Grazing Characteristics and Growth Efficiencies at Two Different Temperatures for Three Nanoflagellates Fed with Vibrio Bacteria at Three Different Concentrations

Small inocula of one of the flagellates Paraphysomonas imperforata, Pteridomonas danica, and Cafeteria roenbergensis were added to suspensions of the bacterium Vibrio natriegens at each of three concentrations between 107 and 108 cells ml-1 and incubated at each of the temperatures 10 degrees C and 25 degrees C. Samples were taken at intervals for counting the flagellates and bacteria to determine the timing of the maximum of flagellate numbers and the concentrations at that time. Measurements of the protein concentration of the suspensions during incubation were used to determine the gross growth efficiency (GGE) or yield of flagellate grazing in each experiment. The most effective grazer was Pteridomonas, followed by Paraphysomonas, with Cafeteria being least effective, as judged by the threshold bacterial concentrations at which flagellate multiplication ceased, which were about 2 x 105, 2 x 106, and 2 x 107, respectively, and by the finding that Pteridomonas consumed 99%, Paraphysomonas about 95%, and Cafeteria only 60-70% of the available bacteria in the experiments. Peak concentrations of flagellates were reached later at the lower temperature, but the numbers of flagellates produced and of bacteria eaten were of a similar order at the two temperatures and the GGE was only slightly higher at the lower temperature. The time taken to reach peak flagellate numbers changed little with a threefold increase in bacterial concentrations, but the GGE increased and the numbers of bacteria eaten to produce one flagellate decreased when the bacterial concentration was increased. The three flagellates show clear evidence of niche specialization in differences in thresholds of bacterial prey concentration.     

Regeneration of Phosphorus and Nitrogen by Four Species of Heterotrophic Nanoflagellates Feeding on Three Nutritional States of a Single Bacterial Strain

Three physiological states of a single bacterial strain, namely, balanced, phosphorus-rich, and nitrogen-rich bacteria, were obtained by culturing a bacterial strain in chemostats under three different nutrient regimens. Each was shown to be distinctly different in elemental composition with respect to C/N/P ratio. These bacteria were fed to four species of heterotrophic nanoflagellates in batch culture grazing experiments, and the percent regeneration efficiencies of bacterium-bound nitrogen and phosphorus by the flagellates were compared. All flagellate species regenerated comparable amounts of nitrogen, which was thought to be due to their similar internal C/N ratios. There was, however, interspecies variation with regard to phosphorus regeneration: the two faster-growing species (Paraphysomonas imperforata and Bodo designis) released significantly more phosphorus than the two slower-growing species (Stephanoeca diplocostata and Jakoba libera). The observed differences were thought to have been influenced by a combination of life cycle strategies and internal C/P ratios.     

Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization

A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with beta- and gamma-Proteobacteria -- Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagellates Bodo saltans and Goniomonas sp., and the ciliate Cyclidium glaucoma. Both flagellate species preferably ingested A. hydrophila over P. fluorescens, while C. glaucoma showed no clear preferences. Differences were found in the digestion of bacterial prey with B. saltans digesting significantly faster P. fluorescens compared to two other protists. The field study was conducted in a reservoir as part of a larger experiment. We monitored changes in the bacterial prey composition available compared to the bacteria ingested in flagellate food vacuoles. Bacteria detected by probe HGC69a (Actinobacteria) and R-BT065 were negatively selected by flagellates. Bacteria detected by probe CF319a were initially positively selected but along with a temporal shift in bacterial cell size, this trend changed to negative selection during the experiment. Overall, our analysis of protistan food vacuole content indicated marked effects of flagellate prey selectivity on bacterioplankton community composition.     

Direct and indirect evidence of size-selective grazing on pelagic bacteria by freshwater nanoflagellates

Size-selective grazing of three heterotrophic nanoflagellates (with cell sizes of 21, 44, and 66 mum) isolated from Lake Arlington, Texas was examined by using a natural mixture of fluorescence labelled lake bacteria. Sizes of ingested bacteria in food vacuoles were directly measured. Larger bacterial cells were ingested at a frequency much higher than that at which they occurred in the assemblage, indicating preferential flagellate grazing on the larger size classes within the lake bacterioplankton. Water samples were collected biweekly from June through September, 1989, fractionated by filtration, and incubated for 40 h at in situ temperatures. The average bacterial size was always larger in water which was passed through 1-mum-pore-size filters (1-mum-filtered water) (which was predator free) than in 5-mum-filtered water (which contained flagellates only) or in unfiltered water (in which all bacterivores were present). The increase of bacterial-cell size in 1-mum-filtered water was caused by a shift in the size structure of the bacterioplankton population. Larger cells became more abundant in the absence of flagellate grazing.     

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Complex flagellar motions and swimming patterns of the flagellates Paraphysomonas vestita and Pteridomonas danica

Most flagellates with hispid flagella, that is, flagella with rigid filamentous hairs (mastigonemes), swim in the direction of the flagellar wave propagation with an anterior position of the flagellum. Previous analysis was based on planar wave propagation showing that the mastigonemes pull fluid along the flagellar axis. In the present study, we investigate the flagellar motions and swimming patterns for two flagellates with hispid flagella: Paraphysomonas vestita and Pteridomonas danica. Studies were carried out using normal and high-speed video recording, and particles were added to visualize flow around cells generating feeding currents. When swimming or generating flow, P. vestita was able to pull fluid normal to, and not just along, the flagellum, implying the use of the mastigonemes in an as yet un-described way. When the flagellum made contact with food particles, it changed the flagellar waveform so that the particle was fanned towards the ingestion area, suggesting mechano-sensitivity of the mastigonemes. Pteridomonas danica was capable of more complex swimming than previously described for flagellated protists. This was associated with control of the flagellar beat as well as an ability to bend the plane of the flagellar waveform.     

RGD肽介导的MR分子探针在体外结直肠癌细胞的显像及对其生物学行为的影响

目的：探讨RGD肽介导的MR分子探针在体外结直肠癌细胞的MRJ显像及对其生物学行为的影响。方法：利用纳米技术构建靶向RGD荧光纳米MR、探针,利用荧光倒置相、差显微镜观察该探针与LOVO细胞结合情况;体外磁共振成像（MRI）显像;利用细胞克隆实验测其增殖活性;流式细胞术检测其细胞周期、凋亡。结果：荧光相差显微镜示该RGD肽介导的MR分子探针能特异性与LOVO细胞结合;体外MRI成像示靶向RGD组T1信号强度高于非靶向组及对照组（P〈0．05）;该探针作用24h后的LOVO细胞增殖活性降低,细胞分裂周期发生变化,阻滞在S＋G2M期的细胞比例上升,细胞凋亡率与其他两组相比有显著增加（P〈0．05）。结论：该RGD肽介导的MR分子探针能与结直肠癌LOVO细胞靶向结合,能增强MR1的显像效果,并对肿瘤细胞具有一定的抑制作用．     

Strain-specific differences in the grazing sensitivities of closely related ultramicrobacteria affiliated with the Polynucleobacter cluster

Ultramicrobacteria (cell volume < 0.1 microm(3)) are the numerically dominant organisms in the plankton of marine and freshwater habitats. Flagellates and other protists are assumed to be the most important predators of these ultramicrobacteria as well as of larger planktonic bacteria. However, due to controversial observations conducted previously, it is not clear as to whether fractions of the ultramicrobacteria are resistant to flagellate predation. Furthermore, it is not known if closely related bacteria vary significantly in their sensitivity to flagellate predation. We investigated the sensitivity of ultramicrobacteria affiliated with the cosmopolitan Polynucleobacter cluster to grazing by Spumella-like nanoflagellates. Laboratory grazing experiments with four closely related (> or =99.6% 16S rRNA gene sequence similarity) bacteria and three closely related (100% 18S rRNA gene sequence similarity) flagellates were performed. In comparison to larger bacteria, predation on the ultramicrobacterial Polynucleobacter strains was weak, and the growth of the predating flagellates was slow. Specific clearance rates ranged between 0.14 x 10(5) and 2.8 x 10(5) units of predator size h(-1). Feeding rates strongly depended on the flagellate and bacterial strain (P < 0.001). Grazing mortality rates of the three flagellate strains investigated varied for the same prey strain by up to almost fourfold. We conclude that (i) ultramicrobacteria affiliated with the Polynucleobacter cluster are not protected from grazing, (ii) strain-specific variations in grazing sensitivity even between closely related bacteria are high, and (iii) strain-specific differences in predator-prey interaction could be an important factor in the evolution and maintenance of microbial microdiversity.     

Prey selectivity and the influence of prey carbon:nitrogen ratio on microflagellate grazing

We investigated the influence of prey species and nutritional value, in terms of carbon:nitrogen (C:N) ratio, on prey selection by the predatory microflagellate Paraphysomonas vestita. Experiments were conducted with two phytoplankton prey species of similar diameter to remove size-specific grazing effects. Live cells of both low and high C:N ratio (ranging from 4.8 to 14; N-replete and N-deplete, respectively) were offered to the predator either individually or in combination. By utilising analytical flow cytometry, we were able to enumerate the two prey species and, hence, study selective predation in the mixed-prey assemblage. In single prey experiments, the maximum observed ingestion rates were found to be higher, at all prey C:N ratios, when Isochrysis galbana was the prey item when compared to Pavlova lutheri, whilst maximum specific predator division rates were similar for both prey. Ingestion rates were influenced by prey nutrient status, higher values being observed with N-replete than N-deplete prey. When the two prey species were presented to P. vestita as a mixture, I. galbana was ingested more rapidly than P. lutheri, although ingestion was found to be suppressed when compared to when this was the sole prey species. Conversely, the presence of I. galbana did not influence the rate of ingestion of P. lutheri. P. vestita was, therefore, able to modify its rate of ingestion on the basis of prey type and prey C:N ratio and to discriminate between alternative prey of similar size in mixed-prey assemblages.     

Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica , on strains of marine Synechococcus . Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas , 9 for Pteridomonas ) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.     

Obstacles to flow cytometric analysis of rumen microbial samples

Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.     

Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Successful predation of filamentous bacteria by a nanoflagellate challenges current models of flagellate bacterivory

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 micro m. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.     

Synergistic and antagonistic effects of viral lysis and protistan grazing on bacterial biomass, production and diversity

In a mesotrophic reservoir, we examined the effects on the bacterioplankton of distinct consumers of bacteria, viruses and heterotrophic nanoflagellates, both alone and combined in an experiment using natural populations and in situ incubations in dialysis bags. Ribosomal RNA-targeted probes were employed as well as 16S RNA gene based PCR denaturing gradient gel electrophoresis (DGGE) to enumerate bacterial groups and assess bacterial community composition. We employed probes for Actinobacteria (HGC69a probe), Cytophaga-Flavobacterium-Bacteroidetes bacteria (CF319a probe), BET42a probe ( Betaproteobacteria ) and a subgroup- Betaproteobacteria (R-BT065 probe). We found consumer-specific effects on bacterial activity and diversity (against a background of CF and BET dominating all treatments) suggesting distinct vulnerabilities to the two sources of mortality. For example, growth rate of Actinobacteria was only positive in the presence of flagellates, while towards the end of the experiment ( T _72−96 h) growth rate of R-BT was only positive in the viruses only treatment. More specific data on how viruses and flagellates influenced Flectobacillus are shown in the companion paper. Highest richness (number of DGGE bands) was found in the virus only treatment and lowest when both consumers were present. In addition, we found suggestions of both antagonistic and synergistic interactions between the two sources of bacterial mortality. Notably, bactivory by flagellates was associated with reductions in bacterial diversity and increases in viral production.     

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc.     

Development of a PNA probe for the detection of the toxic dinoflagellate Takayama pulchella

A peptide nucleic acid (PNA) probe was developed to detect the toxic dinoflagellate, Takayama pulchella TPXM, using fluorescent in situ hybridization (FISH) combined with epifluorescent microscopy and flow cytometry. The PNA probe was then used to analyze HAB samples from Xiamen Bay. The results indicated that the fluorescein phosphoramidite (FAM)-labeled probe (PNATP28S01) [Flu]-OO ATG CCA TCT CAA GA, entered the algal cells easily and bound to the target species specifically. High hybridization efficiency (nearly 100%) was observed. Detection by epifluorescence microscopy and flow cytometry gave comparable results. The fluorescence intensity of the PNA probe hybridized to T. pulchella cells was remarkably higher than that of two DNA probes used in this study and than the autofluorescence of the blank and negative control cells. In addition, the hybridization condition of the PNA probe was easier to control than DNA probes, and when applied to field-collected samples, the PNA probe showed higher binding efficiency to the target species than DNA probes. With the observed high specificity, binding efficiency, and detection signal intensity, the PNA probe will be useful for monitoring harmful algal blooms of T. pulchella.     

Patterns in pelagic and benthic nanoflagellate densities in the coastal upwelling system along the Banc d'Arguin,Mauritania

We studied spatial variation in abundance of marine benthic and pelagic heterotrophic nanoflagellates in relation to abundances of autotrophic flagellates, bacteria and cyanobacteria in an upwelling area off the Banc d'Arguin, Mauritania.There was enormous spatial variation in densities. In the sediments these ranged from 8–219 × 10³ cm^–3 for heterotrophic flagellates. Maximum values are in the range of those for temperate shallow marine bottoms. Low densities (< 20 × 10³) were confined to the deep stations (> 1000 m). Over the shelf (10–100 m depth) densities were high but related to grain size rather than to concurrent upwelling phenomena or to the abundance of benthic macrofauna.Pelagic flagellate abundance appeared to be more indicative of contemporary hydrographic conditions, most obvious by an increase in the ratio heterotrophic/autotrophic nanoflagellates away from the area of most intense upwelling.     

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.