A new method using enhanced green fluorescent protein (EGFP) to determine grazing rate on live bacterial cells by protists

Abstract

A new method was developed for estimating the grazing rate of live bacteria by protists. Bacterial cells (Escherichia coli bearing plasmid pEGFP) expressing enhanced green fluorescent protein (EGFP) were used as a live bacterial tracer. Ciliates (Tetrahymena thermophila) were fed with EGFP-tagged bacterial cells, and the individual cells taken up by the ciliates were detected by epifluorescence microscopy. The EGFP fluorescence was stable during the storage of samples fixed with glutaraldehyde. Comparison of clearance rates based on the uptake of EGFP-tagged live cells and fluorescently-labeled heatkilled cells suggested that the use of heat-killed cells underestimates the clearance rates. We suggest that EGFPtagged bacteria are a useful tracer for determining protist bacterivory in culture and aquatic environments.

Acidotropic probes and flow cytometry: a powerful combination for detecting phagotrophy in mixotrophic and heterotrophic protists

“…number of cells containing food vacuoles) caused by the use of artificial or manipulated prey (Porter 1988, Legrand et al 1996, Bouvier et al 1998, Ishii et al 2002. (2) Transfer of radioactivity from one organism to another or contamination of the mixotrophs' cell membrane by radioactivity without effective ingestion (Legrand et al 1998).…”

“…(2) Transfer of radioactivity from one organism to another or contamination of the mixotrophs' cell membrane by radioactivity without effective ingestion (Legrand et al 1998). (3) To determine whether the particle inside a mixotroph or heterotroph is a parasite or a gamete instead of prey in a food vacuole (Porter 1988, Legrand et al 1996, Bouvier et al 1998, Ishii et al 2002. Since LysoSensor™ Blue is efficient at marking food vacuoles and its fluorescence is not masked by mixotrophs' autofluorescence, this probe might help to identify new mixotrophic species, which are presently known only as strict autotrophs.…”

“…In addition, artificial prey does not allow growth and adaptation of predators to prey (Hammer et al 2001) and some protists can egest the particles 2 or 3 min after ingestion (Weisse 2002), leading to unrealistic ingestion rates. (2) Human counting errors (Marie et al 2004) enhanced by the difficulties of identifying phagotrophy in algae species (Porter 1988, Legrand et al 1996, Bouvier et al 1998, Ishii et al 2002, Rose et al 2004, and questionable detection of phagotrophy (Stoecker 1999) also lead to under-or overestimations of the occurrence of phagotrophy among phytoplanktonic species.…”

New tracer to estimate community predation rates of phagotrophic protists

“…Green and red fluorescent protein labeled cells have since increased in popularity (Parry et al 2001, Ishii et al 2002, Fu et al 2003, Worden et al 2006, Tuorto 2008, but these tracers have not been tested thoroughly for their validity as prey analogs, and most GFP-labeled cultured organisms that have been used in feeding experiments were much larger than cells found in natural prokaryote communities. Here we improve on this concept of a GFP-labeled tracer by using a more suitable cell size.…”

Molecular biogeochemistry of modern and ancient marine microbes

“…While rates of grazing-induced mortality of picocyanobacteria have been measured in situ (Sherr et al, 1987;Hall et al, 1993;Ishii et al, 2002;Massana et al, 2002;Worden and Binder, 2003;An-Yi et al, 2007), the specific identity of the grazers feeding on these cells has not been studied. In the present work, we have used a modification of a RNA stable isotope probing technique (RNA-SIP) (Radajewski et al, 2000;Manefield et al, 2002;Lueders et al, 2004) to identify eukaryotic cells that consume Prochlorococcus and Synechococcus in surface waters at the Hawaii Ocean Time Series (HOT) station ALOHA.…”