Synergistic digestibility effect by planktonic natural food and habitat renders high digestion efficiency in agastric aquatic consumers.

A digestibility enhancing effect of natural food on stomachless fish model (Cyprinus carpio) was verified by fluorogenic substrate assays of enzymatic activities in experimental pond carp gut flush and planktonic food over a full vegetative season. Then compared with size-matched conspecific grown artificially (tank carp) and an advanced omnivore species possessing true stomach (tilapia, Oreochromis niloticus). Results suggested activities of digestive enzymes (except amylolytic) were significantly higher in pond carp (p ≤ 0.05) than in the size-matched tank carp. Even compared to tilapia, pond carp appeared superior (p < 0.05; proteolytic or chitinolytic activities) or comparable (p > 0.05; phosphatase or cellulolytic activities). Amylolytic, chitinolytic, and phosphatases activities in pond carp gut significantly increased (p ≤ 0.01) over season. Several orders-of-magnitude higher enzymatic activities were detected in planktonic natural food than expressed in carp gut. Amino acid markers in planktonic food revealed a higher share of zooplankton (microcrustaceans), but not phytoplankton, synchronized with higher activities of complex polysaccharide-splitting enzymes (cellulolytic and chitinolytic) in fish gut. Periods of clear water phase low in chlorophyll-a and nutrients, but high in certain zooplankton (preferably cladocerans), may create a synergistic digestibility effect in pond carp. We conclude aquatic ecosystem components (natural food, water, microbiota) enhance fishes' hydrolyzing capabilities of C/N/P macromolecules and even their complex polymers such as cellulose, chitin, and maybe phytate (to be validated), to the extent that being stomachless is not an issue. Aquatic nutritional ecologists may consider that laboratory-based understandings of digestibility may underestimate digestion efficiency of free-ranging fish in ponds or lakes.

Accelerated recovery of lake macroinvertebrates in the third decade since the reversal of acidification.

Chemical reversal from acidification has been progressing in European freshwaters since the late 1980s, responding to successful control of atmospheric pollution by acidifying emissions. However, biological recovery is often delayed after improvements in water composition. We studied macroinvertebrate recovery from acidification in eight glacial lakes in the Bohemian Forest (central Europe) between 1999 and 2019. The chemical composition of these lakes reflects a complex of environmental changes, dominated by a very steep decline in acid deposition and, currently, by elevated nutrient leaching following climate-induced tree dieback within their catchments. Temporal trends in species richness, abundance, species traits and community composition were evaluated with regard to water chemistry, littoral habitat properties and fish colonisation. The results showed accelerated recovery of macroinvertebrates following two decades of gradual improvement in water composition and slowly progressing biological rehabilitation. We observed a significant increase in macroinvertebrate species richness and abundance, coupled with distinct changes in community composition, the extent of changes varying between lakes, reflecting different littoral habitat properties (vegetated vs. stony) and water chemistry. Overall, the communities shifted toward more specialised (grazers, filterers, and phytophilous species) and acid-tolerant taxa at the expense of detritivorous, eurytopic and acid-resistant taxa. Where fish reappeared, open-water taxa declined greatly. Compositional changes were likely driven by the combined effects of water chemistry reversal, rehabilitation of habitat conditions and fish colonisation. Despite favourable trends, communities in recovering lakes still lack several biotic elements, particularly less vagile, acid-sensitive taxa and specialised herbivores known from the regional species pool. It is expected that future progress in lake recovery will be further promoted or inhibited by stochastic colonisation or disturbance events.

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.

Insight into Unprecedented Diversity of Cyanopeptides in Eutrophic Ponds Using an MS/MS Networking Approach.

Man-made shallow fishponds in the Czech Republic have been facing high eutrophication since the 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physicochemical properties of water and its aquatic community composition, leading to harmful algal bloom formation. In our current study, we characterized the phytoplankton community across three eutrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprising non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data coupled with a dereplication strategy. This MS networking approach, coupled with dereplication, on the online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin, and epidolastatin 12. We applied the binary logistic regression to estimate the CNPs producers by correlating the GNPS data with the species abundance. The usage of the GNPS web platform proved a valuable approach for the rapid and simultaneous detection of a large number of peptides and rapid risk assessments for harmful blooms.

The Ability of Tetrahymena utriculariae (Ciliophora, Oligohymenophorea) to Colonize Traps of Different Species of Aquatic Carnivorous Utricularia.

The host specificity of the recently described ciliate species Tetrahymena utriculariae was tested in a greenhouse growth experiment, which included 14 different species of aquatic Utricularia as potential host plants. We confirmed the high specificity of the interaction between U. reflexa and T. utriculariae, the former being the only tested host species able to maintain colonization for prolonged time periods. We conclude that this plant-microbe relationship is a unique and specialized form of digestive mutualism and the plant-microbe unit a suitable experimental system for future ecophysiological studies.

Correction to: Hunters or farmers? Microbiome characteristics help elucidate the diet composition in an aquatic carnivorous plant.

AbstractFollowing publication of the original article [1], the author reported an error in Fig. 3.

Hunters or farmers? Microbiome characteristics help elucidate the diet composition in an aquatic carnivorous plant.

BACKGROUND: Utricularia are rootless aquatic carnivorous plants which have recently attracted the attention of researchers due to the peculiarities of their miniaturized genomes. Here, we focus on a novel aspect of Utricularia ecophysiology-the interactions with and within the complex communities of microorganisms colonizing their traps and external surfaces. RESULTS: Bacteria, fungi, algae, and protozoa inhabit the miniature ecosystem of the Utricularia trap lumen and are involved in the regeneration of nutrients from complex organic matter. By combining molecular methods, microscopy, and other approaches to assess the trap-associated microbial community structure, diversity, function, as well as the nutrient turn-over potential of bacterivory, we gained insight into the nutrient acquisition strategies of the Utricularia hosts. CONCLUSIONS: We conclude that Utricularia traps can, in terms of their ecophysiological function, be compared to microbial cultivators or farms, which center around complex microbial consortia acting synergistically to convert complex organic matter, often of algal origin, into a source of utilizable nutrients for the plants.

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.

Light availability may control extracellular phosphatase production in turbid environments.

Extracellular phosphatase production by phytoplankton was investigated in the moderately eutrophic Lipno reservoir, Czech Republic during 2009 and 2010. We hypothesized that production of extracellular phosphatases is an additional mechanism of phosphorus acquisition enabling producers to survive rather than to dominate the phytoplankton. Hence, we examined the relationship between light availability and phosphatase production, as light plays an important role in polymictic environments. Bulk phosphatase activity was measured using a common fluorometric assay, and the production of phosphatases was studied using the Fluorescently Labelled Enzyme Activity technique, which enabled direct microscopic detection of phosphatase-positive cells. In total, 29 taxa of phytoplankton were identified during both years. Only 17 taxa from the total number of 29 showed production of extracellular phosphatases. Species dominating the phytoplankton rarely produced extracellular phosphatases. In contrast, taxa exhibiting phosphatase activity were present in low biomass in the phytoplankton assemblage. Moreover, there was a significant relationship between the proportion of phosphatase positive species in samples and the Z(eu):Z(mix) ratio (a proxy of light availability). A laboratory experiment with different light intensities confirmed the influence of light on production of phosphatases. Our seasonal study confirmed that extracellular phosphatase production is common in low-abundance populations but not in dominant taxa of the phytoplankton. It also suggested the importance of sufficient light conditions for the production of extracellular phosphatases.

Dinitrogen fixation associated with shoots of aquatic carnivorous plants: is it ecologically important?

BACKGROUND AND AIMS: Rootless carnivorous plants of the genus Utricularia are important components of many standing waters worldwide, as well as suitable model organisms for studying plant-microbe interactions. In this study, an investigation was made of the importance of microbial dinitrogen (N2) fixation in the N acquisition of four aquatic Utricularia species and another aquatic carnivorous plant, Aldrovanda vesiculosa. METHODS: 16S rRNA amplicon sequencing was used to assess the presence of micro-organisms with known ability to fix N2. Next-generation sequencing provided information on the expression of N2 fixation-associated genes. N2 fixation rates were measured following (15)N2-labelling and were used to calculate the plant assimilation rate of microbially fixed N2. KEY RESULTS: Utricularia traps were confirmed as primary sites of N2 fixation, with up to 16 % of the plant-associated microbial community consisting of bacteria capable of fixing N2. Of these, rhizobia were the most abundant group. Nitrogen fixation rates increased with increasing shoot age, but never exceeded 1·3 µmol N g(-1) d. mass d(-1). Plant assimilation rates of fixed N2 were detectable and significant, but this fraction formed less than 1 % of daily plant N gain. Although trap fluid provides conditions favourable for microbial N2 fixation, levels of nif gene transcription comprised <0·01 % of the total prokaryotic transcripts. CONCLUSIONS: It is hypothesized that the reason for limited N2 fixation in aquatic Utricularia, despite the large potential capacity, is the high concentration of NH4-N (2·0-4·3 mg L(-1)) in the trap fluid. Resulting from fast turnover of organic detritus, it probably inhibits N2 fixation in most of the microorganisms present. Nitrogen fixation is not expected to contribute significantly to N nutrition of aquatic carnivorous plants under their typical growth conditions; however, on an annual basis the plant-microbe system can supply nitrogen in the order of hundreds of mg m(-2) into the nutrient-limited littoral zone, where it may thus represent an important N source.

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).

Forest die-back modified plankton recovery from acidic stress.

We examined long-term data on water chemistry of Lake Rachelsee (Germany) following the changes in acidic depositions in central Europe since 1980s. Despite gradual chemical recovery of Rachelsee, its biological recovery was delayed. In 1999, lake recovery was abruptly reversed by a coincident forest die-back, which resulted in elevated terrestrial export of nitrate and ionic aluminum lasting ~5 years. This re-acidification episode provided unique opportunity to study plankton recovery in the rapidly recovering lake water after the abrupt decline in nitrate leaching from the catchment. There were sudden changes both in lake water chemistry and in plankton biomass structure, such as decreased bacterial filaments, increased phytoplankton biomass, and rotifer abundance. The shift from dominance of heterotrophic to autotrophic organisms suggested their substantial release from severe phosphorus stress. Such a rapid change in plankton structure in a lake recovering from acidity has, to the best of our knowledge, not been previously documented.

Ecological implications of organic carbon dynamics in the traps of aquatic carnivorous Utricularia plants.

Rootless aquatic carnivorous Utricularia exude up to 25% of their photosynthates into the trap lumen, which also harbours a complex microbial community thought to play a role in enhancing Utricularia nutrient acquisition. We investigated the composition of organic carbon in the trap fluid, its availability for microbial uptake, the influence of plant nutrient status and trap age on its biodegradability, and the composition of prokaryotic assemblages within the traps of three aquatic Utricularia species. Using ion chromatography and basal respiration rate measurements we confirmed that up to 30% of total dissolved organic carbon in Utricularia trap fluid in oligotrophic conditions was easily biodegradable compounds commonly found in plant root exudates (mainly glucose, fructose and lactate). The proportion of these compounds and their microbial utilisation decreased with increasing mineral nutrient supply and trap age. Fluorescence in situ hybridisation analyses showed that microbial trap assemblages are dominated by alpha and beta Proteobacteria, and that the assemblage composition is affected by changes in the ambient mineral nutrient supply. We suggest that organic carbon dynamics within the traps, involving both the plant and associated microbial assemblages, underlies the acquisition of key nutrients by Utricularia and may help explain the evolutionary success of the genus.

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 Utricularia's 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.

Modulation of microbial predator-prey dynamics by phosphorus availability: growth patterns and survival strategies of bacterial phylogenetic clades.

We simultaneously studied the impact of top-down (protistan grazing) and bottom-up (phosphorus availability) factors on the numbers and biomasses of bacteria from various phylogenetic lineages, and on their growth and activity parameters in the oligo-mesotrophic Piburger See, Austria. Enhanced grazing resulted in decreased proportions of bacteria with high nucleic acid content (high-NA bacteria) and lower detection rates by FISH. There was a change in the composition of the bacterial assemblage, whereby Betaproteobacteria were heavily grazed while Alphaproteobacteria and Cytophaga-Flavobacterium-Bacteroides were less affected by predators. Changes in bacterial assemblage composition were also apparent in the treatments enriched with phosphorus, and even more pronounced in the incubations in dialysis tubes (allowing relatively free nutrient exchange). Here, Betaproteobacteria became dominant and appeared to act as successful opportunistic competitors for nutrients. In contrast, Actinobacteria did not respond to surplus phosphorus by population growth, and, moreover, maintained their small size, which resulted in a very low biomass contribution. In addition, significant relationships between high-NA bacteria and several bacterial phylogenetic clades were found, indicating an enhanced activity status. By combining several single-cell methods, new insight is gained into the competitive abilities of freshwater bacteria from a variety of phylogenetic lineages under contrasting sets of bottom-up and top-down constraints.

Maximum growth rates and possible life strategies of different bacterioplankton groups in relation to phosphorus availability in a freshwater reservoir.

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.

Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake.

Activity of extracellular acid phosphatases was measured at single-cell level in bacterioplankton groups defined by their morphology and size, in acidified mountain Lake Certovo, during the 2003 season, with a method based on use of the substrate ELF97 phosphate which provides fluorescent precipitates upon hydrolysis by phosphatases. The bacterial cell-associated precipitates were quantified by image analysis. A specific, conspicuous, apparently homogeneous morphotype of curved cells of approximately 5 microm average length, despite its low total biomass (average of 4%), contributed significantly (in average by 31%) to the total bacterioplankton phosphatase activity in Lake Certovo (ranging from 1.0 to 12.7 micromol l(-1) h(-1), using ELF97 phosphate as a substrate). Bacterial filaments (> 10 microm), although comprising in average 85% of bacterioplankton biomass, contributed to the total bacterioplankton activity only by 45%. Biomass-specific activity of extracellular (cell-surface) phosphatases of the main bacterioplankton morphotypes increased in the order filaments < cocci and rods < curved cells. The biomass-specific activity of bacterioplankton extracellular phosphatases (0-300 nmol microgC(-1) h(-1)) was generally highest in the spring and decreased gradually during summer. These changes could result from seasonal changes in the phosphorus status of the lake and from subsequent regulation of enzyme expression by bacteria.

Long-term studies (1871-2000) on acidification and recovery of lakes in the Bohemian Forest (central Europe).

This paper evaluates long-term changes in the atmospheric depositions of S and N compounds, lake water quality, and biodiversity at eight glacial lakes in the Bohemian Forest over the past 130 years. This time interval covers (i) the 'background' pre-acidification status of the lakes, (ii) a period of changes in the communities that can be partly explained by introduction of fish, (iii) a period of strong lake acidification with its adverse impacts on the communities, (iv) the lake reversal from acidity, which includes the recent status of the lakes. The lake water chemistry has followed-with a characteristic hysteresis-both the sharp increase and decline in the deposition trends of strong anions. Remarkable changes in biota have mirrored the changing water quality. Fish became extinct and most species of zooplankton (Crustacea) and benthos (Ephemeroptera and Plecoptera) retreated due to the lake water acidification. Independent of ongoing chemical reversal, microorganisms remain dominant in the recent plankton biomass as well as in controlling the pelagic food webs. The first signs of the forthcoming biological recovery have already been evidenced in some lakes, such as the population of Ceriodaphnia quadrangula (Cladocera) returning into the pelagial of one lake or the increase in both phytoplankton biomass and rotifer numbers in another lake.