Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-m treatment) or enhance (<5-m treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazerfree" (0.8-m-filtered) treatment, subject only to a relatively low mortality rate (ϳ17% day ؊1 ) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-m filtrate), grazing mortality was ϳ200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

Bacteria of the genus Limnohabitans, more precisely the R-BT lineage, have a prominent role in freshwater bacterioplankton communities due to their high rates of substrate uptake and growth, growth on algal-derived substrates and high mortality rates from bacterivory. Moreover, due to their generally larger mean cell volume, compared to typical bacterioplankton cells, they contribute over-proportionally to total bacterioplankton biomass. Here we present genetic, morphological and ecophysiological properties of 35 bacterial strains affiliated with the Limnohabitans genus newly isolated from 11 non-acidic European freshwater habitats. The low genetic diversity indicated by the previous studies using the ribosomal SSU gene highly contrasted with the surprisingly rich morphologies and different patterns in substrate utilization of isolated strains. Therefore, the intergenic spacer between 16S and 23S rRNA genes was successfully tested as a fine-scale marker to delineate individual lineages and even genotypes. For further studies, we propose the division of the Limnohabitans genus into five lineages (provisionally named as LimA, LimB, LimC, LimD and LimE) and also additional sublineages within the most diversified lineage LimC. Such a delineation is supported by the morphology of isolated strains which predetermine large differences in their ecology.

Phagotrophic protists are diverse and abundant in aquatic and terrestrial environments, making them fundamental to the transfer of matter/energy within their respective food webs. Recognising their grazing impact is essential to evaluate the role of protists in ecosystems, and this includes appreciating prey selectivity. Efforts have been made by groups and individuals to understand selective grazing behaviour by protists: many approaches and perspectives have been pursued, not all of which are compatible. This article, which is not a review, is the product of our discourse on this subject at the SAME 10 meeting. It is the work of individuals, assembled for their breadth of backgrounds, approaches, views, and expertise. Firstly, to communicate ideas and approaches, we develop a framework for selective feeding processes and suggest 6 steps: searching, contact, capture, processing, ingestion, digestion. We then separate study approaches into 2 categories: (1) those examining whole organisms at the community, population, and individual levels, and (2) those examining physiology and molecular attributes. Finally, we explore general problems associated with the field of protistan selective feeding (e.g. linking food selection into food webs and modeling). We do not present all views on any one topic, nor do we cover all topics; instead, we offer opinions and suggest avenues for continued study. Overall, this paper should stimulate further discourse on the subject and provide a roadmap for the future.

We investigated net growth rates of distinct bacterioplankton groups and heterotrophic nanoflagellate (HNF) communities in relation to phosphorus availability by analysing eight in situ manipulation experiments, conducted between 1997 and 2003, in the canyon-shaped Rímov reservoir (Czech Republic). Water samples were size-fractionated and incubated in dialysis bags at the sampling site or transplanted into an area of the reservoir, which differed in phosphorus limitation (range of soluble reactive phosphorus concentrations--SRP, 0.7-96 microg l-1). Using five different rRNA-targeted oligonucleotide probes, net growth rates of the probe-defined bacterial groups and HNF assemblages were estimated and related to SRP using Monod kinetics, yielding growth rate constants specific for each bacterial group. We found highly significant differences among their maximum growth rates while insignificant differences were detected in the saturation constants. However, the latter constants represent only tentative estimates mainly due to insufficient sensitivity of the method used at low in situ SRP concentrations. Interestingly, in these same experiments HNF assemblages grew significantly faster than any bacterial group studied except for a small, but abundant cluster of Betaproteobacteria (targeted by the R-BT065 probe). Potential ecological implications of different growth capabilities for possible life strategies of different bacterial phylogenetic lineages are discussed.

'In late summer 1993 an intensive study was carried out on protozoan grazing in the epilimnion and metalimnion of the eutrophic Rimov Reservoir in south Bohemia. On average, -70% of bacterial production was consumed by heterotrophic flagellates and -20% by ciliates. Ciliate numbers increased from 5 to 70 cells ml l over the 5-week study period. Ciliates ~30 pm in size were numerically dominant in both layers and included Halteria grandinella and Strobilidium hexakinetum (Oligotrichida), Cyrtolophosis mucicola (Cyrtolophosida), Cinetochilum margaritaceum (Scuticociliatida), Urotricha spp., and Coleps sp. (Prostomatida). Ciliate species-specific grazing rates on bacteria and picocyanobacteria were determined. The highest individual cell grazing rates, 4,200 bacteria and 560 picocyanobacteria cell-' h-l, were observed in Vorticella aquadulcis-complex. Oligotrichs ingested on average 360-2,130 bacteria and 76

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.

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.

Planktonic bacteria may represent a substantial nutrient resource available to support the next higher trophic level in the microbial loop (heterotrophic flagellates). In this work we examined the utilization of different size classes of bacteria by flagellated protozoan predators of various sizes. The emphasis was to determine if prey-size selection was a function of predator size. Pseudomonas sp. was grown in chemostats under conditions to yield "large" (mean size, 1.19 pm') or "small" individuals (mean size, 0.36 pm3). Cells were fluorescently labeled (DTAF), mixed in various proportions, and fed to four protozoans ranging in size from 21 to 119 pm3. Bacteria in food vacuoles were enumerated and measured. Size distributions of both offered and taken prey were compared, and a selectivity index was calculated. Protozoans of all four sizes preferred large bacteria (between 0.8 and 1.2 pm').