Heterotrophic flagellate biodiversity and community structure in freshwater streams

The species diversity and community structure of heterotrophic flagellates in watercourses from different geographical regions were investigated. Forty-one species have been identified. The species diversity of cercomonads, kinetoplastids, choanoflagellates, and flagellates is the highest. Bodo saltans, B. designis and Goniomonas truncata are the most common species. Planktonic communities in different streams are most similar in terms of species composition. The highest similarity within benthic cenoses was noted between geographically distant communities. Communities of heterotrophic flagellates seem to be highly heterogenous systems; species distribution depends mainly on the type of habitat and not on geographical factors.     

Fine Structure of Bodo curvifilus Griessmann (Kinetoplastida: Bodonidae)*

SYNOPSIS. Bodo curvifilus Griessmann conforms in its fine structure to the criteria proposed for the genus Bodo, including the presence of subpellicular microtubules, a single large kinetoplast-mitochondrion, emergence of the 2 heterodynamic flagella from a subapical flagellar pocket, and the presence of a paraxial rod associated with the axoneme of each flagellum. B. curvifilus possesses cytoplasmic bodies which resemble endosymbiotic bacteria. These are similar to those found in Bodo saltans. Bodo curvifilus can be distinguished ultrastructurally from Bodo caudatus and B. saltans by the presence in B. curvifilus of a hitherto unreported structure, “the microtubular prism,”consisting of a bundle of 19 microtubules. In cross section, 15 of these microtubules form a cross-linked prismatic array. This microtubular bundle originates near the flagellar pocket and extends for several micrometers into the body of the organism where it follows the periphery of the cell and the long finger-like projections of the kinetoplast-mitochondrion.     

Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.Subject terms: Microbial ecology, Bacteria     

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

Comparison of the genomes of free‐living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free‐living to a parasitic life style has resulted in the loss of approximately 50% of protein‐coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP‐PFK which is homologous to the bacterial PP_i‐PFKs rather than to the canonical eukaryotic ATP‐PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose‐2,6‐bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain‐factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.     

Mitosis of the free-living flagellate Bodo saltans strain Ps+ (Kinetoplastidea, Bodonida)

Mitosis of the free-living flagellate Bodo saltans of the Ps+ strain characterized by the presence of prokaryotic cytobionts in the perinuclear space was studied. Division of B. saltans Ps+ nuclei occurs by the closed intranuclear type of mitosis without condensation of chromosomes. At the initial stages of nuclear division, consecutive anlage of two spatially separated microtubular spindles begins. The spindle containing about 20 microtubules appears first, then, at an angle of 30–40° to it, the second spindle containing half as many microtubules is formed. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one—with 2 pairs. The kinetochores of B. saltans Ps+ have a pronounced laminar structure. Both spindles rest with their ends directly on the internal membrane of the nuclear envelope and form 4 well-pronounced poles. The equatorial phase of mitosis in B. saltans Ps+ is not revealed. The divergence of sister kinetochores towards the poles occurs independently in each spindle. At the elongation phase of mitosis, the poles of both spindles are united in pairs to form a single bipolar structure composed of two loose bundles of microtubules. At this stage of nuclear division, the kinetochores reach the poles of the subspindles and cease to be visible. At subsequent nuclear division stages the nucleus acquires a dumbbell shape. During the reorganization phase the sister nuclei are separated. In the perinuclear space of the interphase nuclei of B. saltans Ps+, 1–2 prokaryotic cytobionts are present. In the course of mitosis, these organisms divide intensively, such that their number can reach 20 and more per nucleus. During separation of sister nuclei, the “excessive” cytobionts are released into the cytoplasmic vacuoles formed by external membranes of the nuclear envelope.     

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.     

Role of Microniches in Protecting Introduced Rhizobium leguminosarum biovar trifolii against Competition and Predation in Soil

The importance of microniches for the survival of introduced Rhizobium leguminosarum biovar trifolii cells was studied in sterilized and recolonized sterilized loamy sand and silt loam. The recolonized soils contained several species of soil microorganisms but were free of protozoa. Part of these soil samples was inoculated with the flagellate Bodo saltans, precultured on rhizobial cells. The introduced organisms were enumerated in different soil fractions by washing the soil, using a standardized washing procedure. With this method, free organisms and organisms associated with soil particles or aggregates >50 μm were separated. The total number of rhizobia was influenced slightly (silt loam) or not at all (loamy sand) by the recolonization with microorganisms or by the addition of flagellates alone. However, when both flagellates and microorganisms were present, numbers of rhizobia decreased drastically. This decrease was more than the sum of both effects separately. Nevertheless, populations of rhizobia were still higher than in natural soil. In the presence of flagellates, higher percentages of rhizobia and other microorganisms were associated with soil particles or aggregates >50 μm than in the absence of flagellates. In recolonized soils, however, the percentages of particle-associated rhizobia were lower than in soils not recolonized previous to inoculation. Thus, the presence of other microorganisms hindered rhizobial colonization of sites where they are normally associated with soil particles or aggregates.     

Channeling of bacterioplanktonic production toward phagotrophic flagellates and ciliates under different seasonal conditions in a river

The objective of this study was to analyze the flux of biomass through the communities of bacteria and phagotrophic protists in the cold and warm conditions occurring seasonally in Butrón River. Bacterial and heterotrophic protistan (flagellate and ciliate) abundance was determined by epifluorescence direct counts; protistan grazing on planktonic bacteria was measured from fluorescently labeled bacteria uptake rates; and the estimate of bacterial secondary production was obtained from [³H]thymidine incorporation rates. The abundance of bacterial, flagellate, and ciliate communities was similar during cold and warm situations. However, we observed that estimates of dynamic parameters, i.e., secondary bacterial production and protistan grazing, in both situations were noticeably different. In the warm situation, grazing rates of flagellates and ciliates (bacteria per protist per hour) were, respectively, 7 times and 18 times higher than those determined in the cold situation, and the grazing rates of the protistan communities (bacteria per protists present in 1 ml of water per hour) increased up to 5 times in the case of flagellates and 42 times in the case of ciliates. Estimates of bacterial secondary production were also higher during the warm situation, showing a ninefold increase. The percentage of bacterial production preyed upon by flagellates or ciliates was not significantly different between the two conditions. These results showed that in the different conditions of a system, the flux of biomass between the trophic levels may be quite different although this process may not be reflected in the abundance of each community of bacteria, flagellates, and ciliates. Offprint requests to: J. Iriberri.     

Comparison of growth efficiencies of protozoa growing on bacteria deposited on surfaces and in suspension

Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.     

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.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Changes in biological characteristics of freshwater heterotrophic flagellates and cladocerans under the effect of metal oxide nano- and microparticles

Data on the effect of nano- and microparticles of cerium and titanium dioxides and zinc oxide on the survival rate and reproduction of nanoflagellates and ceriodaphnia are given. The inhibitory effect of metal oxide nanoparticles on nanoflagellates Bodo saltans Ehrenberg manifested itself in a decrease in their population after 3–4 days of exposure. This effect was found to increase with agent concentrations. In the case of Ceriodaphnia affinis Lilljeborg, their reproductive characteristics showed a significant correlation with the concentrations of nanosize particles of the substances under consideration. Unlike zinc oxide, the microrparticles of titanium and cerium dioxides were less toxic than the respective nanoparticles.     

The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture

The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (_max, 0.17 ± 0.03 h^–1; K_s, 1.1 ± 0.2 × 10⁷ bacteria mL^–1) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-¹⁴C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC₅₀) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC₅₀ values of 4392 (± 167) M and 2770 (± 653) M, respectively. The first-order rate constant for benzene degradation (k₁, 0.48 ± 0.12 day^–1) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 ± 0.13 fmol cell^–1 h^–1) than in their absence (0.26 ± 0.03 fmol cell^–1 h^–1). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.     

Two strains of Pseudomonas fluorescens bacteria differentially affect survivorship of waxworm (Galleria mellonella) larvae exposed to an arthropod fungal pathogen,Beauveria bassiana

Two strains of Pseudomonas fluorescens were found contaminating a biopesticide used in a previous study against Varroa destructor infestations in honey bee hives. In that study, the biopesticide, a formulation of a fungal pathogen of arthropods, Beauveria bassiana, failed to have any negative impact on the mite infestation despite successful results in previous studies using uncontaminated batches of the same biopesticide. The objective of the present research was to determine whether the bacteria may have interfered with the infectivity and/or virulence of B. bassiana in a simplified system; positive results in that system would then provide a rationale for further work under more complex conditions. Galleria mellonella late instar larvae treated topically with both a bacterial suspension of 6.8 to 7.0×10⁷ cfu/ml and a fungal suspension of 2.5×10⁷ or 2.5×10⁸ B. bassiana conidia/ml showed, in the case of one of the bacterial strains, significantly increased survivorship compared to larvae treated with just the B. bassiana suspension. When larvae were immersed in a bacterial suspension prior to application of B. bassiana suspension using a spray tower, a significant positive effect of the same P. fluorescens strain on larval survivorship was observed at 2.5×10⁸ conidia/ml. Neither the bacterial suspensions alone nor blank control solutions had any effect on larval survivorship. These results show that an interaction between the bacteria and the pathogen may explain some of the results from the prior field trial.     

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.     

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.     

Protection of Rhizobium by bentonite clay against predation by flagellates in liquid cultures

Abstract Bentonite clay strongly hampered the activity of the flagellate Bodo soltans in liquid culture, and thereby improved the survival of Rhizobium . A possible coating of bacteria and/or flagellates did not seem to play a part in protecting rhizobia from flagellates. Bentonite did not release any substances toxic to B. saltans during the incubation period. It is suggested that, in liquid cultures, bentonite clay increases the minimum level to which rhizobia can be predated upon, thereby giving rise to the presence of higher rhizobial cell concentrations at the end of the incubation period.     

Zooplankton-mediated changes of bacterial community structure

Enclosure experiments in the mesotrophic Schöhsee in northern Germany were designed to study the impact of metazooplankton on components of the microbial food web (bacteria, flagellates, ciliates). Zooplankton was manipulated in 500-liter epilimnetic mesocosms so that either Daphnia or copepods were dominating, or metazooplankton was virtually absent. The bacterial community responded immediately to changes in zooplankton composition. Biomass, productivity, and especially the morphology of the bacteria changed drastically in the different treatments. Cascading predation effects on the bacterioplankton were transmitted mainly by phagotrophic protozoans which had changed in species composition and biomass. When Daphnia dominated, protozoans were largely suppressed and the original morphological structure of the bacteria (mainly small rods and cocci) remained throughout the experiment. Dominance of copepods or the absence of metazoan predators resulted in a mass appearance of bacterivorous protists (flagellates and ciliates). They promoted a fast decline of bacterial abundance and a shift to the predominance of morphologically inedible forms, mainly long filaments. After 3 days they formed 80–90% of the bacterial biomass. The results indicate that metazooplankton predation on phagotrophic protozoans is a key mechanism for the regulation of bacterioplankton density and community structure.Correspondence to: K. Jürgens.     

Size-selective grazing of coastal bacterioplankton by natural assemblages of pigmented flagellates,colorless flagellates,and ciliates

Fluorescently-labelled bacteria (FLB) were used to study the feeding strategies of a natural assemblage of estuarine protozoans and to examine whether the protozoan grazing could account for the in situ size structure of the bacterioplankton. The FLB, DTAF-stained enterococci, ranging in volume from 0.01 to 0.30 × 10^–1 µm³, were added to a natural planktonic assemblage at a density of 5.5% of the natural bacterioplankton. Initial densities (individuals ml^–1) were as follows: total natural bacteria, 2.2 × 10⁶; FLB, 1.2 × 10⁵; pigmented flagellates, 300; colorless flagellates, 250; and ciliates, 30. FLB consumption rates were determined by examining the contents of protozoan food vacuoles, and the long-term effect of grazing (over a period of 100 hours) was determined by monitoring the decline in the FLB density in experimental vessels. The average consumption rates of FLB by pigmented flagellates were similar to those by flagellates that lacked chloroplasts (0.9 and 0.6 FLB protozoan^–1 hour^–1, respectively). The ciliates consumed bacteria at an average rate that was 17-fold higher (per cell) than flagellates, and they displayed a greater preference for larger bacteria than did the flagellates. FLB of the mid-size classes (0.025–0.100 µm³) were heavily grazed by the entire protozoan assemblage; the smallest (<0.025 µm³) and the largest (>0.100 µm³) FLB escaped protozoan grazing. This had a profound effect on the resulting size distribution of FLB. At the end of a 100-hour incubation, the percentage of mid-size FLB (0.025 to 0.100 µm³) decreased 2.0–2.2-fold, while the percentage of the smallest and the largest FLB increased 2.0–2.5-fold. Resultant densities of FLB were consistent with initial clearance rates determined for the protozoan groups. The grazing rates of protozoans on FLB were species-specific; whereas some species consumed FLB, others did not demonstrate bacterivory. The results suggest that protozoan grazing has a major effect on the size distribution of coastal bacterioplankton. By selectively feeding on a particular size-class of bacteria, planktonic ciliates may consume 15–90% day^–1 of the standing stock of largest size classes of bacterioplankton. Thus, ciliates, which were present in low abundance in the field, could not balance the production of the entire bacterial community, but they may strongly influence the portion of the bacterial community represented by the largest bacterial class. The direct effect of flagellates (e.g., grazing) was limited to smaller bacteria.Offprint requests to: M. P. Shiaris.