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

Summary

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.

Abstract

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.

Introduction

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 functio....

Protocol

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....

Results

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^{10,16,17,18,22}.......

Discussion

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.......

Materials

List of materials used in this article

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