Double strand RNA delivery system for plant-sap-feeding insects

Abstract

Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests.

Fig 1. Delivery of nutrients through green beans.

(A) Certified organic green beans were immersed in ddH₂O or ddH₂O solution with green food coloring for a period of 3 hrs. Transport of green food coloring was observed in the green bean encircled by the oval area at the exposed calyx. (B) BMSB feeding bioassay. 3 animals were placed in a magenta jar with 3 greens beans immersed in either 2 ml microcentrifuge tube containing ddH₂O or a solution of ddH₂O and green food coloring. (C) BMSB placed in magenta jars are able to pierce through the green beans and reach the diet with their stylets. (D) BMSB excreta observed on day 2 and 3 post ingesting a solution of ddH₂O and green food coloring through green beans.

Fig 2. Comparison of natural and artificial diets.

Six diets were compared to assess optimal rearing of BMSB. Each magenta jar contained one BMSB and test diet as follows; (A) BMSB reared on green beans, (B) BMSB reared on green beans immersed in 300 μl of ddH₂O, (C) BMSB reared on artificial gypsy moth diet, (D) BMSB reared on artificial diet formulated for BMSB consisting of applesauce and 2% agar, (E) BMSB reared on artificial diet formulated for BMSB consisting of applesauce and 8% agar and, (F) BMSB reared on artificial diet formulated for BMSB consisting of green bean puree.

Fig 3. Effect of various diets on BMSB nymph growth.

BMSB nymphs 5 each were allowed to feed for a period of 4 weeks during which their body masses were recorded. Diets consisting of green beans; green beans immersed in water; artificial diet for gypsy moth; artificial diet of applesauce and 2% agar; artificial diet of applesauce and 8% agar and, artificial diet consisting of green bean puree. The plot has been normalized to the control diet of green beans. Data was expressed as mean ± SEM. A one way analysis of variance (ANOVA) was performed to test for statistical significance of data, P-value of 0.00048.

Fig 4. Analysis of dsRNA delivered through green beans.

(A) dsRNA of LacZ gene (lane 2), JHAMT (lane 3), and Vg (lane 4), were obtained after PCR products from genomic DNA were amplified with primers containing T7 promoter sequence. These fragments were further transcribed using T7 RNA polymerase; the obtained in vitro transcribed dsRNA was confirmed by electrophoresis on 1% agarose and visualized by staining with Sybr Gold® (Life technologies) alongside a DNA ladder (Lane 1). (B) Total RNA extracted from green beans immersed in 5 μg of dsRNA for 1 day (Lanes 2–4) or 6 days (Lanes 5–7) was subjected to cDNA synthesis. 2 μg of this total RNA was subjected to RNase A digestion (0.5 μg/μl final) for 1 hr (Lanes 8–13). RT-PCR was performed on representative cDNAs of LacZ, JHAMT and Vg genes following electrophoresis on a 1% agarose gel and visualized by staining with Sybr Gold (Life technologies) (Lanes 2–13) alongside a DNA ladder (Lanes 1 and 15).

Fig 5. Quantitative RT-PCR analysis of transcript levels after RNAi-mediated depletion of genes in BMSB.

Total RNA from 3 individual BMSB 4^th instar nymphs fed on JHAMT (A) 5μg (0.017 μg/μl), (B) 20μg (0.067 μg/μl) and Vg (C) 5μg (0.017 μg/μl) dsRNAs in 300 μl of ddH₂O delivered through green beans was isolated and the levels of transcripts were measured by qPCR. LacZ RNAi (Mock) served as a negative control. The 18s RNA was used as an internal standard to correct for differences in RNA recovery from tissues. Results are from three biological replicates, and error bars indicate SEM. A one way analysis of variance (ANOVA) was performed to test for statistical significance of data that indicate significant differences at P < 0.0001 level.

Fig 6. Harlequin bug (M. histrionica) feeding on green beans.

(A) Certified organic green beans were immersed in ddH₂O or ddH₂O solution with green food coloring for a period of 3 hrs. 3 each of 4^th instar HB were allowed to resume feeding on these beans after 24 hr starvation in each magenta vessel. (B) HB feeding bioassay day 2. (C) & (D) Some animals were observed to molt on day 3 of feeding. (E) & (F) Animals were observed to be feeding on green beans on days 3 and 4 respectively. (G) & (H) Green colored excreta was observed on day 5 and 6 of feeding HB with green beans immersed in solution of water and green food coloring.

Fig 7. Pea aphid (A. pisum) feeding on green beans.

(A) Certified organic green beans were immersed in ddH₂O or ddH₂O solution with green food coloring for a period of 3 hrs. 15 animals each of pea aphids were allowed to resume feeding on these beans after a 24 hr starvation. (B) & (C) pea aphid feeding bioassay. Animals feeding on beans immersed in either ddH₂O or a solution of ddH₂O and green food coloring respectively. (D) Excreta droplets were barely observed on day 3 of feeding pea aphids with green beans immersed in water. (E) Green colored excreta observed on day 3 of feeding pea aphids with green beans immersed in solution of water and green food coloring.

Fig 8. Harlequin bug (M. histrionica) feeding on baby collard greens.

(A) Organically grown baby collard greens were washed with sodium hypochlorite. The petioles of these leaves were then immersed in ddH₂O or ddH₂O solution with green food coloring for a period of 3 hrs. Three each of 4^th instar HB nymph were allowed to resume feeding on these collard greens after 24 hr starvation in each magenta vessel. (B) HB feeding bioassay day 1. (C) HB feeding bioassay day 1 containing ddH₂O solution with green food coloring, (D) Day 3 of feeding. (E) Excreta was observed on day 3 of feeding HB with collard greens immersed in water. (F) Green excreta was observed on day 3 of feeding HB with collard greens immersed in solution of water and green food coloring.