Table 1.
The siRNA oligos used in this study.
Figure 1.
The silencing of Ts-pmy mRNA mediated by siRNA.
The adult and larval T. spiralis were transfected with FITC-labeled control siRNA by electroporation (A). Uptake of FITC-labeled siRNA into adult worms (a) and larvae (b) 18 hours after electroporation under a fluorescent microscope. No fluorescence was observed in the untreated group (c). The relative levels of Ts-pmy mRNA in parasites (larvae and adults) 6 days after being electroporated with different siRNAs, as determined with qRT-PCR (B). All of the assays were performed in triplicate, and the data are presented as the mean ± SE. *p<0.05 compared with control siRNA.
Figure 2.
The silencing of Ts-pmy mRNA in larvae treated with siRNA1743.
The relative level of Ts-pmy mRNA in larvae following electroporation or soaking with various concentrations of siRNA1743 for 6 days, as determined with qRT-PCR (A). The better siRNA suppressive effect in treated larvae was observed when a longer incubation was applied after electroporation (B). All of the assays were performed in triplicate, and the data are presented as the mean ± SE. *p<0.05 compared with control siRNA.
Figure 3.
The silencing of Ts-pmy mRNA mediated by dsRNA.
The relative level of Ts-pmy mRNA expression in larvae soaked with different dsRNAs (50 ng/µl) for 6 days (A). The relative level of Ts-pmy mRNA in larvae following electroporation or soaking with various concentrations of dsRNA-PF3 for 6 days, as measured with qRT-PCR (B). Specific suppression of Ts-pmy and Ts87 mRNA expression in larvae 6 days after being soaked with 50 ng/µl dsRNA-PF3 or Ts87 dsRNA (C). All of the assays were performed in triplicate, and the data are presented as the mean ± SE. *p<0.05 compared with the control dsRNA-treated group.
Figure 4.
The silencing of Ts-PMY protein expression mediated by siRNA or dsRNA.
Western blot with specific antibodies showing the specific inhibition of Ts-PMY protein expression in extracts of T. spiralis larvae (A, B) and adult worms (C, D) induced by dsRNA-PF3 (A, C) or siRNA1743 (B, D). Graphs on the right show the relative protein levels measured by densitometry from three independent experiments. *p<0.05 compared with the control dsRNA/siRNA-treated group.
Figure 5.
Suppression of Ts-pmy mRNA expression by siRNA1743 caused defects in larval molting.
A. The larvae were electroporated with 8 µM siRNA1743 (a) or control siRNA (b) and incubated for 18 hours. The untreated larvae (c) were used as a negative control. The larvae with cuticle sheath at the end(s) were counted as molting ones. B. The molting percentage for each treatment was calculated. All of the assays were performed in triplicate. *p<0.05 compared with the control siRNA-treated group.
Figure 6.
Suppression of Ts-pmy mRNA expression by siRNA1743 caused surface damage and defected viability of treated larvae.
The larvae treated with siRNA1743 for 6 day were stained with SYTOX for 15 min and then observed under fluorescence microscopy (A) or measured with a fluorescence microplate reader at 485 nm excitation and 530 nm emission (B). All of the assays were performed in triplicate. *p<0.05 compared with the control siRNA-treated group.
Table 2.
Recovery of adult worms, muscle larvae and newborn larvae of T. spiralis from mice infected with larvae transfected with siRNA1743 by electroporation.