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

Abstract

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.

Insights into foraminiferal influences on microfabrics of microbialites at Highborne Cay, Bahamas

“…Importantly, it is certainly feasible that other heterotrophic protists including flagellates and ciliates and even metazoans were also associated with changes in Neoproterozoic stromatolite occurrences. The concentrations of the microtubule inhibitor colchicine that was used in our ∼6-mo-long microfabric experiment, however, is not thought to prevent ciliate and metazoan activities (33).…”

Comparison among the microbial communities in the lake, lake wetland, and estuary sediments of a plain river network

“…In a plain river network region, several types of sediment exist in different regions, such as lakes, wetlands, and estuaries. As sediment microbes from these regions affect entire aquatic systems (Shimeta & Cook, ; Shtarkman et al., ), understanding microbial communities in different basin regions is crucial.…”

Predator-prey interactions in aerobic granulation systems

“…To better understand the role of protozoan predation in sequencing batch reactors (SBRs), eukaryotic metabolic inhibitors were added to both floccular and granular sludge in mini-SBRs that were designed and fabricated for aerobic granulation. Thiram, which is a eukaryotic inhibitor, has been shown to be effective against the natural assemblages of ciliates and flagellates in marine sediments (Shimeta and Cook 2011). Inhibition refers to either metabolic inactivation or death.…”

“…To better investigate the role of protozoan predation in granule formation and maintenance of structural integrity, thiram was added into mSBRs that contained either floccular or granular sludge. Eukaryotic inhibitors are generally used for the inhibition of protozoan predators to study the rate of predation on bacteria, their effects on bacterial growth and their influence on bacterial activities (Ramirez andAlexander 1980, Shimeta andCook 2011). Thiram was selected as it was demonstrated to be highly effective against natural assemblages of ciliates (Shimeta and Cook 2011).…”

“…Eukaryotic inhibitors are generally used for the inhibition of protozoan predators to study the rate of predation on bacteria, their effects on bacterial growth and their influence on bacterial activities (Ramirez andAlexander 1980, Shimeta andCook 2011). Thiram was selected as it was demonstrated to be highly effective against natural assemblages of ciliates (Shimeta and Cook 2011). Other inhibitors such as cycloheximide and nystatin caused an initial decline in ciliate count which was followed by a bloom of tolerant ciliates species (Shimeta and Cook 2011).…”