Oral Delivery of dsRNA to Inhibit Gene Expression in Insects

 

Entry of dsRNA into cells is the first step in one of the most powerful tools in contemporary molecular biology: transcript knockdown via RNA interference (RNAi). RNAi-based transcript knockdown has been used in insects of numerous Orders and will, without doubt, continue to be a very important part of the reverse-genetics toolbox in insects. For the most part, dsRNA has been administered to insects by microinjection into hemolymph. While effective, this approach has limitations, not the least of which is the tedium it involves, and the smaller the insect species, the greater the challenge presented by injection. Feeding of dsRNA is a more attractive approach than hemolymph injection because it is non-invasive and also opens the possibility of developing new methods of pest control. However, the stability of dsRNA during or after oral delivery remains a large problem for this approach. To increase the stability of dsRNA and enhance their cellular uptake, polymeric nanoparticles have been used for nucleic acid delivery in RNAi-based gene therapeutics. The Avila-Flores Research Group is combing two threads of research – the use of BAPCs to facilitate the uptake of double-stranded nucleic acids; and the desirability of obtaining efficient transcript knockdown in insects by feeding dsRNA.

 

We demonstrated the ability of BAPCs to deliver dsRNA orally, and inhibit gene function in insect species from two different Orders and with different feeding mechanisms: Tribolium castaneum, the red flour beetle fed with solid flour diet; and the pea aphid, Acyrthosiphon pisum, fed in artificial liquid diet. The gene transcripts tested (BiP and Armet) are part of the unfolded protein response (UPR) and suppressing their translation resulted in lethality. In Tribolium, we also suppressed the expression of the Vermillion gene, that acts in the developing eye with its  transcript encoding the protein required for the development of  normal eye color. Ingestion of dsVermillion-RNA in complex with BAPCs during larval stages gave rise to adults with white (non-colored) eyes at a rather high frequency (about 50% with n = 20), thus verifying the systemic nature of the RNAi effect created by ingestion of dsRNA/BAPC complexes (Fig. 1). These results show that complexation of dsRNA with BAPCs greatly enhances the oral delivery of dsRNA over dsRNA alone in the diet.

Figure 1. Effects of feeding dsVermillion-RNA -/+ BAPCs in Tribolium. Feeding of BAPCs-dsVermillion complexes (as a “supplement” to the flour diet of Tribolium larvae) resulted in the absence of Vermillion color (in the eye) in treated insects, right panel.

We are currently applying this technology to different species such as Popillia japonica (Japanese beetle). The Japanese beetle is an invasive and generalist herbivore. Japanese beetle adults and larvae attack >300 species in >70 plant families including the major field crops in the US (corn, soybeans, and cotton). Our aim is to evaluate if BAPCs/dsRNA can provide effective plant protection against Popillia japonica by simple applying these formulations on plant leaves. We are also studying the bio-distribution of fluorescence-labeled dsRNAs with and without BACPs in different species, in order to shed a light on how dsRNA is transported from cell to cell to induce systemic RNAi (Fig. 2).

Figure 2. Localization of fluorescently labeled Armet-dsRNA in Tribolium larvae 8 hr after the feeding. The fluorescence was shown as magenta on the bright-field background. All pictures were captured in the same condition in a LSM700 confocal microscope. (A) midgut; (B) Fat body (C) Malpighian tubule, (D) to (F) are same tissues in the Tribolium fed with labeled Armet-dsRNA alone. Scale bar: 20 mM