Direct experimental manipulation of intestinal cells in Ascaris suum, with minor influences on the global transcriptome

Abstract

Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes.

Keywords: A. suum; Intestine; Methodology; Nematode; RNAi; dsRNA.

Fig. 1

Overall experimentation workflow. Computational target prioritization, including utilizing existing Caenorhabditis elegans datasets (A) was used to identify target Ascaris suum genes for use in the cannulation experiment (B). Priority targets from RNA interference (RNAi) knockdown experiments (C) were used to optimize the experimentation (D). Finally, global transcriptome responses to heterogeneous small interfering dsRNA (hsiRNA) was performed to identify gene sets of interest.

Fig. 2

Counts of genes differentially regulated by double-stranded (ds)RNA treatment (and not control) between 0 and 24 h in both the anterior and posterior Ascaris suum intestine. Genes (A) upregulated and (B) downregulated by hpo-19 RNA interference (RNAi) treatment and not control, in the anterior and posterior intestine are identified. Genes (C) upregulated and (D) downregulated by ceh-20 RNAi treatment and not control, in the anterior and posterior intestine are identified. Genes (E) upregulated (F) downregulated by RNAi treatment and not control in both the anterior and posterior intestine are compared for the two target genes. GO, Gene Ontology.

Fig. 3

Real-time (RT) PCR results in the anterior and posterior Ascaris suum intestine, after performing double-stranded (ds)RNA + heterogeneous small interfering dsRNA (hsiRNA) RNA interference (RNAi) treatment against the target genes (A) hpo-19 and (B) ceh-20. Off-target genes ddx-23, sre-12 and drr-1 were used as controls. Error bars represent S.D., and significance values were calculated according to two-tailed t-tests with unequal variance.

Fig. 4

Real-time (RT) PCR experimentation to optimize double-stranded (ds)RNA delivery in the cannulated Ascaris suum experimental system. (A) The effect on hpo-19 knockdown as a result of utilizing different dsRNA preparations in the anterior and posterior A. suum intestine. (B) The effect on hpo-19 knockdown as a result of different heterogeneous small interfering dsRNA (hsiRNA) doses in the anterior and posterior A. suum intestine. (C) The effect on hpo-19 knockdown in the intestine and reproductive tissue as a result of dsRNA and hsiRNA delivery in the intrapseudocoelomic (IP) injection experimental design. Error bars represent S.D., and significance values were calculated according to two-tailed t-tests with unequal variance. RNAi, RNA interference.