Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests

Abstract

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[ N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal- Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to S. frugiperda larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

Figure 1

(a) Structure, number-average molecular weight (M_n), and dispersity (Đ) of pGPMA. (b) SEC trace of pGPMA. (c) Gel electrophoresis of pGPMA-dsRNA IPECs. Numbers indicate polymer/dsRNA weight ratio. (d) Proposed morphological changes in IPEC structure between pH = 7.4 and pH = 10.

Figure 2

Sf9 cells treated with Cy5-labeled dsRNA (red) complexed with pGPMA after (a, top row) 24 h or (b, bottom row) 48 h. Nuclei were stained with DAPI (blue). Scale bars = 5 μm. (c) Cell viability assay of pGPMA after 48 h employing polymer concentrations identical to the indicated weight ratios used in IPECs. Cell viability was determined relative to the untreated control. Error bars represent the standard deviation from triplicate experiments.

Figure 3

(a) Expression of CDC27 determined by RT-qPCR in Sf9 cells following incubation with pGPMA complexed with either CDC27- or control-dsRNA. Numbers indicate polymer/dsRNA weight ratios. Values are normalized to CDC27 expression in respective control (KIF-dsRNA-treated) samples. Errors bars represent SEM. (b) Expression of CDC27 determined by RT-qPCR in Sf9 cells following incubation with CDC27 dsRNA complexed with either pGPMA (8×) or Lipofectamine 3000. Values are normalized relative to respective untreated controls. Error bars represent SEM. (c) RT-qPCR quantification of CDC27-dsRNA transfected by pGPMA, Lipofectamine 3000, or untreated control. Values are relative to zero. Error bars represent SEM. For plots a–c, groupings indicated with asterisks (∗) were found to be significantly different after Tukey analysis.

Figure 4

(a) Expression of V-ATPase mRNA in midgut tissue from second instar fall armyworm larvae fed with pGPMA complexed with either V-ATPase dsRNA or GFP dsRNA determined by RT-qPCR. Letters indicate individual animals. Days between feeding and harvesting are indicated in parentheses. Values are normalized to V-ATPase expression in control sample. Error bars represent SEM. (b) Percent survival of second and third fall armyworm larvae fed pGPMA complexed with dsRNA targeting V-ATPase (N = 25) or control dsRNA (N = 31). (c) Image of fall armyworm larval gut after feeding with pGPMA complexed with dsRNA targeting GFP or (d) sfV-ATPase. Scale bars =2 mm.