Delivery of lethal dsRNAs in insect diets by branched amphiphilic peptide capsules

Abstract

Development of new and specific insect pest management methods is critical for overcoming pesticide resistance and collateral off-target killings. Gene silencing by feeding dsRNA to insects shows promise in this area. Here we described the use of a peptide nano-material, branched amphiphilic peptide capsules (BAPCs), that facilitates cellular uptake of dsRNA by insects through feeding. The insect diets included dsRNA with and without complexation with BAPCs. The selected insect species come from two different orders with different feeding mechanisms: Tribolium castaneum and Acyrthosiphon pisum. The gene transcripts tested (BiP and Armet) are part of the unfolded protein response (UPR) and suppressing their translation resulted in lethality. For Acyrthosiphon pisum, ingestion of BiP-dsRNA associated with BAPCs led to the premature death of the aphids (t_1/2=4-5days) compared to ingestion of the same amounts of free BiP-dsRNA (t_1/2=11-12days). Tribolium castaneum was effectively killed using a combination of BiP-dsRNA and Armet-dsRNA complexed with BAPCs; most dying as larvae or during eclosion (~75%). Feeding dsRNA alone resulted in fewer deaths (~30%). The results show that complexation of dsRNA with BAPCs enhanced the oral delivery of dsRNA over dsRNA alone.

Keywords: Acyrthosiphon pisum; Armet; BAPCs; BiP; Oral delivery; Tribolium castaneum; dsRNA.

Figure 1.

(A) 5 × 5 μm AFM image analysis of the BACP nanoparticles (40 μM). (B) 5 × 5 μm AFM image analysis of the BACP- dsRNA complexes (40 μM and 1 μg respectively) (C) Schematic representation of BACPs- dsRNA interactions.

Figure 2. (DLS) and (ZP) analyses for different BAPCs-DNA formulations.

(A) Size (z-average) and (B) zeta potential. Data are based on two independent experiments. Different formulations were tested keeping the amount of dsRNA constant (1 μg) and varying the BACPs concentration. Differences between values were compared by ANOVA using Bonferroni as post-test. Statistical significance: (**) P < 0.01; (****) P < 0.0001. Non-statistical significance (ns) was considered when P > 0.05.

Figure 3.. Survival curves showing the effect of BAPCs complexed with BiP-dsRNA in Acyrthosiphon pisum.

The upper panel shows the survival rates for dsRNA at the indicated concentrations plus and minus the BAPCs. The bottom panel represents the RNA-free diet without and without BAPCs. Data is based on three independent experiments, n =20 for each experiment. Statistical significance: (****) p < 0.0001 versus control groups treated with naked BiP-dsRNA. Non-statistical significance (ns) was considered when p > 0.05. Differences between values were compared by Log-rank (Mentel-Cox) test.

Figure 4:

Pea aphid transcript levels of BIP-mRNA isolated from gut. Shown are the transcript levels of the BiP-mRNA when the insects (n = 20 per group) were fed BiP-dsRNA with and without complexation with BAPCs. Time zero represents the mRNA levels of insects prior to placing them on the diet. Data represent mean values +SD of three experiments combined.

Figure 5.. Survival curves showing the effect of BAPCs complexed with dsArmet + BiP-dsRNA in Tribolium castaneum.

Early-instar larvae were fed wheat-flour/yeast extract diet supplemented with Armet- or BiP-dsRNA singly or together (500 ng of each) with or without BAPCs. Day 1 represents the first day of feeding. After 6 days, the insects were transferred to the standard diet, lacking dsRNA or BAPC additions. Diseased Tribolium were counted on a daily basis. The number of insects on Day 1 were 35 (Diet alone); 30 (water plus CaCl₂) 50 (BAPCs-only control); 30 (Armet-dsRNA alone); 30 (BiP-dsRNA alone); 30 (BIP-dsRNA/BAPC complexes); 30 (BIP- + Armet-dsRNAs/BAPC complexes) and 45 (no BAPCs and no dsRNA). Pupation started over a range of days, averaging about day 30 with eclosion (emergence of adults from pupae) over a range starting about Day 40. No deaths occurred in the adult stage. Statistical significance: (*) p < 0.05, (**) p < 0.01, versus control groups treated with naked BiP-dsRNA or BiP-Armet-dsRNA. Non-statistical significance (ns) was considered when p > 0.05. Differences between values were compared by Log-rank (Mentel-Cox) test.

Figure 6.. 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.

Figure 7.. 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 μM