Aquatic quillworts, Isoëtes echinospora and I. lacustris under acidic stress-A review from a temperate refuge.

Quillworts (Isoëtes) represent highly specialized flora of softwater lakes, that is, freshwater ecosystems potentially sensitive to acidification. In this paper, we combine a review of previous studies and our new results to address unrecognized reproduction strategies of quillworts to overcome long-term environmental stresses. These strategies play an important role in the plant's ability to overcome atmospheric acidification of freshwaters, protecting the plants until their environment can recover. Environmental drivers of recovery of Isoëtes echinospora and I. lacustris were studied in two acidified lakes in the Bohemian Forest (Central Europe). Both populations survived more than 50 years of severe acidification, although they failed to recruit new sporelings. Their survival depended entirely on the resistance of long-living adult plants because the quillworts do not grow clonally. During the past two decades, a renewal of I. echinospora population inhabiting Plesné Lake has been observed, while no such renewal of I. lacustris, dwelling in Cerné Lake, was evident, despite similar changes in water composition occurring in both lakes undergoing advanced recovery from acidification. Our in vitro experiments revealed that the threshold acidity and toxic aluminium concentrations for sporeling survival and recruitment success differed between I. echinospora (pH ≤ 4.0 and ≥300 µg L-1 Al at pH 5) and I. lacustris (pH ≤ 5.0 and ≥100 µg L-1Al at pH 5). The higher sensitivity of I. lacustris to both stressors likely stems from its year-long germination period and underlines the risk of exposure to chronic or episodic acidification in recovering lakes. By contrast, the shorter germination period of I. echinospora (2-3 months) enables its faster and deeper rooting, protecting this quillwort from periodic acidification during the next snowmelt. Our study brings novel insights into widely discussed environmental issues related to the long-term degradation of softwater lakes, which represent important hotspots of pan-European biodiversity and conservation efforts.

CARD-FISH and prey tracer techniques reveal the role of overlooked flagellate groups as major bacterivores in freshwater hypertrophic shallow lakes.

Heterotrophic nanoflagellates (HNF) and ciliates are major protistan planktonic bacterivores. The term HNF, however, describes a functional guild only and, in contrast to the morphologically distinguishable ciliates, does not reflect the phylogenetic diversity of flagellates in aquatic ecosystems. Associating a function with taxonomic affiliation of key flagellate taxa is currently a major task in microbial ecology. We investigated seasonal changes in the HNF and ciliate community composition as well as taxa-specific bacterivory in four hypertrophic freshwater lakes. Taxa-specific catalyzed reporter deposition-fluorescence in situ hybridization probes assigned taxonomic affiliations to 51%-96% (average ±SD, 75 ± 14%) of total HNF. Ingestion rates of fluorescently labelled bacteria unveiled that HNF contributed to total protist-induced bacterial mortality rates more (56%) than ciliates (44%). Surprisingly, major HNF bacterivores were aplastidic cryptophytes and their Cry1 lineage, comprising on average 53% and 24% of total HNF abundance and 67% and 21% of total HNF bacterivory respectively. Kinetoplastea were important consumers of bacteria during summer phytoplankton blooms, reaching 38% of total HNF. Katablepharidacea (7.5% of total HNF) comprised mainly omnivores, with changing contributions of bacterivorous and algivorous phylotypes. Our results show that aplastidic cryptophytes, accompanied by small omnivorous ciliate genera Halteria/Pelagohalteria, are the major protistan bacterivores in hypertrophic freshwaters.

Low Specific Phosphorus Uptake Affinity of Epilithon in Three Oligo- to Mesotrophic Post-mining Lakes.

Epilithon contributes to phosphorus (P) cycling in lakes, but its P uptake traits have been rarely studied. We measured the chemical composition of epilithon and its inorganic P uptake kinetics using isotope 33P in three deep oligo- to mesotrophic post-mining lakes in April, July, and October 2019. Over the sampling period, epilithon biomass doubled, while the P content in biomass dropped to 60% of the April values, and the seasonal changes in P content expressed per epilithon area were only marginal and statistically not significant. High epilithic C:P molar ratios (677 on average) suggested strong P deficiency in all investigated lakes. Regarding the kinetic parameters of phosphorus uptake, maximum uptake velocity (V max , seasonal range 1.9-129 mg P g OM-1 h-1) decreased by an order of magnitude from April to October, while half-saturation constant (K S , seasonal range 3.9-135 mg P L-1) did not show any consistent temporal trend. Values of epilithic specific P uptake affinity (SPUA E , seasonal range 0.08-3.1 L g OM-1 h-1) decreased from spring to autumn and were two to four orders of magnitude lower than the corresponding values for seston (SPUA sest ), which showed an opposite trend. Considering our results, we suggest a possible mechanism underlying a stable coexistence of planktonic and epilithic microorganisms, with plankton prospering mostly in summer and autumn and epilithon in winter and spring season. Additionally, a phenomenon of reversible abiotic P adsorption on epilithon was observed.

Recovery of freshwater microbial communities after extreme rain events is mediated by cyclic succession.

Small lakes and ponds occupy an enormous surface area of inland freshwater and represent an important terrestrial-water interface. Disturbances caused by extreme weather events can have substantial effects on these ecosystems. Here, we analysed the dynamics of nutrients and the entire plankton community in two flood events and afterwards, when quasi-stable conditions were established, to investigate the effect of such disturbances on a small forest pond. We show that floodings result in repeated washout of resident organisms and hundredfold increases in nutrient load. Despite this, the microbial community recovers to a predisturbance state within two weeks of flooding through four well-defined succession phases. Reassembly of phytoplankton and especially zooplankton takes up to two times longer and features repetitive and adaptive patterns. Release of dissolved nutrients from the pond is associated with inflow rates and community recovery, and returns to predisturbance levels before microbial compositions recover. Our findings shed light on the mechanisms underlying functional resilience of small waterbodies and are relevant to global change-induced increases in weather extremes.

Cascading effects in freshwater microbial food webs by predatory Cercozoa, Katablepharidacea and ciliates feeding on aplastidic bacterivorous cryptophytes.

Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-µm filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-µm fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55-90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-µm treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-µm treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.

Changing environmental conditions underpin long-term patterns of phytoplankton in a freshwater reservoir.

Environmental changes can exert strong pressure on freshwater biota and lead to unwanted alterations of local communities and deterioration of ecosystem services. Disentangling the links between environmental and community changes is, therefore, essential to understand and predict the impact of human activities on freshwater ecosystems. This is particularly relevant for man-made freshwater reservoirs that represent a nexus between anthropogenic, environmental, and biotic effects. Reservoir food webs depend strongly on phytoplankton dynamics, which are affected by abiotic conditions, nutrient availability and grazing pressure by zooplankton. We studied the effects of relevant environmental drivers (hydrochemistry, hydrodynamics and zooplankton) on the composition, diversity and community stability of main morpho-functional phytoplankton groups over 32 years in the Rímov Reservoir (Czech Republic). Environmental conditions in the reservoir are characterised by three distinct periods (1983-89, 1990-99, and 2000-14) defined by shifts and breakpoints in temporal trends in reservoir hydrochemistry and hydraulic conditions, and we examined if and how phytoplankton responded to these abrupt changes. We found significant differences in phytoplankton composition among the three periods. Phytoplankton underwent a substantial compositional shift towards a dominance of pennate diatoms. Time-lag analysis of dissimilarity in phytoplankton composition revealed higher and stochastic annual variations until 1999, followed by a lower variability and divergence in phytoplankton composition in subsequent years. Changes in overall phytoplankton assemblage and most abundant morpho-functional phytoplankton groups were driven mainly by hydrochemical (total nitrogen) and hydrodynamic variables (inflow rate, surface level and mixing depth) and less by zooplankton dynamics. These results suggest that phytoplankton are driven primarily by nutrient input and water regime, both of which can be appropriately managed to support valuable ecosystem services provided by phytoplankton in freshwater reservoirs.

Diel changes and diversity of pufM expression in freshwater communities of anoxygenic phototrophic bacteria.

The anoxygenic phototrophic bacteria (APB) are an active component of aquatic microbial communities. While DNA-based studies have delivered a detailed picture of APB diversity, they cannot provide any information on the activity of individual species. Therefore, we focused on the expression of a photosynthetic gene by APB communities in two freshwater lakes (Cep lake and the Rímov Reservoir) in the Czech Republic. First, we analyzed expression levels of pufM during the diel cycle using RT-qPCR. The transcription underwent a strong diel cycle and was inhibited during the day in both lakes. Then, we compared DNA- (total) and RNA-based (active) community composition by sequencing pufM amplicon libraries. We observed large differences in expression activity among different APB phylogroups. While the total APB community in the Rímov Reservoir was dominated by Betaproteobacteria, Alphaproteobacteria prevailed in the active library. A different situation was encountered in the oligotrophic lake Cep where Betaproteobacteria (order Burkholderiales) dominated both the DNA and RNA libraries. Interestingly, in Cep lake we found smaller amounts of highly active uncultured phototrophic Chloroflexi, as well as phototrophic Gemmatimonadetes. Despite the large diversity of APB communities, light repression of pufM expression seems to be a common feature of all aerobic APB present in the studied lakes.

An Experimental Insight into Extracellular Phosphatases - Differential Induction of Cell-Specific Activity in Green Algae Cultured under Various Phosphorus Conditions.

Extracellular phosphatase activity (PA) has been used as an overall indicator of P depletion in lake phytoplankton. However, detailed insights into the mechanisms of PA regulation are still limited, especially in the case of acid phosphatases. The novel substrate ELF97 phosphate allows for tagging PA on single cells in an epifluorescence microscope. This fluorescence-labeled enzyme activity (FLEA) assay enables for autecological studies in natural phytoplankton and algal cultures. We combined the FLEA assay with image analysis to measure cell-specific acid PA in two closely related species of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta) isolated from two acidic lakes with distinct P availability. The strains were cultured in a mineral medium supplied with organic (beta-glycerol phosphate) or inorganic (orthophosphate) P at three concentrations. Both strains responded to experimental conditions in a similar way, suggesting that acid extracellular phosphatases were regulated irrespectively of the origin and history of the strains. We found an increase in cell-specific PA at low P concentration and the cultures grown with organic P produced significantly higher (ca. 10-fold) PA than those cultured with the same concentrations of inorganic P. The cell-specific PA measured in the cultures grown with the lowest organic P concentration roughly corresponded to those of the original Coccomyxa population from an acidic lake with impaired P availability. The ability of Coccomyxa strains to produce extracellular phosphatases, together with tolerance for both low pH and metals can be one of the factors enabling the dominance of the genus in extreme conditions of acidic lakes. The analysis of frequency distribution of the single-cell PA documented that simple visual counting of 'active' (labeled) and 'non-active' (non-labeled) cells can lead to biased conclusions regarding algal P status because the actual PA of the 'active' cells can vary from negligible to very high values. The FLEA assay using image cytometry offers a strong tool in plankton ecology for exploring P metabolism.

Multiple long-term trends and trend reversals dominate environmental conditions in a man-made freshwater reservoir.

Man-made reservoirs are common across the world and provide a wide range of ecological services. Environmental conditions in riverine reservoirs are affected by the changing climate, catchment-wide processes and manipulations with the water level, and water abstraction from the reservoir. Long-term trends of environmental conditions in reservoirs thus reflect a wider range of drivers in comparison to lakes, which makes the understanding of reservoir dynamics more challenging. We analysed a 32-year time series of 36 environmental variables characterising weather, land use in the catchment, reservoir hydrochemistry, hydrology and light availability in the small, canyon-shaped Rímov Reservoir in the Czech Republic to detect underlying trends, trend reversals and regime shifts. To do so, we fitted linear and piecewise linear regression and a regime shift model to the time series of mean annual values of each variable and to principal components produced by Principal Component Analysis. Models were weighted and ranked using Akaike information criterion and the model selection approach. Most environmental variables exhibited temporal changes that included time-varying trends and trend reversals. For instance, dissolved organic carbon showed a linear increasing trend while nitrate concentration or conductivity exemplified trend reversal. All trend reversals and cessations of temporal trends in reservoir hydrochemistry (except total phosphorus concentrations) occurred in the late 1980s and during 1990s as a consequence of dramatic socioeconomic changes. After a series of heavy rains in the late 1990s, an administrative decision to increase the flood-retention volume of the reservoir resulted in a significant regime shift in reservoir hydraulic conditions in 1999. Our analyses also highlight the utility of the model selection framework, based on relatively simple extensions of linear regression, to describe temporal trends in reservoir characteristics. This approach can provide a solid basis for a better understanding of processes in freshwater reservoirs.

The Limnohabitans Genus Harbors Generalistic and Opportunistic Subtypes: Evidence from Spatiotemporal Succession in a Canyon-Shaped Reservoir.

We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Rímov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Rímov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.

Distribution and ecological preferences of the freshwater lineage LimA (genus Limnohabitans) revealed by a new double hybridization approach.

The ecological relevance and factors shaping dynamics of Limnohabitans sp. have been largely studied by fluorescence in situ hybridization with a 16S rRNA probe targeting the R-BT group (lineages LimBCDE), but not lineage LimA. Consequently, ecology and distribution of LimA remained unknown. We developed a double hybridization strategy using a novel 23S rRNA probe specifically targeting LimA and LimE that in combination with the existing R-BT probe can discriminate LimA populations. This technique was applied for more than 1000 samples from 46 freshwater sites including long-term data sets from oligo-mesotrophic Lake Zurich, CH and meso-eutrophic Rímov reservoir, CZ. LimA was ubiquitously distributed and highly abundant. Observed ecological preferences of LimA in Lake Zurich were in general similar to already reported for Limnohabitans with highest numbers in surface waters during growing seasons. Three times higher densities of LimA were detected in Rímov reservoir, where they were significantly more abundant at the riverine zone especially after flood events that introduced fresh terrestrial DOM (dissolved organic matter). Moreover, statistical analyses of biological and physicochemical parameters obtained from small dynamic water bodies confirmed a correspondence between LimA and allochthonous DOM, in opposite to R-BT that was more related to algal primary production.

Photochemical cleaving of allochthonous organic-metal complexes contributes to phosphorus immobilization in surface waters.

The photochemical transformation of terrestrial dissolved organic carbon (DOC) in surface waters exposed to UV radiation causes the precipitation of metal (Al and Fe) bearing complexes with high phosphorus sorption capacities. To better elucidate this process, a series of laboratory experiments was performed with stream and river waters with pH range from 3.5 to 8.2 and concentrations of dissolved reactive phosphorus from 2 to 142 µg L-1. Samples were filtered (0.4 µm) and UV (350 nm) irradiated for 24 h at 68 W m-2, i.e. under conditions equivalent to ∼2 summer days of natural solar radiation. Irradiated samples and dark controls were then spiked with 33P-phosphate and the kinetics of P adsorption on freshly formed particles was determined after separation by ultracentrifugation. Up to 68% of the added P was removed from the solution within 48 h of the spike. The P sorption was pH dependent, with the maximum sorption ability at pHs of 6-7. We hypothesize that this process can importantly contribute to the immobilization and lower bioavailability of P in the inlet areas of (especially circum-neutral) lakes due to the intensive photochemical degradation of allochthonous DOC-metal complexes.

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.

3D reconstruction of TMJ after resection of the cyst and the stress-strain analyses.

The aim of this article is 3D analysis of the temporomandibular joint (TMJ) patient, who underwent surgery, during which the right TMJ was resected along with the ramus of mandible and consequently the joint was reconstructed with subtotal replacement. The main goal is to give a suitable formulation of mathematical model, which describes the changes of stresses in TMJ incurred after the surgery. The TMJ is a complex, sensitive and highly mobile joint which works bilaterally so each side influences the contralateral joint and because of this the distribution of the stresses is changed in the healthy joint as well. Detailed knowledge about function these are necessary for clinical application of temporomandibular joint prosthesis and also help us estimate the lifetime of the prosthesis a possibilities of alteration in the contra lateral joint components. The geometry for the 3D models is taken from the CT scan date and its numerical solution is based on the theory of semi-coercive unilateral contact problems in linear elasticity. This article provides medical part with case report, discretion of treatment, than the methods of mathematical modeling and his possibilities are described and finally results are reported.

Detection of extracellular phosphatase activity at the single-cell level by enzyme-labeled fluorescence and flow cytometry: the importance of time kinetics in ELFA labeling.

ELF97 phosphate (ELF-P) is a useful compound for assessing the phosphorus-related status of planktonic aquatic populations. The technique has been successfully applied to phytoplankton and more recently to heterotrophic prokaryotes in both freshwater and marine samples. We have used a recently developed protocol that enables the detection by flow cytometry of ELF alcohol (ELFA), the product of ELF-P hydrolysis. This protocol allows for identification of the fraction of cells able to express phosphatase activity (i.e., ELFA-labeled). This protocol is also very valuable in the study of time kinetics in this ELFA-labeling. The percentage of ELFA-labeled cells, the relative median ELFA fluorescence per cell, and the absolute ELFA fluorescence were determined in both freshwater (lake) and marine samples. The incubation time necessary to reach a stable percentage of active cells with maximal fluorescence intensity varied widely among samples. We highlight very subtle but important problems of discrimination between active and nonactive cells and of estimation of per-cell activity and we underline the importance of studying time kinetics of ELFA-labeling to determine the appropriate incubation time and thus making sample comparisons more relevant. Working on time kinetics of ELFA-labeling is promising for phosphomonoester hydrolysis rate determination at single cell level.

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry.

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.

Distribution of aerobic anoxygenic phototrophs in temperate freshwater systems.

The presence of aerobic anoxygenic phototrophs (AAPs) was recently reported from various marine environments; however, there is little information regarding their distribution in fresh waters. We surveyed a number of freshwater systems in central Europe, by infra-red fluorometry, infra-red epifluorescence microscopy, fluorescence emission spectroscopy and pigment analyses. AAPs were found to be abundant in several oligotrophic and mesotrophic lakes (50-400 ng of bacteriochlorophyll a l(-1), 10-80% of bacterial biomass), while in more eutrophized water bodies they represented a negligible part of the total microbial community (< 1%). The observed freshwater AAPs were morphologically diverse and different from previously observed marine species. Under temperate European climatic conditions, AAP populations undergo strong seasonal changes in terms of both abundance and species composition, with the maximum biomass in summer and the minimum in winter. In the mountain lakes Certovo and Plesné, AAPs contributed more than one half of total bacterial biomass during their summer maximum. These results show that photoheterotrophic bacteria represent an important part of the microbial community in many freshwater systems.

APPLICATION OF THE PDMPO TECHNIQUE IN STUDYING SILICA DEPOSITION IN NATURAL POPULATIONS OF FRAGILARIA CROTONENSIS (BACILLARIOPHYCEAE) AT DIFFERENT DEPTHS IN A EUTROPHIC RESERVOIR(1).

At weekly intervals from July to October 2006, we measured silica deposition in the summer diatom assemblage at various depths in the eutrophic Rímov 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.

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.

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.