Figure 1.
Western blot analyses of SDS-PAGE separated extracts of Schistosoma mansoni sporocysts.
Larvae were cultured for 7 days in CBSS containing GFP, GST26, or Prx1/2 dsRNA, followed by probing with specific anti-GST26 (Fig. 1A), anti-Prx1 (Fig. 1B) or sample loading control anti-α tubulin antibodies. Using anti-α tubulin reactivity to normalize sample loads, scanning densitometry was used to quantify immunoreactive GST26 (Fig. 1C) and Prx1 (Fig. 1D) intensities in specific dsRNA-treated vs. GFP dsRNA control sporocyst groups. Both GST26 and Prx protein levels were significantly knocked down by 80% and 50%, respectively, when compared to the GFP dsRNA treatment. ** P<0.01; *** P<0.001; N = 3.
Figure 2.
Representative confocal epifluorescent photomicrographs of Schistosoma mansoni sporocysts showing immunolocalization of anti-GST26 and anti-Prx1 antibodies after cultivation in medium containing GST26, Prx or control GFP dsRNA.
Fluorescence specific to anti-GST26 reactivity (green) observed in GST26 dsRNA-treated larvae (Fig. 2B) was noticeably reduced compared to the nonspecific GFP dsRNA control sporocysts (Fig. 2A), consistent with the high protein knockdown (∼80%) seen in Western blot analysis. By contrast, little difference in fluorescence levels was observed between the nonspecific GFP dsRNA control-treated (Fig. 2C) and Prx1/2 dsRNA-treated (Fig. 2D) sporocysts, reflecting the relatively small decrease (∼50%) observed in immunoblot protein levels. N = 3.
Figure 3.
Graphic representation of the effect of exogenous H2O2 exposure on Schistosoma mansoni sporocysts following treatment with dsRNAs for GFP (specificity control), SOD, GPx, GST26, GST28 and Prx1/2.
Double-stranded RNA-reated sporocysts were exposed to 50 µM H2O2 for 4, 24 and 48 hrs (stippled, grey and black bars, respectively). Knockdown of larval GPx, GST26, GST28 and Prx1/2 antioxidants increased sporocyst mortality after 24 and 48 hr under oxidative stress conditions when compared to GFP dsRNA-treated or no treatment controls. Note that sporocysts treated with SOD dsRNA showed no difference in susceptibility to H2O2 oxidation at any of the time points compared to controls. **P<0.001; ***P<0.0001; N = 4.
Figure 4.
Effects of catalase on H2O2-mediate killing of GPx, GST26, GST28, SOD, Prx1/2 dsRNA-treated and control GFP dsRNA-treated Schistosoma mansoni sporocyst in vitro.
After 7 days of dsRNA incubation sporocysts were exposed to H2O2 alone, catalase alone or catalase combined with H2O2 for 48 hr followed by evaluation of sporocyst death using propidium iodide staining. Significant increases in H2O2–mediated mortality was abrogated in the presence of bovine catalase [H2O2+catalase] showing that H2O2 was the primary source of larval killing in antioxidant dsRNA-treated sporocysts, with the exception of SOD, *P<0.05; **P<0.001; ***P<0.0001; N = 6.
Figure 5.
In vitro cell-mediated cytotoxicity (CMC) assay results.
Schistosoma mansoni sporocysts, cultured for 7 days in medium containing antioxidant (GPx, Prx1/2, GST26 and 28, SOD) or control GFP dsRNA were incubated for 24 hr with plasma-free hemocytes from the susceptible NMRI snail strain of Biomphalaria glabrata followed by assessment of larval mortality by propidium iodide staining. Co-culture of GST26, GST28 and Prx1/2 dsRNA-treated sporocysts with snail hemocytes resulted in small, but significant increases in percent larval mortality when compared to GFP dsRNA controls. The GPx dsRNA-treatment exhibited a nonsignificant increase in larval killing, while mortality of SOD dsRNA-treated sporocysts showed no difference compared to GFP dsRNA controls. *P≤0.04; N = 4.