ribosomal RNA (SSU rRNA) gene sequence of the isolates was determined and aligned with previously published sequences of members belonging to the Chrysophyceae sensu stricto. Phylogenetic analysis of the 28 new sequences confirmed their position within the Chrysophyceae sensu stricto and positioned them within different clades. Most of the sequences grouped within clade C and formed several subclusters separated from each other by green taxa including flagellates belonging to Ochromonas, Dinobryon, Poterioochromonas and others. All soil isolates clustered together (subcluster C1) with the soil strain Spumella elongata and the undescribed soil strain 'Spumella danica'. Aquatic isolates were affiliated with at least two branches (C2 and C3). Sequence similarity to the closest related member of the Chrysophyceae ranged between 92% and 99.6%, sequence divergence among the 'Spumella-like' flagellates was as high as 10%. We conclude that (i) the 'Spumella-like' flagellates are a diverse group both in terms of sequence dissimilarity between isolates and in terms of the number of genotypes, (ii) Spumella and Ochromonas are polyphyletic, and (iii) based on the SSU rRNA gene no biogeographical restriction of certain branches could be observed even though different ecotypes may be represented by the same genotype.

Base Sequence↗

The predatory soil flagellate Heteromita globosa stimulates toluene biodegradation by a Pseudomonas sp.

A model food chain was established to investigate the influence of grazing by flagellates on bacteria degrading toluene in batch culture. The rate of toluene consumed by a Pseudomonas sp. strain PS+ (max. 0.37 fmol cell(-1) h(-1)) was significantly higher in the presence of the bacterivorous flagellate Heteromita globosa (max. 1.38 fmol cell(-1) h(-1)). A maximum increase of up to 7.5 times was observed in the rate of toluene consumed by these bacteria during exponential growth of this flagellate. Carbon conversion efficiency (CCE) of bacteria to flagellate biomass was estimated to be 33.4% based on measured biovolumes and published values for carbon contents. However, the CCE for toluene-derived carbon was lower (max. 4.9%) when calculations were based on incorporation of [ring-U-(14)C]toluene into biomass of flagellates grazing on labelled bacteria. The findings suggest a potential role for flagellates in bioremediation processes.

Animals↗

A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.

The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites.

Animals↗

Preferential expression of one beta-tubulin gene during flagellate development in Physarum.

The microbial eukaryote Physarum polycephalum displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophila. We have identified beta-tubulin cDNAs from Physarum that are derived from the betA beta-tubulin locus and encode beta 1A tubulin. We have also identified a partial cDNA for the unlinked betB beta-tubulin gene, which encodes beta 1B tubulin. The polypeptide sequences encoded by betA and betB show 99% identity, but the nucleotide sequences show only 85% identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete beta-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB beta-tubulin genes.

Amino Acid Sequence↗

Colloidal and Dissolved Organic Matter Excreted by a Mixotrophic Flagellate during Bacterivory and Autotrophy.

Excretion of dissolved and colloidal organic carbon by a mixotrophic flagellate, the chrysophyte Poterioochromonas malhamensis, was studied. Flagellates were incubated either with C-labeled bacteria or with inorganic C, in order to compare organic exudates originating from primary production with exudates originating from ingested bacteria. Colloids of >0.02 mum constituted a larger fraction of the exudates originating from ingested bacteria, compared with exudates derived from primary production. Flagellate feeding on bacteria specifically labeled in different cell components was compared. Cell wall components gave rise to less colloidal organic carbon than did other cell constituents. To investigate the degradability of flagellate C-exudates, they were added to lake water and mineralization to CO(2) was monitored. Bacterially derived exudates were more recalcitrant than exudates originating from photosynthesis. The results support the hypothesis that bacterial utilization of labile organic compounds, followed by flagellate bacterivory and exudation, results in a transformation of labile organic matter into more recalcitrant forms.

Journal Article↗

Contrasting bacterial strategies to coexist with a flagellate predator in an experimental microbial assemblage.

We studied predator-induced changes within a slowly growing mixed microbial assemblage that was sustained by algal exudates in a continuous cultivation system. In situ hybridization with fluorescent monolabeled oligonucleotide probes was used for a tentative community analysis. This method also allowed us to quantify the proportions of predators with ingested bacteria of different taxonomic groups. In addition, we determined grazing rates on bacteria with fluorescently labelled prey. Bacteria belonging to the alpha and beta subdivisions of the phylum Proteobacteria ((alpha)- and (beta)-Proteobacteria, respectively) showed very different responses to the addition of a bacterivorous flagellate, Bodo saltans. Within one day, filamentous protist-inedible bacteria developed; these belonged to the (beta)-Proteobacteria and constituted between 8.7 and 34% of bacteria from this subgroup. Total abundance of (beta)-Proteobacteria decreased from 3.05 x 10(sup6) to 0.23 x 10(sup6) cells ml(sup-1), and estimated cell division rates were low. Other morphologically inconspicuous protist-edible bacteria belonging to the (alpha)-Proteobacteria were found to respond to predation by an increase in growth rate. Although these bacteria were heavily grazed upon, as on average >85% of flagellate cells had ingested (alpha)-Proteobacteria, they numerically dominated after the addition of B. saltans (mean, 1.35 x 10(sup6) cells ml(sup-1)). It was thus mainly those fast-dividing strains of (alpha)-Proteobacteria that supported the growth of the flagellate population. We conclude that bacteria in mixed assemblages can adopt at least two distinct strategies as a reaction to intense flagellate predation: to outgrow predation pressure or to develop inedible, inactive filaments. Since these strategies occurred within 24 h after the addition of the flagellate, we hypothesize that chemical stimuli released by the predator may have triggered bacterial responses.

Journal Article↗

Examination of bacterial flagellation by dark-field microscopy.

A method is described for visualizing unstained bacterial flagella by dark-field light microscopy. Since individual filaments can be seen, a genus such as Salmonella, which is peritrichously flagellated, can readily be distinguished from a polarly flagellated genus such as Pseudomonas. Polarly flagellated bacteria generally swim much faster than peritrichously flagellated bacteria, and turn by abrupt reversals. The differences in flagellation and motility provide diagnostic criteria that may be useful in clinical microbiology.

Bacteria↗

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Actins↗

Metamorphosis of coeloblastula performed by multipotential larval flagellated cells in the calcareous sponge Leucosolenia laxa.

The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.

Animals↗

Isolation, cultivation and partial characterization of a kinetoplastid flagellate from the hindgut of the water bug, Lethocerus indicus.

A new species of kinetoplastid flagellates was isolated from the hindgut of the giant water bug, Lethocerus indicus, and successfully cultured in modified glycerol beef extract medium. Light and electron microscopic studies as well as growth in various culture media are described. The flagellates were in three different shapes and sizes in the hindgut of the host as well as in the culture medium. Each form of the parasite contained a kinetoplast which was located near the basal body of the flagellate. The ultrastructural features of this species exhibit great similarity to other species of Bodo. The flagellate ingested bacteria, grew in culture, and was identical with that observed in the hindgut of the bugs. The name Bodo indica has been proposed for this kinetoplastid flagellate.

Animals↗