Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte)

Abstract

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

Figure 1. dsRNA DvSnf7 direct toxicity in Western Corn Rootworm (WCR, Diabrotica virgifera virgifera) larvae.

A) Growth inhibition in second instar larvae fed on diet overlaid with 1000 ng/mL DvSnf7 dsRNA for 5 days compared to larvae fed with control diet containing similar concentration of GFP dsRNA. Scale bar  = 1 mm. Mortality of WCR larvae to varying exposure times of DvSnf7 dsRNA incorporated into diet bioassay at B) 50 ng and C) 1000 ng of dsRNA/mL diet. Open columns represents the mortality of 12-day continuous feeding.

Figure 2. Uptake of dsRNA by Western Corn Rootworm (WCR, Diabrotica virgifera virgifera) by midgut cells.

(A) Cy3-labeled 240 bp DvSnf7 dsRNA is taken up by WCR midgut cells (b–c), while DvSnf7 siRNAs are not (e–f, h–i). Nuclei DAPI staining was used to visualize midgut cells (a, d, g, j). Controls with Cy-3 dye alone do not show intracellular incorporation (k–l); Scale Bar: 50 µm.(B) Diet bioassays confirmed that labeled 240 bp dsRNA retains activity, while siRNAs are inactive. Percent mortality was determined 12 days after continuous exposure of WCR neonates to 100 ng/mL of dsRNA/siRNA. (C) Real-time RT-PCR of midgut tissue cultures exposed to 1 µg/100 µL of insect medium of dsRNA/siRNA. Snf7 mRNA levels are reduced after exposure to DvSnf7 240 bp dsRNA, while no mRNA reduction is observed after tissue incubation with siRNAs or control medium. Stars represent values significantly different from controls (p = 0.05; t-test).

Figure 3. DvSnf7 mRNA and DvSNF7 protein suppression by RNAi.

DvSnf7 dsRNA causes suppression at mRNA (A) and protein (B–D) levels in Western Corn Rootworm (WCR, Diabrotica virgifera virgifera). (A) Real-time RT-PCR results showing significant decrease in DvSnf7 mRNA expression in insects fed with 60 ng/mL of DvSnf7 240 bp dsRNA continuously for one and five days. Insects fed with control diets containing water or GFP dsRNA do not show mRNA suppression. (B) Western blot results using anti-SNF7 antibody showed DvSNF7 protein suppression in insects fed with DvSnf7 240 bp dsRNA after 5 days, which was confirmed by quantification of the Western blot by densitometry (C) as well as ELISA (D). Stars represent values significantly different from controls (p = 0.05; t-test).

Figure 4. RNAi effect spreads to other tissues beyond the midgut in Western Corn Rootworm (WCR, Diabrotica virgifera virgifera).

DvSnf7 mRNA levels were decreased in isolated midgut and carcass tissues of second instar larvae fed for 24 hours with 1000 ng/mL diet of DvSnf7 240 bp dsRNA, as assessed by Real-time RT-PCR. Further mRNA suppression is observed in the carcass and midgut at days 3 and 5 post-feeding. Means followed by same letter are not significantly different (p = 0.05; t-test).