Phospholipase D expression and function during encystation. The potential role of PLD in encystation was examined by observing the changes in expression and enzyme activity, and the effect of inhibition of PLD on encystation efficiency. (a) Expression of two E. invadens PLD genes. FPKM values for EIN_017100 and EIN_196230 at each encystation time point are shown. (b) PLD enzyme activity was measured using the Amplex Red Phospholipase D kit (Molecular Probes). Relative activity (measured as fluorescence at 585 nm and normalized to trophozoite activity) is shown for each time point. Error bars indicate ± standard deviation. (c) Inhibition of PLD decreases encystation efficiency. Encysting cultures of E. invadens were either untreated, treated with 0.6% n-butanol (an inhibitor of PLD activity) or with 0.6% t-butanol (which has no effect on PLD activity). Significant reduction of encystation efficiency (P < 0.01) was seen with n-butanol treatment, when compared to untreated parasites, but efficiency did not change with addition of t-butanol. Error bars indicate ± standard deviation. Insets show representative images of n-butanol and t-butanol treated samples, stained with calcofluor, which labels cysts. Note the higher percentage of positively staining cells in the t-butanol treated sample. (d) Inhibition of E. invadens PLD by n-butanol. To confirm that the effect of n-butanol on encystation was due to inhibition of PLD, enzyme activity of trophozoite lysate was measured in the presence of either n- or t-butanol. Relative activity (measured as fluorescence at 585 nm and normalized to activity in untreated lysate) is shown for each condition. Error bars indicate ± standard deviation.