Jaroslav Vrba

Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture

ABSTRACT We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyteCryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the classActinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacteriumsp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.

Hysteresis in Reversal of Central European Mountain Lakes from Atmospheric Acidification

Extremely high emissions of S and N compounds in Central Europe (both ∼280 mmol m-2 yr-1) declined by ∼70and ∼35%, respectively, during the last decade. Decreaseddeposition rates of SO4-2, NO3-, and NH4+ in the region paralleled emission declines. The reduction in atmospheric inputs of S and N to mountain ecosystemshas resulted in a pronounced reversal of acidification in the Tatra Mountains and Bohemian Forest lakes. Between the 1987–1990and 1997–1999 periods, concentrations of SO4-2 and NO3- decreased (average ± standard deviation) by 22±7 and 12±7 μmol L-1, respectively, in theTatra Mountains, and by 19±7 and 15±10 μmol L-1, respectively, in the Bohemian Forest. Their decrease was compensated in part (1) by a decrease in Ca2+ + Mg2+ (17±7 μmol L-1) and H+ (4±6 μmol L-1), and an increase in HCO3-(10±10 μmol L-1) in the Tatra Mountains lakes, and (2) by a decrease in Al (7±4 μmol L-1), Ca2+ + Mg2+ (9±6 μmol L-1), and H+ (6±5 μmol L-1), in Bohemian Forest lakes. Despite the rapid decline in lake water concentrations of SO4-2 and NO3- in response to reduced S and N emissions, their present concentrations in some lakes are higher than predictionsbased on observed concentrations at comparable emission rates during development of acidification. This hysteresis in chemical reversal from acidification has delayed biological recovery of the lakes. The only unequivocal sign of biological recovery hasbeen observed in Černé Lake (Bohemian Forest) where a cladoceran species Ceriodaphnia quadrangular has recentlyreached its pre-acidification abundance.

Size Selective Feeding in Cyclidium glaucoma (Ciliophora, Scuticociliatida) and Its Effects on Bacterial Community Structure: A Study from a Continuous Cultivation System

Three aspects of size selective feeding by the scuticociliate Cyclidium glaucoma were studied in continuous cultivation systems. Firstly, grazing-induced changes in abundance, biomass, and size structure of a bacterial community were investigated. Secondly, we studied possible grazing-protection mechanisms of bacteria as a response to permanent presence of the predator. And finally, we were looking for potential feedback mechanisms within this predator-prey relationship, i.e., how the ciliate population reacted to a changed, more grazing-protected bacterial community. The first stage of the cultivation system consisted of the alga Cryptomonas sp. and the accompanying mixed bacterial community. These organisms were transferred to two second stage vessels, a control stage without ciliates and a second one inoculated with C. glaucoma. After the first week, the abundance of bacteria in the latter decreased by 60% and remained stable until the end of the experiment (65 d), whereas bacterial biomass was less affected (393 mg C L-1 during days 0-7, 281 mg C L-1 afterwards). The mean bacterial cell volume doubled from 0.089 mm3 to 0.167 mm3, which was mainly due to increasing cell widths. During the whole investigation period formation of colonies or filaments was not observed, but we found a clear feedback of ciliates on bacterial size. An increase in bacterial cell volume was always followed by a decline of the predator population, resulting in a yet undescribed type of microbial predator-prey relation. Literature and our own data on the optimal food size range grazed by C. glaucoma showed that bacterial cell width rather than length was responsible for that observed phenomenon. Finally, we suggest that uptake rates of spherical latex beads give only limited information on truly ingestible prey volumes and that prey geometry should be considered in future studies on size selective feeding of protists.

Utricularia carnivory revisited: plants supply photosynthetic carbon to traps

The rootless, aquatic Utricularia species belong to the largest and most cosmopolitan carnivorous plant genus. Populations of Utricularia plants are an important component of many standing, nutrient-poor, and humic waters. Carbon (C) allocation is an aspect of Utricularias ecophysiology that has not been studied previously and there is considerable uncertainty about the functional and ecological benefit of the trap-associated microbial community and the potential role played by C exudation in enhancing plant-microbe interactions. A 13C-labelling experiment was conducted in greenhouse conditions to determine the C allocation between plant tissues of increasing age and trap fluid in two Utricularia species. Both species allocated a majority of the newly fixed C into the fast growing shoot apex (46.1+/-8.6% in U. vulgaris and 56.1% in U. australis). Carbon allocation rapidly decreased with increasing age of the shoot, constituting only 8.0+/-4.0% and 6.7% of the total newly fixed C in the oldest analysed segments in U. vulgaris and U. australis, respectively. In the trap-bearing shoot segments, the ratio of C exuded into the trap fluid to that in plant tissues increased markedly with age--in the oldest analysed segments twice as much newly fixed C was allocated into the trap fluid than the plant tissue. Overall, a significant amount of the newly fixed C, approximately 25% (U. vulgaris) and 20% (U. australis), was allocated to the trap fluid. The importance of C exudation for the development of the microbial community associated with the traps as well as for the growth and ecology of aquatic Utricularia is discussed.

Extracellular phosphatase activity of freshwater phytoplankton exposed to different in situ phosphorus concentrations

Extracellular phosphatase production and biomass change were investigated in phytoplankton species transplanted from the phosphorus-limited dam area of a eutrophic reservoir and exposed to the phosphorus-sufficient inflow part and vice versa. Extracellular phosphatase activity was studied using the enzyme-labelled fluorescence (ELF) technique, allowing for direct microscopic detection of enzyme activity and, moreover, its quantification using image cytometry. Several phytoplankton species (e.g. Anabaena planctonica, Microcystis aeruginosa, Fragilaria crotonensis, Ankyra ancora and Planktosphaeria gelatinosa) regulated phosphatase activity according to external phosphorus concentration. On the contrary, picocyanobacteria and several green algae (Coelastrum microporum, Crucigeniella sp., Pediastrum tetras, and Staurastrum planctonicum) did not produce extracellular phosphatases at all. The species-specific extracellular phosphatase activity of F. crotonensis, A. ancora, and P. gelatinosa ranged between 0.02 and 3.5 fmol μm−2 h−1.

Biological recovery of the Bohemian Forest lakes from acidification

A limnological survey of eight small, atmospherically acidified, forested glacial lakes in the Bohemian Forest (Šumava, Böhmerwald) was performed in September 2003. Water chemistry of the tributaries and surface layer of each lake was determined, as well as species composition and biomass of the plankton along the water column, and littoral macrozoobenthos to assess the present status of the lakes. The progress in chemical reversal and biological recovery from acid stress was evaluated by comparing the current status of the lakes with results of a survey four years ago (1999) and former acidification data since the early 1990s. Both the current chemical lake status and the pelagic food web structure reflected the acidity of the tributaries and their aluminium (Al) and phosphorus (P) concentrations. One mesotrophic (Plešné jezero) and three oligotrophic lakes (Černé jezero, Čertovo jezero, and Rachelsee) are still chronically acidified, while four other oligotrophic lakes (Kleiner Arbersee, Prášilské jezero, Grosser Arbersee, and Laka) have recovered their carbonate buffering system. Total plankton biomass was very low and largely dominated by filamentous bacteria in the acidified oligotrophic lakes, while the mesotrophic lake had a higher biomass and was dominated by phytoplankton, which apparently profited from the higher P input. In contrast, both phytoplankton and crustacean zooplankton accounted for the majority of plankton biomass in the recovering lakes. This study has shown further progress in the reversal of lake water chemistry as well as further evidence of biological recovery compared to the 1999 survey. While no changes occurred in species composition of phytoplankton, a new ciliate species was found in one lake. In several lakes, this survey documented a return of zooplankton (e.g., Cladocera: Ceriodaphnia quadrangula and Rotifera: three Keratella species) and macrozoobenthos species (e.g., Ephemeroptera and Plecoptera). The beginning of biological recovery has been delayed for ∼20 years after chemical reversal of the lakes.

Massive occurrence of heterotrophic filaments in acidified lakes: seasonal dynamics and composition

We documented permanent presence of heterotrophic filaments in three acidified lakes (pH<5) in the Bohemian Forest. Due to acidification, crustacean zooplankton were absent in all but one lake. In terms of carbon flow, microorganisms were thus almost exclusive players in the pelagic food webs. Variety of extremely long (>100 microm) heterotrophic filaments occurred in the lakes. The filaments usually accounted for >50% of total heterotrophic microbial biomass in the pelagic zone (medians of the total biomass: 82-108 microg C l(-1)), except for anoxic bottom layers and for episodic appearance of Daphnia longispina in one lake. Seasonal filament formation was mainly induced and maintained by grazing of mixotrophic flagellates (Dinobryon spp.) on small unicellular bacteria in the absence of cladoceran filtration. The filaments were less active than unicellular suspended bacteria. Fluorescence in situ hybridisation revealed that usually <50% of total bacteria were targeted with oligonucleotide probes for Eubacteria.

Metal and proton toxicity to lake zooplankton: A chemical speciation based modelling approach

The WHAM-FTOX model quantifies the combined toxic effects of protons and metal cations towards aquatic organisms through the toxicity function (FTOX), a linear combination of the products of organism-bound cation and a toxic potency coefficient for each cation. We describe the application of the model to predict an observable ecological field variable, species richness of pelagic lake crustacean zooplankton, studied with respect to either acidification or the impacts of metals from smelters. The fitted results give toxic potencies increasing in the order H(+) < Al < Cu < Zn < Ni. In general, observed species richness is lower than predicted, but in some instances agreement is close, and is rarely higher than predictions. The model predicts recovery in agreement with observations for three regions, namely Sudbury (Canada), Bohemian Forest (Czech Republic) and a subset of lakes across Norway, but fails to predict observed recovery from acidification in Adirondack lakes (USA).

Evaluation of Extracellular, High-affinity β-N-acetylglucosaminidase Measurements from Freshwater Lakes: An Enzyme Assay to Estimate Protistan Grazing on Bacteria and Picocyanobacteria

Protistan community grazing rates upon both bacterioplankton and autotrophic picoplankton were estimated using fluorescently-labeled prey and by measurement of extracellular hydrolysis of 4-methylumbelliferyl (MUF) β-N-acetylglucosaminide in a eutrophic reservoir and an oligo-mesotrophic lake during phytoplankton blooms. In addition, enzyme methods were optimized in bacterivorous flagellate cultures by two enzyme assays, based on fluorometric detection of protistan digestive activity, which were compared and calibrated independently against flagellate bacterivory. Enzymatic hydrolyses of MUF β-N,N′,N″-triacetylchitotriose and MUF β-N-acetylglucosaminide were measured in cell-free (sonicated) and whole-cell (unsonicated) samples. The hydrolysis of both substrates, using the whole-cell enzyme assay at in situ pH, was correlated significantly with total grazing rate of Bodo saltans. Thus the whole-cell enzyme assay with MUF β-N-acetylglucosaminide was used for freshwater samples. High-affinity (Km < 1 μmol 1−1) and low-affinity (Km > 100 μmol 1−1) enzymes were distinguished kinetically in most samples from both systems studied. Activities (Vmax) of the high-affinity enzyme varied from 0.24 to 1.43 nmol 1−1 h−1. Protistan community grazing on bacterioplankton was in the range of 0.15–1.36 μg C 1−1 h−1. both for lake and reservoir, the differences being observed in grazing on picocyanobacteria (lake, 0.03-0.22 μg C 1−1 h−1. reservoir, 0.35–1.56 μg C 1−1) h−1. The enzyme activities were correlated significantly with the protistan grazing both on bacterioplakton (rs = 0.62, P < 0.001) and total procaryotic picoplankton (the sum of organic carbon grazed from bacteria and picocyanobacteria, rs = 0.73, P < 0.001) in the eutrophic reservoir. Weaker relationships (rs = 0.42) with a lower slope were found for the oligo-mesotrophic lake. Ingestion rate studies are time-consuming and the digestive enzyme assay with MUF β-N-acetylglucosaminide presents a rapid alternative for estimating total protistan prokaryotic picoplanktivory in freshwaters.

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: Čertovo jezero (CT), Prášilské jezero (PR), and Plešné jezero (PL) in the Bohemian Forest (Šumava, 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.