Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm 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 "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% 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-microm filtrate), grazing mortality was approximately 200% 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.     

Altering the balance between bacterial production and protistan bacterivory triggers shifts in freshwater bacterial community composition

Bacterivorous protists are known to induce changes in bacterial community composition (BCC). We hypothesized that changes in BCC could be related quantitatively to a measure of grazing: the ratio of bacterial mortality to growth rate. To test this hypothesis, we analyzed time-course changes in BCC, protistan grazing rate, and bacterial production from 3 in situ studies conducted in a freshwater reservoir and three laboratory studies. In the field experiments, samples were manipulated to yield different levels of protistan bacterivory and incubated in dialysis bags. Laboratory investigations were continuous cultivation studies in which different bacterivorous protists were added to bacterial communities. BCC was assessed using 4–6 different rRNA-targeted oligonucleotide probes for community analysis. Change in BCC (Δ BCC) was estimated as the sum of changes in the proportions of the two phylogenetic groups that showed the largest shifts. Analysis of a set of 22 estimates of shifts in the ratio of grazing to production rate over periods of 48–72 h and Δ BCC showed that Δ BCC was positively and tightly correlated (r ² = 0.784) with shifts in the ratio of grazing mortality to cell production. While the nature of a shift in BCC is unpredictable, the magnitude of the change can be related to changes in the balance between bacterial production and protistan grazing. This revised version was published online in August 2006 with corrections to the Cover Date.     

Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics

ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.     

A key role of aluminium in phosphorus availability,food web structure,and plankton dynamics in strongly acidified lakes

We studied extracellular acid phosphatase activity (AcPA) of planktonic microorganisms, aluminium (Al) speciation, and phosphorus (P) cycling in three atmospherically acidified (pH of 4.5–5.1) mountain forest lakes: ?ertovo jezero (CT), Prá?ilské jezero (PR), and Ple?né jezero (PL) in the Bohemian Forest (?umava, Böhmerwald). Microorganisms dominated pelagic food webs of the lakes and crustacean zooplankton were important only in PR, with the lowest Al concentrations (193 µg L^?1) due to 3–4 times lower terrestrial input. The lakes differed substantially in Al speciation, i.e., in the proportion of ionic and particulate forms, with the highest proportion of ionic Al in the most acid CT (pH = 4.5). The P concentration in the inlet of PL (mean: 22.9 µg L^?1) was about five times higher than in CT and PR (3.9 and 5.1 µg L^?1, respectively). Average total biomass of planktonic microorganisms in PL (593 µg C L^?1) was, however, only ～2-times higher than in CT and PR (235 and 272 µg C L^?1, respectively). Enormous AcPA (means: 2.17–6.82 µmol L^?1 h^?1) and high planktonic C : P ratios suggested severe P limitation of the plankton in all lakes. Comparing 1998 and 2003 seasons, we observed changes in water composition (pH and Al speciation) leading to a significant increase in phytoplankton biomass in the lakes. The increase in the seston C : P ratio during the same time, however, indicates a progressive P deficiency of the lakes. The terrestrial Al inputs, together with in-lake processes controlling the formation of particulate Al, reduced P availability for planktonic microorganisms and were responsible for the differences in AcPA. At pH < 5, moreover, ionic Al forms caused inhibition of extracellular phosphatases. We postulate that both particulate and ionic Al forms affect P availability (i.e., inhibition of extracellular phosphatases and inactivation of P), specifically shape the plankton composition in the lakes and affect plankton recovery from the acid stress.     

Chlorophyll content of Plešné Lake phytoplankton cells studied with image analysis

Using image analysis, chlorophyll autofluorescence was measured in single cells of green alga Monoraphidium dybowskii and in filaments of cyanobacteria (Pseudanabaena sp. and Limnothrix sp.) in the vertical profile of small acidified mountain lake Ple?né jezero (Ple?né Lake) from May to November of 2003. Cell chlorophyll autofluorescence was converted to cell chlorophyll content using a conversion factor determined by comparing the total autofluorescence of phytoplankton in a microscope field with spectrophotometrically determined total chlorophyll concentration; the conversion factor did not differ between epilimnion (0.5 m depth) and hypolimnion (9 m depth). Vertical patterns of chlorophyll concentration and of cellular chlorophyll content depended on water column mixing: during the period of stable thermal stratification, a metalimnetic peak in total chlorophyll concentration was present and cellular chlorophyll contents in the metalimnion and hypolimnion were notably elevated compared to the surface. Monotonous vertical profiles of both total chlorophyll concentration and cell chlorophyll content were typical for the period of water column overturn. During the stratification period, hypolimnetic Monoraphidium cell chlorophyll content was on average twice as high (maximum difference 2.7-fold) compared to surface values (of 3.2–12.9 fg µm^?3), while in filamentous cyanobacteria (surface cell chlorophyll content of 2.2–13.3 fg µm^?3), the difference was much higher — six-fold on average, with an 11.6-fold maximum value. The values measured with image analysis in 2003 were compared to unpublished values of total phytoplankton biomass-specific chlorophyll concentrations obtained using manual phytoplankton biomass determination and spectrophotometric chlorophyll measurement in 1998 at the same locality. Good agreement was found in seasonal patterns and vertical profiles of chlorophyll between both seasons.     

APPLICATION OF THE PDMPO TECHNIQUE IN STUDYING SILICA DEPOSITION IN NATURAL POPULATIONS OF FRAGILARIA CROTONENSIS (BACILLARIOPHYCEAE) AT DIFFERENT DEPTHS IN A EUTROPHIC RESERVOIR1

At weekly intervals from July to October 2006, we measured silica deposition in the summer diatom assemblage at various depths in the eutrophic ?ímov Reservoir (Czech Republic) using PDMPO, the 2‐(4‐pyridyl)‐5{[4‐(2‐dimethylaminoethyl‐aminocarbamoyl)‐methoxy]phenyl}oxazole labeling technique. Fluorescence microscopy coupled with image analysis allows quantifying silicon (Si) deposition over time and a simple distinction between cells that are actively depositing Si and those that are not. Diatom assemblage was exclusively dominated by Fragilaria crotonensis Kitton, which formed pronounced subsurface maxima (2–6.5 m). Concentrations of the main nutrients (Si and phosphorus, P) were low over the whole season; however, at depth, the nutrient availability was higher than at the surface. Fragilaria silica deposition rates were eight times higher at the surface than at depth. Half the population was involved in silica deposition at the surface, while only 20% active cells were doing so at depth. At the surface, silica deposition was limited by P deficiency; the effect of dissolved Si (DSi) was not statistically significant. Silica deposition at depth was significantly constrained by low light availability despite the 1% average light attenuation at depth, which is supposed sufficient for photosynthesis. This study represents the first attempt to employ the PDMPO technique coupled with quantitative image analysis of PDMPO fluorescence in freshwater ecology. On the basis of our results, PDMPO probe appears to be an appropriate proxy for the study of resource limitation in natural diatom populations.     

Grazer and virus-induced mortality of bacterioplankton accelerates development of Flectobacillus populations in a freshwater community

We present a detailed analysis of the effects of distinct bacterial mortality factors, viral lysis and heterotrophic nanoflagellates (HNF) bacterivory, associated with the development of filamentous Flectobacillus populations. Reservoir bacterioplankton communities were subjected to additions of both HNF and viruses together, or HNF alone, and then incubated in situ in dialyses bags. For distinct bacterial groups, mortality or growth stimulation was analysed by examining bacterial prey ingested in HNF food vacuoles with fluorescence in situ hybridization (FISH) and via FISH with microautoradiography (MAR-FISH). We also developed a semi-quantitative MAR-FISH-based estimation of relative activities of Flectobacillus populations (targeted by the R-FL615 probe). Bacterial groups vulnerable to HNF predation (mainly clusters of Betaproteobacteria), or discriminated against (Actinobacteria), were detected. Bacterial lineages most vulnerable to virus-lysis (mainly the Betaproteobacteria not targeted by the R-BT065 probe, of the Polynucleobacter cluster) were identified by comparing treatments with HNF alone to HNF and viruses together. Filaments affiliated with the Flectobacillus cluster appeared in both treatments, but were about twice as abundant, long and active as in incubations with viruses and HNF as compared with HNF alone. Viruses appeared to selectively suppress several bacterial groups, perhaps enhancing substrate availability thus stimulating growth and activity of filamentous Flectobacillus.     

Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.     

Seasonal strengths of the abiotic and biotic drivers of a zooplankton community

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