Numerical and Thermal Response of the Bacterivorous Ciliate Colpidium kleini, a Species Potentially at Risk of Extinction by Rising Water Temperatures.

We investigated the food-dependent growth and thermal response of the freshwater ciliate Colpidium kleini using numerical response (NR) experiments. This bacterivorous ciliate occurs in lotic water and the pelagial of lakes and ponds. The C. kleini strain used in this work was isolated from a small alpine lake and identified by combining detailed morphological inspections with molecular phylogeny. Specific growth rates (rmax) were measured from 5 to 21 °C. The ciliate did not survive at 22 °C. The threshold bacterial food levels (0.3 - 2.2 × 106 bacterial cells mL-1) matched the bacterial abundance in the alpine lake from which C. kleini was isolated. The food threshold was notably lower than previously reported for C. kleini and two other Colpidium species. The threshold was similar to levels reported for oligotrich and choreotrich ciliates if expressed in terms of bacterial biomass (0.05 - 0.43 mg C L-1). From the NR results, we calculated physiological mortality rates at zero food concentration. The mean mortality (0.55 ± 0.17 d-1) of C. kleini was close to the mean estimate obtained for other planktonic ciliates that do not encyst. We used the data obtained by the NR experiments to fit a thermal performance curve (TPC). The TPC yielded a temperature optimum at 17.3 °C for C. kleini, a maximum upper thermal tolerance limit of 21.9 °C, and a thermal safety margin of 4.6 °C. We demonstrated that combining NR with TPC analysis is a powerful tool to predict better a species' fitness in response to temperature and food.

Temperature-dependent resistance to starvation of three contrasting freshwater ciliates.

We investigated the temperature-dependent response to starvation of three contrasting freshwater ciliates (Ciliophora). The cyst-forming algivorous species Meseres corlissi and the bactivorous species Glaucomides bromelicola, which cannot form cysts, co-occur in the reservoirs (tanks) of tree bromeliads. The mixotrophic species Coleps spetai is common in many lakes. We hypothesized that the ciliates' different traits and life strategies would affect their survival rates and temperature sensitivity under food depleted conditions. We measured the decline of the ciliate populations in microcosm experiments at different temperatures for several days. We used an imaging flow cytometer to size the ciliates and documented their morphological and physiological changes in response to starvation. We found that the cyst-forming species had the highest mortality rates but may endure long-term starvation by encystment. The sympatric, non-encysting species suffered the lowest mortality rates and could survive for more than three weeks without food. The mixotrophic species had intermediate mortality rates but showed the highest phenotypic plasticity in response to starvation. A significant fraction of the C. spetai population appeared unaffected by starvation, suggesting that the endosymbionts provided some resources to the host cells. The mean mortality rate per day of all three species increased with temperature by 0.09 °C-1.

Functional ecology of planktonic ciliates: Measuring mortality rates in response to starvation.

Population dynamics of aquatic ciliates are controlled "bottom-up" via food supply and "top-down" by grazing and parasitism. While intrinsic growth rates of ciliates under saturating food conditions have been studied in some detail, mortality rates induced by starvation have received little attention thus far. To this end, we examined the response of three algivorous freshwater ciliate species to starvation using three different optical methods. Two of these methods, i.e. ciliate mortality rates (δ) estimated from (i) numerical response experiments and (ii) the rate of decline (ROD) in cell numbers, investigated the response of the ciliate population using conventional light microscopy. The third method, imaging cytometry using a FlowCAM instrument, monitored single cells during the starvation experiment. Like light microscopy, the FlowCAM approach estimated δ based on ROD in the experimental containers. However, imaging cytometry also measured the relative cellular chlorophyll a content in the ciliates' food vacuoles as a proxy for the nutritional status of the cells. The linear decline of the cellular chl. a yielded an independent estimate of δ that was similar to δ calculated from ROD. Additionally, the FlowCAM measurements revealed a high degree of phenotypic plasticity of the ciliates when exposed to starvation.

Top-down control of planktonic ciliates by microcrustacean predators is stronger in lakes than in the ocean.

Planktonic ciliates are major components of pelagic food webs in both marine and freshwaters. Their population dynamics are controlled 'bottom-up' by prey availability and 'top-down' by microcrustacean predators. In oceans, copepods are the main ciliate predators while in lakes cladocerans are the typical predators. The efficacy by which these functionally different predators control ciliate population dynamics is debated. We, therefore, investigated experimentally the grazing of three microcrustacean predators with different feeding modes on five freshwater ciliates. We then performed a meta-analysis to assess if our findings can be generalised for aquatic ecosystems. We hypothesized that top-down control is stronger in lakes than in the ocean. We find that: (i) average ingestion rates of marine and freshwater microcrustaceans do not differ; (ii) clearance rates of freshwater cladocerans decrease with ciliate size but increase with ciliate size in freshwater copepods; (iii) clearance rates of the marine microcrustaceans is unrelated to ciliate cell size. These findings have implications for the functioning of freshwater and marine food webs: (i) the ciliate-microcrustacean link is stronger in lakes than in the ocean, and (ii) globally top-down control of ciliates is unlikely in the ocean.

Ecology of planktonic ciliates in a changing world: Concepts, methods, and challenges.

Plankton ecologists ultimately focus on forecasting, both applied and environmental outcomes. We review how appreciating planktonic ciliates has become central to these predictions. We explore the 350-year-old canon on planktonic ciliates and examine its steady progression, which has been punctuated by conceptual insights and technological breakthroughs. By reflecting on this process, we offer suggestions as to where future leaps are needed, with an emphasis on predicting outcomes of global warming. We conclude that in terms of climate change research: (i) climatic hotspots (e.g. polar oceans) require attention; (ii) simply adding ciliate measurements to zooplankton/phytoplankton-based sampling programs is inappropriate; (iii) elucidating the rare biosphere's functional ecology requires culture-independent genetic methods; (iv) evaluating genetic adaptation (microevolution) and population composition shifts is required; (v) contrasting marine and freshwaters needs attention; (vi) mixotrophy needs attention; (vii) laboratory and field studies must couple automated measurements and molecular assessment of functional gene expression; (viii) ciliate trophic diversity requires appreciation; and (ix) marrying gene expression and function, coupled with climate change scenarios is needed. In short, continued academic efforts and financial support are essential to achieve the above; these will lead to understanding how ciliates will respond to climate change, providing tools for forecasting.

Container volume may affect growth rates of ciliates and clearance rates of their microcrustacean predators in microcosm experiments.

We studied the effect of volume in small containers (microcosms) on five common planktonic freshwater ciliates and three zooplankton species, namely Daphnia sp., the calanoid copepod Eudiaptomus sp., and the cyclopoid copepod Cyclops sp. We measured ciliate specific growth rates and their loss rates due to microcrustacean predation in short-term experiments. We hypothesized that container volume ranging from 10 to 200 mL would not affect the activity of our prey and predator species. We found that the response to volume was species-specific; growth rates of three ciliate species were sensitive to volume. However, the volume effect was not unequivocal because different timing of the microcosm experiments (block effects) may have caused random bias due to varying morphological and/or physiological conditions of the ciliates and their predators. For predator clearance rate, the volume effect was insignificant in the filter-feeding Daphnia and Eudiaptomus but was significant for the predatory copepod Cyclops, which was hampered in the smallest experimental containers. Total crustacean clearance rates averaged over all treatments appeared unaffected by predator species, while ciliate species significantly affected the results. Our growth and clearance rates are close to previous findings with the same or similar planktonic prey and predator species.

Functional Ecology of Two Contrasting Freshwater Ciliated Protists in Relation to Temperature.

We conducted microcosm experiments with two contrasting freshwater ciliates on functional traits (FTs) related to their growth rate (numerical response, NR) and ingestion rate (functional response, FR) over a range of ecologically relevant temperatures. Histiobalantium bodamicum and Vorticella natans are common planktonic ciliates but their abundance, swimming behavior, and temperature tolerance are different. In contrast to most sessile peritrich species, the motile V. natans is not strictly bacterivorous but also voraciously feeds upon small algae. We observed three main alterations in the shape of NR of both species with temperature, that is, change in the maximum growth rate, in the initial slope and in the threshold food level needed to sustain the population. Similarly, maximum ingestion rate, gross growth efficiency (GGE), and cell size varied with temperature and species. These findings caution against generalizing ciliate performance in relation to the ongoing global warming. Our results suggest that V. natans is the superior competitor to H. bodamicum in terms of temperature tolerance and bottom-up control. However, the abundance of V. natans is usually low compared to H. bodamicum and other common freshwater ciliates, suggesting that V. natans is more strongly top-down controlled via predation than H. bodamicum. The taxonomic position of V. natans has been debated. Therefore, to confirm species and genus affiliation of our study objects, we sequenced their small subunit ribosomal RNA (SSU rDNA) gene.

Chemically labeled toxins or bioactive peptides show a heterogeneous intracellular distribution and low spatial overlap with autofluorescence in bloom-forming cyanobacteria.

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

Light affects picocyanobacterial grazing and growth response of the mixotrophic flagellate Poterioochromonas malhamensis.

We measured the grazing and growth response of the mixotrophic chrysomonad flagellate Poterioochromonas malhamensis on four closely related picocyanobacterial strains isolated from subalpine lakes in central Europe. The picocyanobacteria represented different pigment types (phycoerythrin-rich, PE, and phycocyanin-rich, PC) and phylogenetic clusters. The grazing experiments were conducted with laboratory cultures acclimated to 10 µmol photon/m2/sec (low light, LL) and 100 µmol photon/m2/sec (moderate light, ML), either in the dark or at four different irradiances ranging from low (6 µmol photon/m2/sec) to high (1,500 µmol photon/m2/sec) light intensity. Poterioochromonas malhamensis preferred the larger, green PC-rich picocyanobacteria to the smaller, red PE-rich picocyanobacterial, and heterotrophic bacteria. The feeding and growth rates of P. malhamensis were sensitive to the actual light conditions during the experiments; the flagellate performed relatively better in the dark and at LL conditions than at high light intensity. In summary, our results found strain-specific ingestion and growth rates of the flagellate; an effect of the preculturing conditions, and, unexpectedly, a direct adverse effect of high light levels. We conclude that this flagellate may avoid exposure to high surface light intensities commonly encountered in temperate lakes during the summer.

Temperature × light interaction and tolerance of high water temperature in the planktonic freshwater flagellates Cryptomonas (Cryptophyceae) and Dinobryon (Chrysophyceae).

Using microcosm experiments, we investigated the interactive effects of temperature and light on specific growth rates of three species each of the phytoplanktonic genera Cryptomonas and Dinobryon. Several species of these genera play important roles in the food web of lakes and seem to be sensitive to high water temperature. We measured growth rates at three to four photon flux densities ranging from 10 to 240 µmol photon · m-2  · s-1 and at 4-5 temperatures ranging from 10°C to 28°C. The temperature × light interaction was generally strong, species specific, and also genus specific. Five of the six species studied tolerated 25°C when light availability was high; however, low light reduced tolerance of high temperatures. Growth rates of all six species were unaffected by temperature in the 10°C-15°C range at light levels ≤50 µmol photon · m-2 · s-1 . At high light, growth rates of Cryptomonas spp. increased with temperature until the temperature optimum was reached and then declined. The Dinobryon species were less sensitive than Cryptomonas spp. to photon flux densities of 40 µmol photon · m-2 · s-1 and 200 µmol photon · m-2  · s-1 over the entire temperature range but did not grow under a combination of very low light (10 µmol photon · m-2  · s-1 ) and high temperature (≥20°C). Among the three Cryptomonas species, cell volume declined with temperature and the maximum temperature tolerated was negatively related to cell size. Since Cryptomonas is important food for microzooplankton, these trends may affect the pelagic carbon flow if lake warming continues.

Rapid detection and quantification of the potentially toxic cyanobacterium Planktothrix rubescens by in-vivo fluorometry and flow cytometry.

We combined profiling of the bloom-forming and potentially toxigenic cyanobacterium Planktothrix rubescens using a multiparameter probe equipped with a phycoerythrin sensor (in vivo fluorometry, IVL) in Lake Mondsee, Austria, with flow cytometric live analyses of discrete samples taken from several depths in the upper 20 m of the water column. Results obtained by IVL and acoustic flow cytometry (AFC) were compared to microscopic analyses of integrated (0-21 m) water samples using fixed material. This comparison was made because the integrated samples are used for the Austrian monitoring programme according to the EU Water Framework Directive. We demonstrate that AFC provides quantitative analyses of the filaments of P. rubescens that are significantly correlated to IVF and microscopic analyses, allowing rapid (within hours) and more precise calculation of P. rubescens biomass than estimates derived from IVL. Our analysis shows that vertically integrated water samples provide unreliable information on the concentration of P. rubescens in the upper surface waters and on the peak concentration of P. rubescens within the water column. We conclude that the protocol that we developed is superior to the current monitoring practice.

Functional diversity of aquatic ciliates.

This paper first reviews the concept of functional diversity in general terms and then applies it to free-living aquatic ciliates. Ciliates are extremely versatile organisms and display an enormous functional diversity as key elements of pelagic food webs, acting as predators of bacteria, algae, other protists and even some metazoans. Planktonic ciliates are important food for zooplankton, and mixotrophic and functionally autotrophic species may significantly contribute to primary production in the ocean and in lakes. The co-occurrence of many ciliate species in seemingly homogenous environments indicates a wide range of their ecological niches. Variation in space and time may foster co-occurrence and prevent violating the competitive exclusion principle among ciliates using the same resources. Considering that many ciliates may be dormant and/or rare in many habitats, ciliate species diversity must be higher than can be deduced from simple sampling techniques; molecular methods of identification clearly point to this hidden diversity. From a functional point of view, the question is how much of this diversity represents redundancy. A key challenge for future research is to link the ecophysiological performance of naturally co-occurring ciliates to their functional genes. To this end, more experimental research is needed with with functionally different species.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Functional ecology of aquatic phagotrophic protists - Concepts, limitations, and perspectives.

Functional ecology is a subdiscipline that aims to enable a mechanistic understanding of patterns and processes from the organismic to the ecosystem level. This paper addresses some main aspects of the process-oriented current knowledge on phagotrophic, i.e. heterotrophic and mixotrophic, protists in aquatic food webs. This is not an exhaustive review; rather, we focus on conceptual issues, in particular on the numerical and functional response of these organisms. We discuss the evolution of concepts and define parameters to evaluate predator-prey dynamics ranging from Lotka-Volterra to the Independent Response Model. Since protists have extremely versatile feeding modes, we explore if there are systematic differences related to their taxonomic affiliation and life strategies. We differentiate between intrinsic factors (nutritional history, acclimatisation) and extrinsic factors (temperature, food, turbulence) affecting feeding, growth, and survival of protist populations. We briefly consider intraspecific variability of some key parameters and constraints inherent in laboratory microcosm experiments. We then upscale the significance of phagotrophic protists in food webs to the ocean level. Finally, we discuss limitations of the mechanistic understanding of protist functional ecology resulting from principal unpredictability of nonlinear dynamics. We conclude by defining open questions and identifying perspectives for future research on functional ecology of aquatic phagotrophic protists.

Ciliates and the rare biosphere-community ecology and population dynamics.

Application of deep sequencing technologies to environmental samples and some detailed morphological studies suggest that there is a vast, yet unexplored rare ciliate biosphere, tentatively defined in terms of operational taxonomic units. However, very few studies complemented molecular and phylogenetic data with morphological and ecological descriptions of the species inventory. This is mainly because the sampling effort increases strongly with decreasing species abundance. In spite of this limited knowledge, it is clear that species that are rare under certain environmental conditions (temporal rare biosphere) may become abundant when the physical, chemical, and biological variables of their habitat change. Furthermore, some species may always be present in low numbers if their dispersal rates are exceedingly high (accidental rare biosphere). An intriguing question is whether there are some species that are always rare, i.e., in every suitable environment. This permanent rare biosphere is conceptually different from the temporal rare biosphere. This review characterizes typical aquatic habitats of the rare ciliate biosphere, portrays different scenarios under which some or even many species may be permanently rare (background fauna), and identifies some fundamental questions that need to be addressed to achieve a better understanding of the population dynamics of the rare ciliate biosphere.