Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.
Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.
Heterotrophic prokaryotes are a key biological component in aquatic systems and account for a significant fraction of plankton biomass1,2,3. Factors that control their abundance, diversity, and activity are crucial for understanding their role in biogeochemical cycling (i.e., the fate of organic carbon and other nutrients and flow of energy from prokaryotes to higher trophic levels). Protozoan grazing is one of these important factors. Bacterivory of heterotrophic nanoflagellates and ciliates imposes a strong top-down control over prokaryotic abundance, community function, structure, diversity, and even cellular morphology and growth rate of particular bacterial groups4,5,6. In some systems, protists serve as the major cause of bacterial mortality6,7.
The standard approach used to assess protozoan bacterivory, which has been used for some time now, involves the use of fluorescently labeled bacteria (FLB) as prey analogues and epifluorescence microscopy. Cell-specific uptake rates can be determined by quantifying the number of labeled prey particles in protistan food vacuoles over a selected time course8. There are several advantages to this approach. Tracer is added to natural samples with natural predator and prey assemblages. There is minimum sample manipulation prior to incubation, minimum sample alteration by the added FLB tracer, and incubation times are short to ensure sound results obtained under close to in situ conditions. Alternatively, in environments with low numbers of bacterivorous protists or zooplankton (e.g., offshore marine systems), disappearance rates of FLB added to samples in low amounts (2%-3% tracer) can be detected via flow cytometry in long-term (12-24 h) incubation experiments. Then, numbers of FLB at the start and end points (integrating the impact of all bacterivores) are quantified by flow cytometry (for details, see previous publication9). However, such a parameter only represents total aggregated bacterivory rates that cannot be directly attributed to any particular protistan and zooplankton grazer groups or species.
Overall, quantifying the protistan species- or morphotype-specific bacterial mortality rates in the aquatic environment accurately and with ecological meaning can be challenging. Some protists are selective grazers, and the size and cell shape of the added FLB tracer may distort natural rates of prey ingestion10,11. Moreover, protistan activity and metabolism are highly temperature-sensitive12; therefore, the amount of added FLB tracer needs to be carefully manipulated for each individual sample type (not only based on the natural abundance, size, and morphology of bacteria and prevailing types of bacterivores, but also on temperature). Most studies focus on bulk protistan grazing activity; however, the bacterivory of specific protistan species often holds a much higher information value and may be preferable. In this case, taxonomic knowledge of the protist species present in a sample and understanding of their behavior is needed. Hence, considerable amounts of time and labor are required to obtain sound results on species-specific rates of bacterivory attributable to a particular protistan group or species.
Despite these difficulties, this approach remains the most suitable tool currently available to assess protistan bacterivory in natural settings. Presented here is a comprehensive, easy-to-follow method for using FLB as a tracer in aquatic microbial ecology studies. All of the mentioned problematic aspects of the approach are accounted for and an improved workflow is described, with two experiments from contrasting environments as well as contrasting ciliate species as examples.
The first case study was conducted in an epilimnetic environment from the mesotrophic Římov water reservoir in the Czech Republic, which shows grazer and bacterial abundances comparable to most surface freshwater bodies (cf.5,7). The second case study was conducted in the highly specific environment inside traps of the aquatic carnivorous plant Utricularia reflexa, which hosts extremely high numbers of both grazing mixotrophic ciliates (Tetrahymena utriculariae) and bacterial cells. Calculations of cell-specific grazing rates and bacterial standing stocks in both sample types are shown. A range of ecological interpretations of the results is then discussed, and examples of possible follow-up studies are finally suggested.
1. Sample collection
2. Fixation of collected samples
3. Sample filtration
4. Enumeration of bacterial numbers on the filters
5. Determining protistan abundance
6. Determining community structure of ciliates in plankton samples
NOTE: Ciliate communities in freshwater habitats are highly diverse14,15,16,18, and their microscopic determination is challenging. Sorting the ciliate groups into functional guilds10,14,16,17 allows for more detailed analysis of different ciliate groups as pelagic bacterivores.
7. Estimating ciliate grazing rates
Example experiment I was run in Římov water reservoir (South Bohemia, CZ), which is a natural site with lower natural in situ predator and prey abundance. Representative data is reported for the omnivorous ciliate species Halteria grandinella, which is an abundant and efficient grazer of picoplankton (<2 µm) particles10,16,17,18,22. Figure 3 shows box-and-whisker plots of numbers of FLB per cell of Halteria sp. from the Římov reservoir (Figure 3A), which was recalculated to rates of bacterial uptake per hour (Figure 3B) detected in four individual experiments conducted in April, May, August, and September. There was high variability in ciliate uptake rates, largely caused by the temporal differences in water temperature.
It should be noted that the Q10 parameter reflects the fact the microbial processes run approximately 2.5x faster with a temperature increase of 10 °C12, which also holds for ciliate uptake rates on bacteria. With this physiological rule in mind, considerably different proportions of FLB and incubation times were used for different seasons (for details, see Figure 3A). Thus, the expected temperature effect was compensated for, and the experimental setting yielded optimized average and median values of uptake rates approximately between 5-10 FLB per ciliate cell. Generally, these amounts of ingested FLB are easily countable (see examples in Figure 2, two left photographs), generating precise estimates of the tracer (mostly being between 1-15 FLB per ciliate) uptake rates. However, due to modified FLB tracer added (%) and different times of sample incubation the absolute values (expressed as number of bacteria grazed ciliate per hour) differed significantly (p < 0.01, Kruskal-Wallis test; followed by Dunn's multiple-comparison test, p < 0.05; see examples in Figure 3B) among the experiments. The data also illustrate typical natural variability in absolute bacterivory rates in the planktonic populations of Halteria grandinella, with a close match of their mean and medium values (Figure 3).
In the presence of highly efficient bacterivorous ciliates in samples, such as peritrichous ciliates, they can become heavily "over-labeled" by FLB in typical tracer amounts of 5%-10% of total bacteria (see right side photograph in Figure 2). This may strongly limit the accurate quantification of ingested FLB. In such cases, it is suggested to run additional parallel incubations with only low amounts of FLB accounting for only 1.5%-3% of total bacteria. However, generally both the tracer amounts as well as incubation times can be manipulated to optimize the number of FLB per cell (Figure 2).
Example experiment II: Displayed is the data from a system with large predator and prey abundances, where only an extremely small sample volume is available to experimentally estimate bacterivory rates of the ciliate Tetrahymena utriculariae25. It is a moderate bacterial grazer living in high abundance exclusively in traps of carnivorous Utricularia reflexa plants26,27. Figure 4 shows box-and-whisker plots of number of FLB per cell of T. utriculariae under different experimental settings (Figure 4A,B) that is recalculated into rates of bacterial uptake per hour (Figure 4C,D) detected in young, mature, and old traps. Interestingly in traps, chloroplast-bearing populations of the ciliate T. utriculariae were detected, while apochloric populations of T. utriculariae were isolated from traps and maintained on mixed bacterial suspension growing on wheat grains in the dark (for details, see Figure 1 in a previous publication26).
The chloroplast-bearing populations live in light-illuminated traps; thus, the chloroplasts can provide an additional organic carbon source and oxygen to the ciliate host. One of the hypotheses tested was that the apochloric ciliate populations grazed bacteria significantly faster, as the bacteria represent the only particulate source of organic carbon available to dark-grown isolated subpopulations of the ciliate.
Indeed, while there were no significant differences in bacterivory rates of the ciliates living in young, mature and old traps of Utricularia reflexa (Figure 4A,C), the apochloric populations of T. utriculariae grazed bacteria significantly (p < 0.01, Kruskal-Wallis test; followed by Dunn's multiple-comparison test, p < 0.05), approximately 3x faster than the chloroplast-bearing ciliates living in young, mature, and old traps (Figure 4C,D). Note that again, both the tracer amounts as well as incubation times (Figure 4A,B, top) were modified to optimize number of FLB per cell (generally between 1-15), with average and median values around 5 FLB/ciliate. These numbers are distinguishable in ciliate food vacuoles and allowed accurate tracer counting. However, expressed in absolute numbers of bacteria grazed per hour, the chloroplast-bearing and apochloric populations grazed approximately 350 and 1,000 bacteria ciliate per hour, respectively. This experimental set-up brought new insights into the metabolic and physiologic traits of two distinct subpopulations of the same ciliate species living under strikingly different environmental constraints25,26,27.
Figure 1: Workflow of using fluorescently labeled bacteria (FLB) to estimate cell- and species-specific grazing rates from the ratio of ingested tracer FLB to total number of natural bacteria in the sample. For more details, see section 7 of the protocol. Please click here to view a larger version of this figure.
Figure 2: Examples of ciliate cells from plankton of a eutrophic fishpond. Examples are shown from the pond with countable FLB in ciliate cells (generally 1-10 tracer FLB per cell, the left two microphotograps) compared to a peritrichous ciliate Pelagovorticella natans (the right side microphotograph). Even during a short, 5 min incubation period, it became "over-labeled" by the tracer FLB, making quantitation of the ingested FLB inaccurate or almost impossible. In this case, it is suggested to decrease the tracer amount to 1.5%-3% of total bacteria. However, generally both the tracer amounts and incubation times can be manipulated to optimize the number of FLB ingested per cell. For more details, see section 7 of the protocol. Please click here to view a larger version of this figure.
Figure 3: Box-and-whisker plots of numbers of FLB per cell of Halteria sp. from the Římov reservoir (Exp I) (A), recalculated to rates of bacterial uptake per hour (B). The data were detected under different seasonal settings, represented by four examples from April to September. The top of panel A shows information on water temperature, different FLB tracers added (%), and different times of sample incubation. It should be noted that the latter two parameters can be modified to optimize number of FLB per cells, with average (full line) and median (dashed line) values approximately between 5-10 FLB per ciliate cell (A). The bars show the 25th and 75th percentiles of all data (50-180 cells inspected) and whiskers stand for the 1th and 99th percentiles. (B) Different small letters indicate significant differences in cell-specific bacterivory rates of Halteria sp. during the studied period. Please click here to view a larger version of this figure.
Figure 4: Box-and-whisker plots of numbers of FLB per cell. Plots are shown of chloroplast-bearing Tetrahymena utriculariae from triplicate treatments of young, mature, and old traps of Utricularia reflexa (Exp II) (A), recalculated to rates of bacterial uptake per hour (C). The data were compared to bacterial uptake rates of the duplicate apochloric populations of T. utriculariae (B,D) isolated from traps but maintained on mixed bacterial suspension growing on wheat grains in dark. On the top of panels A and B, different FLB tracers added (%) and different times of sample incubation are shown. It should be noted that the latter two parameters were modified to optimize number of FLB per cells, with average (full line) and median (dashed line) values approximately between 5-10 FLB per ciliate cell (A,B). The bars show the 5th and 95th percentiles of all data (50-100 cells inspected), and whiskers stand for the 1th and 99th percentiles. Please click here to view a larger version of this figure.
Deciphering trophic interaction in aquatic systems is always challenging28, especially at the nano-plankton scales involving protists and their prey, bacteria. When it comes to nutrient uptake pathways and quantification, the application of methods successfully used at higher trophic levels is less possible, due to the high complexity of biotic interactions. These include, for example, stable isotope labeling approaches. This protocol shows the advantages of using epifluorescence microscopy and fluorescently labeled bacteria as a tracer to track and semi-quantify/estimate the carbon flow (bacterial prey: various protistan grazers including mixotrophic grazers29) pathways through the base of microbial food webs. One such advantage is the high accuracy of the single-cell approach, and the other is the unprecedented resolution regarding grazer community structure and distinguishing between different functional guilds, species (Exp I), and even subpopulations of the same species (Exp II).
Critical steps in the protocol
There are several critical steps in the protocol, which can ensure that advantages of the methodology are utilized to their full potential. First, a basic understanding of the studied environment prior to commencement of the experiment is always beneficial. This includes microscopic screening of the diversity and abundance of potential grazers present, bacterial prey sizes, and prey distribution both 1) in the water column (e.g., a vertical profile from the epilimnion to the hypolimnion) and 2) in the case of canyon-shaped reservoirs, on the dam-inflow transect. Second, careful manipulation with collected samples will ensure representative results. Temperature is an extremely important factor affecting most microbial processes12, including protist grazing rates (Figure 3).
Third, manipulating the amount of tracer added based on the quantification of bacterial cells or type of grazer in the sample will ensure that problems with over-labeling (Figure 2) are eliminated. It should be noted that there is a very broad spectrum in ciliate species-specific uptake rates (for details, see step 7.2); thus, to apply the protocol appropriately, prior knowledge of major ciliate species with their time-course uptake rates is essential. It is strongly advised to run preliminary experiments with different tracer amounts to avoid possible ciliate under-labeling (none or too few FLB are taken up per ciliate cell, yielding statistically unsound data) or over-labeling (appears as large numbers of FLB forming "condense FLB clouds" or flocks in ciliate food vacuoles packed by the tracers, thus severely limiting their precise quantification; see upper right example in Figure 2). It should also be noted that incubation times with FLB are generally shorter than 30 min, since the average digestion time of picoplankton by ciliates is around 1.5 h, and digestion starts (ingested picoplankton cells loses its typical shape and color) approximately after 45-60 min30. Similarly, optimal dilution and distribution of samples on the filter prior to microscopic viewing needs to be achieved for accurate results.
Modifications and troubleshooting
The major steps, possible modifications, and troubleshooting modifications of the technique are illustrated in Figure 1 and Figure 2. Additionally, it should be noted that in cases of high concentrations of detrital particles, phytoplankton cells, or their colonies in plankton, such samples 1) should be correspondingly diluted to achieve a stage at which individual grazer cells can be distinguished on the filter surface and 2) be subjected to quantification of food vacuole contents.
Limitations
The main limitation for successful application of this method lies in the presence of various organic detritus or abundant inorganic/organic particles with attached bacteria or aggregates in amounts that prevent clear sample viewing under the epifluorescence microscope and precise estimation of a tracer amount added. It should be noted that the presented tracer technique works primarily with free (i.e., suspended) bacteria that are not attached to particles. However, based on our own experience and literature references (see previous publications2,4,8,10,16,18,21,26), the presented methodology is suitable for most aquatic environments. Examples of two natural, contrasting systems differing in trophic status, detritus content, and grazer diversity and numbers are provided (Figure 3 and Figure 4).
Significance of the approach with respect to existing methods
Importantly, from knowledge of the abundance of a taxon/taxa of bacterivores and their species-specific bacterivory rates, the bulk bacterivory rate of the protistan taxon (or total ciliate assemblage) can be calculated. If this approach is applied to natural plankton environments concomitantly for both heterotrophic flagellates and ciliates (representing the major grazers of bacterioplankton2,6,7), the protistan grazing-induced turnover time of bacterial populations in a given environment can be estimated16,17,18,22. Such data hold fundamental importance for the estimation of carbon flow dynamics in microbial food webs.
Future applications
There are other specific environments in which this method, with some modifications, may be used successfully. These include activated sludge systems, rumen ecosystems, aquatic sediments, and hypertrophic fishponds17. However, application in these nutrient- and microbes-rich environments requires preliminary tests to optimize the protocol regarding the proper size, morphology, and numbers of tracer FLB that can mimic the typical size distribution and other characteristics of prey bacteria inherent to the environment.
Currently, there is increasing interest in combining this approach with the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), in which the identity of the grazer cell (e.g., heterotrophic flagellate) is detected with a specific FISH-probe and the uptake rate is based on FLB content in food vacuoles of the flagellate cell on the same microscopic slide31. A sophisticated, new approach called double hybridization32 is a combination of FISH probes at the levels of the predator cell and prey bacteria (that are also labeled specifically by a phylogenetic strain, a bacterial lineage-specific FISH probe). The approach is elegant but also time-consuming and requires specific skills and experience31,32, while application of various FLB uptake approach modifications can be more easily adopted for routine use in laboratories.
The authors have nothing to disclose.
This study was supported by the Czech Science Foundation under the research grant 13-00243S and 19-16554S awarded to K. Š. and D. S., respectively. This article was also supported by the project "Biomanipulation as a tool for improving water quality of dam reservoirs" (No CZ.02.1.01/0.0/0.0/16_025/0007417), funded by the European Regional Development Fund, in Operational Programme Research, Development and Education.
0.2-µm pore-size filters | SPI supplies, https://www.2spi.com/ | B0225-MB | Black, polycarbonate track etch membrane filters, diameter approprite for filtering apparatus used |
5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF) | Any brand | ||
Automatic pipettes with adjustable volumes | Any brand, various sizes | ||
Centrifuge | 22 000 x g | ||
Cryovials | Any brand, 2 mL size | ||
DAPI (4´,6-Diamidino-2´-phenylindole dihydrochloride) | Any brand | 1 mg ml-1 | |
Epiflorescence microscope | Magnification from 400 x up to 1000 x | ||
Filters appropriate for viewing in the DAPI and DTAF range | |||
Counting grid in one of the oculars | |||
Filtering apparatus | Usually with a diameter of 25 mm | ||
Formaldehyde | A brand for microscopy | ||
Glutaraldehyde | A brand for microscopy | ||
Immersion oil for microscopy | Specific oil with low fluorescence | ||
Lugol´s solution | Any brand or see comment | Make an alkaline Lugol' solution as follows: Solution 1 – dissolve 10 g of potassium iodide in 20 ml in MQ water, then add 5 g of iodine. Solution 2 – add 5 g of sodium acetate to 50 ml of MQ water. Add the solution 2 to the solution 1 and thoroughly mix | |
Methanol stabilized formalin | Any brand available for microscopy purposes | ||
Microscope slides and cover slips | Any brand produced for microscopy purposes | ||
MQ water for diluting samples | Any brand |
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Phosphate-buffered saline (PBS; pH = 9) | Any brand | 0.05 M Na2HPO4-NaCl solution, adjusted to pH 9 | |
PPi-saline buffer | Any brand | 0.02 M Na4P2O7-NaCl solution. Add 0.53 g Na4P2O7 to 100 ml of MQ water plus 0.85 g NaCl | |
Sampling device | Appropriate for obtaining representative sample | e.g. Friedinger sampler for lake plankton | |
Sodium thiosulfate solution | Any brand | 3% solution is used in the protocol | |
Sonicator | Any brand | 30 W | |
Vortex | Any brand allowing thorough mixing of the solutes and samples | ||
Water bath | Any brand allowing temperature to be maintained at 60 °C |