Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Heterotrophic prokaryotes are a key biological component in aquatic systems and account for a significant fraction of plankton biomass^1,2,3. 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 groups^4,5,6. In some systems, protists serve as the major cause of bacterial mortality^6,7.

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 ingestion^10,11. 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.

Protokół

1. Sample collection

1. Collection of reservoir water sample: the first case study (Exp I; lower natural in situ predator and prey abundance system)
   1. Collect water samples from the desired location at a suitable depth. Keep the samples in a temperature-controlled cooler filled at in situ temperature (avoiding temperature shock; it should be noted that uptake rates of protists are temperature-dependent) during transport to the laboratory.
      NOTE: Our sampling was conducted in the meso-eutrophic canyon-shaped Římov reservoir (South Bohemia, volume 34.5 x 10⁶ m³, maximal depth 43 m, mean retention time 100 days, dimictic). The sampling site was located at a 30 m depth, close to the dam. A mixed sample at a 0.5 m depth was collected by a 2-l Friedinger sampler.
   2. Continue to section 1.2 as soon as possible.
2. Collection of fluid from the traps of carnivorous Utricularia reflexa plants: the second case study (Exp II; a system with high predator and prey abundances, extremely small sample volume)
   1. Gently shake the plants underwater, remove from the cultivation container, and place them on absorbent material to absorb excess water. Depending on the robustness of the plants and size of the traps, choose 8-10 plant individuals.
   2. Divide each shoot into roughly equal parts by counting leaf nodes bearing trapping organs. Each shoot segment will serve to collect mixed samples representing young, middle-aged, and old traps.
   3. Attach a thin glass capillary and Eppendorf vial for sample collection to a peristaltic pump. Proceed to insert the capillary tip into the trap opening. Using the vacuum pump, suck out all the fluid from each trap until 900 ± 100 µL of trap fluid is collected for each trap age category.
   4. Use triplicate subsamples of 200 µL of the pooled trap fluid for protistan grazing experiments. Process these immediately as detailed in section 3. Preserve the remaining ±300 µL of the sample for all other analyses of microbial components living in the fluid, as detailed below (section 2).
   5. Proceed immediately to section 2.

2. Fixation of collected samples

1. Exp I and II: fix water/trap fluid sub-samples for bacterial enumeration (section 4; approximately 20 mL and 0.3 mL, respectively) with formaldehyde for at least 1 h to obtain a 2% final volume:volume concentration in each sample.
   NOTE: Handle formaldehyde exclusively in the fume hood, and wear gloves at all times while manipulating samples.

3. Sample filtration

1. Sample dilution (Exp I): no dilution is needed for the reservoir water samples. (Exp II): dilute the trap fluid sample 10x-100x with particle-free MQ water to achieve a suitable distribution of the target microbes on filter surfaces prior to counting via epifluorescence microscopy.
2. Filter 1-2 mL (Exp I) of the reservoir water or 10-30 μL (Exp II) of the subsample of the trap fluid for bacterial counting onto black 0.2 µm pore-size filters, using a filtration funnel (25 mm diameter).
3. Stain the filters with DAPI (4',6-diamidino-2'-phenylindole dihydrochloride, 0.2% final concentration) for 4 min.
   NOTE: Avoid skin and working surface contamination, and wear gloves.
4. Place the filter with concentrated microbes onto a drop of immersion oil (for fluorescent microscopy) on a microscope slide. Place another oil drop onto the center of the filter and cover with a coverslip, making sure that the oil is distributed evenly.
5. At this point, preferably process samples immediately, or alternatively, store them in the freezer (-20 °C) for several weeks to months until further analysis.

4. Enumeration of bacterial numbers on the filters

1. Place the slide under the epifluorescence microscope (with the filter set corresponding to the fluorochrome DAPI). Place a 10 x 10 counting grid into one of the oculars. Move the slide to a random position.
2. Quantify bacterial cells (blue fluorescence) in the area of the counting grid (under the magnification 1000x). In the counts, include the cells crossing the left and upper edges of the counting grid, while excluding those located across the right and lower edges.
3. Move to another random position and repeat enumeration on at least 10-15 counting grids, amounting to 500 cells counted in total.
4. Establish a conversion factor for a given microscope and magnification, based on knowledge of the ratio of the area of one grid to the total effective filtration area of the filter. Then, divide the total number of cells counted by the number of grids counted, yielding an average number of bacteria per grid.
5. Multiply the latter parameter by the conversion factor established and normalize the resulting number per mL of a sample (depending on the volume of sample filtered) to obtain the total bacterial abundance per mL.

5. Determining protistan abundance

1. Fix the water sample (Exp I) or trap fluid (Exp II) subsamples with either glutaraldehyde (1% final concentration, more suitable for samples with the presence of chlorophyll containing particles to be processed days to weeks after fixation) or using the formol-thiosulfate decolorization technique specified below.
   NOTE: Both preservation techniques prevent egestion of the ingested material from food vacuoles of protists⁸.
2. Regarding the formol-thiosulfate decolorization technique, add 100 µL/1 µL of Lugol’s solution into 20 mL/200 µL of the water/trap fluid subsample (Exp I/Exp II, respectively).
3. Follow immediately with the addition of 0.5 mL/50 µL of borate-buffered formalin then 20 µL/2 µL of 3% sodium thiosulphate (Exp I/Exp II, respectively).
   NOTE: The sodium thiosulphate decolorizes the yellow color of Lugol to enable observation of cells under the epifluorescence microscope⁸.
4. Filter a known volume of sample (depending on the number of target protists) onto 1 µm pore-size black polycarbonate filters.
5. Estimate the number of the protist species is by counting at least 200 cells under magnification 600x using the counting grid (see above).
6. Reduce the volume of the sample under a vacuum in the filtration funnel by low vacuum to approximately 2 mL. Then, release the under-pressure and add the DAPI fluorochrome (4',6-diamidino-2-phenylindole dihydrochloride¹³, 0.2% final concentration) for 2 min.

6. Determining community structure of ciliates in plankton samples

NOTE: Ciliate communities in freshwater habitats are highly diverse^14,15,16,18, and their microscopic determination is challenging. Sorting the ciliate groups into functional guilds^10,14,16,17 allows for more detailed analysis of different ciliate groups as pelagic bacterivores.

1. Evaluate ciliate community structure by combining the following:
   1. DAPI-stained samples in epifluorescence microscopy (to localize the ciliate cells with bright fluorescence of macro- and micro-nuclei of different sizes and morphology) combined with the uptake of fluorescently labeled bacteria (FLB⁸; for details, see below), tracking the ability of ciliates to feed on bacteria.
   2. Live sample observation in selected cases^17,19. For more details of the above approaches and criteria used for grouping ciliates into different taxonomic categories, see previous publications^16,17.
      NOTE: Cited studies have indicated that among ciliates, omnivorous species from Stichotrichia(genera Halteria and Pelagohalteria) and Oligotrichia (namely Rimostrombidium spp.) are the most important pelagic consumers of bacterioplankton in a vast majority of freshwater habitats^10,17,18.

7. Estimating ciliate grazing rates

1. Calculate the uptake rates of ciliates on bacteria based on changes in the average number of tracer [i.e., FLB⁸ per ciliate related to time of incubation (5-15 min)] and tracer amount of FLB added, accounting at most for 5%-15% of total bacteria.
2. To compare uptake rates among different protistan species, normalize the uptake rates as the number of bacteria per ciliate per hour, with calculations based on the actual incubation time and proportion of tracer FLB added.
   NOTE: The general scheme of the FLB method application to estimate both cell- or species-specific and bulk bacterivory rates in natural samples is depicted in Figure 1.
3. Preparation of FLB from bacterial strains indigenous to a freshwater environment⁸
   1. Select a suitable size (mean cell volume = MCV) and morphology of bacteria so that they effectively mimic the typical sizes of bacterioplankton/bacterial cells in the aquatic system being investigated.
      NOTE: For Exp I, a mixture of isolated strains from the study location of the genus Limnohabitans and Polynucleobacter was used (i.e., typical, highly abundant bacterioplankton in lakes and ponds)²⁰. For details on morphology and sizes of the strains, see previous publications^3,17,18,21.
   2. Harvest bacterial cells from culture by centrifugation (5,000 x g) for 15 min at the early stationary phase and mix them at a numerical ratio that yields MCV ± SD of the cells in the mixture corresponding to the typical MCV of bacteria at the chosen location.
   3. Suspend the pellets in 10 mL of phosphate-buffered saline (PBS; pH = 9).
   4. Add 2 mg of the yellow-green fluorescing dye 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF, binds to proteins) to the cell suspension in the phosphate-saline buffer, and incubate in a 60 °C water bath for 2 h.
   5. After the incubation, centrifuge the cells down, decant the DTAF solution, and wash and centrifuge 3x with PBS.
   6. After the final wash, re-suspend the cells in 20 mL of the PP_i-saline buffer.
   7. Vortex the FLB suspension and pipette 1.5 mL aliquots into 2 mL cryo-vials, then keep frozen (at -20 °C) in PP_i-saline buffer until use.
   8. Pre-filter PP_i-saline buffer through a 0.2 µm polycarbonate filter for use in the next step.
   9. To determine FLB concentration, transfer a small aliquot (usually 20-40 µL) to 2 mL of particle-free PP_i-saline buffer, sonicate at 30 W for several 2 s bursts, and filter onto 0.2 µm polycarbonate black filter for enumeration via epifluorescence microscopy (1,000X magnification) under optical filter settings for DTAF (448 nm/520-540 nm).
4. Tracer technique for estimation of ciliate bacterivory
   1. For grazing experiments in natural planktonic habitats, dispense 300 mL samples into well-rinsed 1 L flasks and incubate at in situ temperature for 15 min (to allow protists to recover from the handling shock).
   2. Add FLB tracers to constitute 5%-15% of total bacteria, with the amounts added depending upon the season and water temperature.
      NOTE: There is a very broad, season-dependent spectrum in ciliate species-specific uptake rates spanning several orders of magnitude (i.e., from 10¹-10⁴ bacteria ciliate^-1 per hour)^{10,16,17,18,22,23,24}.
   3. In periods of enhanced occurrence of ciliates with high uptake rates (usually during the summer), also run a parallel incubation with very low FLB additions, constituting only 2%-4% of total bacteria to avoid excessive loading of ciliate vacuoles by tracer FLB (see examples in Figure 2).
   4. Incubate ciliate/plankton samples with FLB for 5-15 min.
   5. There are two possibilities for sample fixation to prevent egestion of the ingested material from food vacuoles of protists⁸. Terminate the incubations by the addition of 1% glutaraldehyde (final concentration that is more suitable for samples with chlorophyll-containing particles, such as algae). Alternatively, use 100 µL/10 µL of Lugol's solution into 20 mL/200 µL of water/trap fluid subsample, followed immediately by the addition of 0.5 mL/10 µL of borate-buffered formalin then 200 µL/2 µL of 3% sodium thiosulphate (Exp I/Exp II, respectively).
5. After adding the fixative, let the samples rest for at least 1 h in the dark at 4 °C to ensure thorough preservation of ciliate cells.
6. Take natural plankton subsamples from 4-30 mL/10-30 μL (Exp I/Exp II, respectively; the volume depends on ciliate abundance) and stain with DAPI (final concentration 0.2% wt/vol; for details, see step 3.2 above).
7. Pass through 1 µm black filters and inspect via epifluorescence microscopy to count ciliates (600x magnification) and enumerate the number of FLB tracers ingested (mostly at 1000x magnification) as detailed in previous publications^2,17. Inspect samples within 7 days after preservation.
8. To estimate total protozoan/species-specific grazing, multiply average uptake rates of all ciliates, or of only the ciliate species as detected by in situ abundance.
9. Example of the calculation of per-cell uptake rates from in situ data from the Římov water reservoir is described as follows:
   1. Assume that the bacterial concentration is 3.55 x 10⁶ bacteria/mL and tracer FLB added is 0.25 x 10⁶ FLB/mL, which yields a sum of 3.8 x 10⁶ bacteria/mL of total bacterial particles (natural bacteria + FLB = 100% of prey particles) available to phagotrophic protists in the natural sample.
      NOTE: The FLB tracers added thus represent 6.58% (a project of 0.25/0.038) of total bacterial particles. The average number of FLB per Halteria sp. is 6.2 FLB in 5 min incubations.
   2. To normalize uptake per hour, use the following calculation: (6.2 x 12)/(6.58/100) = 1131 bacteria per Halteria cell/h.
      NOTE: For more examples of distributions of individual uptake rates of Halteria sp. detected under variable water temperatures, amounts (as percents) of tracer FLB added, and different incubation times with FLB, see Figure 3.

Wyniki

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) particles^10,16,17,18,

Dyskusje

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 fl...

Materiały

Name	Company	Catalog Number	Comments
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
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