Inadequacy of the Eucaryote Inhibitor Cycloheximide in Studies of Protozoan Grazing on Bacteria at the Freshwater-Sediment Interface

Tremaine, Sarah C.; Mills, Aaron L.

doi:10.1128/aem.53.8.1969-1972.1987

Cited by 50 publications

(21 citation statements)

References 26 publications

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“…Cycloheximide, as a eukaryote-specific inhibitor, has been commonly used in studying protozoan predation (Taylor & Pace, 1987). The most commonly used inhibitory concentration is 100 mg L À1 (Sanders & Porter, 1986), while various concentration ranges, for example 25-200 mg L À1 (Tremaine & Mills, 1987), were reported to be effective in inhibiting protozoa. Much higher concentrations of cycloheximide were used previously in sediment studies (Davies et al, 1995), presumably to counter the adsorption of cycloheximide by organic matter in sediment.…”

Section: Discussionmentioning

confidence: 99%

Effects of autochthonous microbial community on the die-off of fecal indicators in tropical beach sand

Fan¹,

Goto²,

Yan³

2010

FEMS Microbiology Ecology

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The recently observed high levels of fecal indicators in beach sand confound beach water monitoring efforts. The high levels of fecal indicators may be caused by the loss or the reduced activities of common environmental stresses controlling die-off in the sand. Microcosm experiments were conducted to compare the effects of biotic stresses from autochthonous sand bacteria, protozoa, and viruses on Escherichia coli and Enterococcus faecalis in two tropical beach sands. The inhibition of protozoan activities by cycloheximide did not significantly affect the die-off of E. coli, indicating that protozoan predation played a limited role in beach sand. The contribution from phage infection to E. coli die-off was also negligible. Consequently, autochthonous bacteria were identified as the predominant biotic stress to the die-off of E. coli in beach sand. Subsequent experiments demonstrated that the beach sand had a very low protozoan concentration and low protozoan growth potential when compared with various environmental samples. Co-culturing of E. coli with autochthonous sand bacterial isolates significantly enhanced E. coli die-off. PCR-denaturing gradient gel electrophoresis analysis revealed a complex sand bacterial community, suggesting that bacterial antagonistic effects may be widespread. The study also found that E. faecalis exhibited a much longer survival in beach sand compared with E. coli.

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Section: Discussionmentioning

confidence: 99%

Effects of autochthonous microbial community on the die-off of fecal indicators in tropical beach sand

Fan¹,

Goto²,

Yan³

2010

FEMS Microbiology Ecology

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“…A literature search yielded over 30 publications (most shown in Table 1), none of which tested all three assumptions thoroughly. Of the studies that did address these assumptions to some extent, most notable are Newell et al (1983), Sherr et al (1986), Taylor et al (1986), Taylor and Pace (1987), Tremaine and Mills (1987), and Tso and Taghon (2006) who evaluated the first and third assumptions in some detail, but only Taylor and Testing assumptions of the eukaryotic inhibitor method for investigating interactions between aquatic protozoa and bacteria, applied to marine sediment Pace (1987) also tested for effects on non-target eukaryotes (phytoplankton).…”

mentioning

confidence: 99%

“…McCambridge and McMeekin 1980;Newell et al 1983;Marino and Gannon 1991), whereas others reported incomplete inhibition (e.g. Sherr et al 1986;Sanders and Porter 1986;Taylor and Pace 1987;Tremaine and Mills 1987;Chabaud et al 2006). In some cases, cycloheximide took at least 48 h (Tremaine and Mills 1987) or even 3 d (Chabaud et al 2006) to inhibit protozoa, which is a problematic time frame for incubation experiments with natural communities.…”

mentioning

confidence: 99%

“…Sherr et al 1986;Sanders and Porter 1986;Taylor and Pace 1987;Tremaine and Mills 1987;Chabaud et al 2006). In some cases, cycloheximide took at least 48 h (Tremaine and Mills 1987) or even 3 d (Chabaud et al 2006) to inhibit protozoa, which is a problematic time frame for incubation experiments with natural communities. Likewise, in some studies cycloheximide was shown to have no direct impact on bacteria (e.g.…”

mentioning

confidence: 99%

“…Likewise, in some studies cycloheximide was shown to have no direct impact on bacteria (e.g. McCambridge and McMeekin 1980;Newell et al 1983;Sherr et al 1986;Bomo et al 2004), whereas in others, it was found to inhibit aerobic or anaerobic bacteria (Taylor and Pace 1987;Tremaine and Mills 1987). Taylor and Pace (1987) strongly advised caution in the use of eukaryotic inhibitors, and Tremaine and Mills (1987) concluded that cycloheximide should not be used in grazing…”

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confidence: 99%

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Testing assumptions of the eukaryotic inhibitor method for investigating interactions between aquatic protozoa and bacteria, applied to marine sediment

Shimeta

Cook

2011

Limnology & Ocean Methods

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Eukaryote-metabolic inhibitors have been applied in published incubation experiments to inhibit protozoans and infer their impacts on bacteria. This method has been used with water-column, sedimentary, biofilm, biofilter, soil, groundwater, wastewater, and sludge samples. The approach rests on assumptions that rarely have been tested: 1) the inhibitor is completely effective against all protozoans; 2) the inhibitor has no effects on other eukaryotes in the system (e.g., microalgae, metazoans) that could influence bacterial activity; and 3) the inhibitor has no effect on bacterial activity. We tested these assumptions for 10 inhibitors used in published studies (amphotericin B, anisomycin, colchicine, cycloheximide, cytochalasin B, fumagillin, griseofulvin, neutral red, nystatin, and thiram) and one combination of inhibitors (colchicine + cycloheximide) using marine sedimentary organisms. Only thiram was completely effective against the natural assemblages of ciliates and flagellates. With some inhibitors, partial inhibition of ciliates was followed by regrowth within 6-24 h or a bloom of a resistant species. Most inhibitors also killed infaunal invertebrates. Five inhibitors were further tested for direct effects on bacteria at concentrations that inhibited ciliates. Each inhibitor altered either esterase activity, denitrification, or both. All inhibitors thus violated one or more assumptions of the method. Conclusions in published work that did not demonstrate the validity of all assumptions underlying this method should not be accepted. Any future attempt to use a eukaryotic inhibitor against protozoans must confirm all assumptions of this method for the particular habitat, species, and processes to which the inhibitor will be applied.

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Protistan Communities in Groundwater

Kinner

2003

Encyclopedia of Environmental Microbiology

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Studies of Protozoa in Soils and the Subsurface Types, Abundance, and Enumeration Methods for Protozoa Protozoan Transport in the Subsurface Protozoan Role in the Subsurface Impact of Protozoa on Contaminant Biodegradation in Aquifers

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Inadequacy of the Eucaryote Inhibitor Cycloheximide in Studies of Protozoan Grazing on Bacteria at the Freshwater-Sediment Interface

Cited by 50 publications

References 26 publications

Effects of autochthonous microbial community on the die-off of fecal indicators in tropical beach sand

Effects of autochthonous microbial community on the die-off of fecal indicators in tropical beach sand

Testing assumptions of the eukaryotic inhibitor method for investigating interactions between aquatic protozoa and bacteria, applied to marine sediment

Protistan Communities in Groundwater

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