Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

The overall goal of these procedures is to demonstrate a natural oral delivery method. For gene-specific dsRNAs, to induce RNA interference in vivo and insect pests. This method can help to answer key questions on how to deliver biomolecules inside of insects, to elucidate the effect RNAi-based Molecular biopesticides, and to control insects in the environment.

The main advantage of this technique is this delivery through nucleic acid mediated gene silencing can be applied to plant sap feeding as well as chewing-insects. Though the methods here demonstrate delivery of double-stranded RNA to hemipteran pests, like the brown marmorated stink bug, harlequin bug, and asian citrus psyllid. They can be applied to other insects.

To begin the protocol, select early 4th Instar brown marmorated stink bug, or BMSB Nymphs, hatched from the egg mass, and starve them for 24 hours prior to dsRNA feeding. Next, select slender, certified organic green beans and wash them with 0.2%Sodium Hypochlorite solution for five minutes. Follow with three double-distilled H2O washes, and allow the beans to air-dry.

Once they are dry, trim the green beans from the calyx end to a total length of 7.5 centimeters using a clean razor blade. Immerse the washed and trimmed green beans in a capless, two milliliter micro-centrifuge tube containing 300 microliters of control solution. Make dilutions of the in vitro synthesized lac Z, JHAMT, or VGDSRNAs, by diluting 5 micrograms, or 20 micrograms, and 300 microliters of RNAs, DNAs-free water to yield final concentrations of 0.017 micrograms per microliter, or 0.067 micrograms per microliter respectively.

Immerse the washed and trimmed green beans in a capless, two milliliter micro-centrifuge tube, containing 300 microliters of dsRNA solution. Wrap and seal the edges of the micro-centrifuge tubes, enclosing the immersed beans to prevent evaporation of the dsRNA solution, and to prevent the animals from entering the micro-centrifuge tube. Position the tubes upright at room temperature for three hours, to allow the dsRNA solution to load throughout the green bean by capillary action.

Place the tubes in clean, polypropylene culture vessels, and place 3 starved 4th Instar BMSB nymphs in the culture vessels. Treat three insects per culture vessel. Each containing three green beans with green food coloring, or dsRNA solution.

Maintain the insects at 25 degrees Celsius and 72%relative humidity, under a 16L, 8D photoperiod in an incubator. Allow the insects to feed on the green beans immersed and absorbed in dsRNA for five days, and replenish them with fresh diets of dsRNA-treated green beans after three days. Select plants or seedlings, and do not water them for two to three days prior to use, to let the soil dry out to damp, but not completely dry.

Using a hand-pumped spray bottle, apply 200 milliliters of dsRNA solution to the lower canopy. Sample the new growth from previously-topped trees after 25 to 40 days, by collecting approximately 10 leaves from the tips of four branches. Extract the total RNA, and analyze it by RT-PCR and qPCR for the presence of the applied dsRNA trigger.

Similarly, topically spray 10 milliliters of dsRNA to the lower region of a seedling, or small-potted tree foliage. Select plants or seedlings, and do not water them for two to three days prior to use, to let the soil dry out, to damp, but not completely dry. Next, add one liter of dsRNA solution to the soil of large-potted plants.

Add one liter of water after one hour. Apply 100 milliliters of dsRNA solution to the soil of one-meter tall potted trees in partially-dry soils. Allow the plants the receive the dsRNA solution as a soil drench, to soak for 30 minutes.

Then apply plain water-only treatment to aid absorption by the roots. Select citrus seedlings, new or approximately 3.5 year-old plants to inject dsRNA using the stem tap, also known as the trunk injections method. Wrap the copper tip of each injector four to six times with a 0.6 centimeter-wide strip of ceiling film to prevent leakage near the tip.

Next, fill the tree trunk injectors with six milliliters of dsRNA solution diluted with DNAs, RNAs-free water. Drill holes in the citrus plants using a 10 milliliter drill bit, and take care not to exceed two centimeters, or about half the diameter of the stem. Then, inject the solution into the tree trunk, and leave the injector in the trunk for six to 10 hours to allow absorption of the dsRNA solution.

Let the insects feed on the cuttings from treated trees at 3, 10, and 30 days post-treatment. Pour out 30 grams of the clay absorbent into a 50 milliliter conical tube to the 35 milliliter mark. Pour 20 milliliters of dsRNA solution diluted in DNAs, RNAs-free water into the tube to wet all the absorbent.

Cap the conical tube, tip the tube to help remove air, and place the tube upright, and let it stand for one to two minutes while the clay particles absorb the solution. Add enough dsRNA-soaked clay into the soil mix to fill a one gallon pot. Mix and turn the soil by hand to thoroughly mix the dsRNA-soaked clay into the soil.

Use the soil to re-pot seedlings selected for the dsRNA treatment. Water the soil with 200 milliliters of plain water, without dsRNA, after 30 minutes to one hour. Follow with 100 milliliters of plain water.

After 24 hours, put the plant on a normal watering schedule. Test four to six leaves of the treated potted plants with clay absorbents and dsRNA each month, post-treatment for dsRNA, by collecting the most apical leaves of new plant growth. QPCR of green bean segments immersed in In vitro synthesized dsRNA, specific to brown marmorated stink bug, or BMSB mRNA, indicated the successful induction of RNAi as the expression of targets JHAMT and Vg was significantly reduced in vivo.

This method of delivery is also effective on another hemipteran pest, the harlequin bug. As indicated by their green colored excreta. After watching these visual demonstrations, you should have a good understanding of how to deliver double-stranded RNA to insects through oral ingestion for inducing RNAi insects in vivo.

The advances being made in RNAi nucelotide technology continues to expand the capabilities of researchers to modulate RNA, gene expression, and protein levels in a wide range of living organisms. This goes from plants, to insects, and the most difficult micro-organisms. So while the cost of production of double strand RNA continues to decline, and methods of delivery and stability continue to improve, the future of RNAi technologies will continue to be used to solve emerging, complex problems.