1. BMSB Rearing

1. Rear BMSB insects as per standard lab practice and previously described⁶³.
2. Raise ACP (D. citri) insects on Citrus macrophylla in a glasshouse (22 °C) and natural light. Use adult ACP, at approximately 5-7 days post eclosion.

2. Selection of Gene Regions and In Vitro Synthesis of dsRNA

1. Select genes specific to BMSB from previously published transcriptome profiles³².
2. Ensure the regions of interest selected vary between 200 to 500 base pairs.
3. Perform polymerase chain reaction (PCR) using the conditions described below to generate fragments associated with the selected gene of interest from genomic DNA. See Table 1 for the gene-specific oligonucleotides.
   1. PCR reaction: In a 0.25 mL PCR tube, combine 5 µL of 10X PCR Buffer, 4 µL of dNTP Mixture (2.5 mM each), 2 µL of DNA template (50 ng/µL), 2.5 µL each of Primers 1 and 2 (10 µM), 0.25 µL of DNA polymerase (5 U/µL), and DNase/RNase free water up to 50 µL.
   2. PCR condition: Cycle the PCR reaction to amplify the region of interest at 95 °C for 3 min followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, 72°C for 1 min. Incubate the reaction at 72 °C for an additional 10 min. Purify the PCR reaction using a purification kit.
4. Amplify the obtained PCR fragments further with gene specific primers flanked with the T7 RNA polymerase promoter sequence (5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA-3') as mentioned earlier⁶².
5. Use LacZ gene as a negative control (mock) for RNAi.
   NOTE: LacZ is a gene that encodes β-galactosidase amplified from Escherichiacoli genomic DNA (the primers used are listed in Table 1).
6. Perform in vitro transcription to yield dsRNA as described earlier⁶².
7. Dissolve and resuspend the resulting dsRNA in 150 µL DNase/RNase free water, measure the concentration, and store at -80 °C for future use.

3. Delivery of dsRNA Using Green Beans

1. Select early 4^th instar BMSB nymphs hatched from the same egg mass and starve them for 24 h prior to dsRNA feeding.
2. Select slender certified organic green beans (Phaseolus vulgaris L.) and wash with 0.2% sodium hypochlorite solution for 5 min.
   NOTE: Slender green beans were selected so that the beans can easily be accommodated in the 2 mL microcentrifuge tubes.
3. Wash 3 times with ddH₂O and allow to air dry.
4. Trim the green beans from the calyx end to a total length of 7.5 cm using a clean razor blade.
5. Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 µL of control solution (a 1:10 dilution of green food coloring (ingredients: Water, Propylene Glycol, Fd&C Yellow 5, Fd&C Blue 1, and Propylparaben as preservative)).
6. Make dilutions of the in vitro synthesized LacZ, JHAMT, or Vg dsRNAs by diluting 5 µg or 20 µg in 300 µL of RNase/DNase free water to yield final concentrations of 0.017 µg/µL or 0.067 µg/µL, respectively.
7. Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 µL of dsRNA solution (from step 3.6).
8. Wrap and seal the edges of the microcentrifuge tubes enclosing the immersed beans to avoid evaporation of the dsRNA solution and to prevent the animals from entering the microcentrifuge tube.
9. Position these tubes in an upright manner at room temperature for 3 h to allow the dsRNA solution to be loaded throughout the green bean by capillary action.
10. Place these tubes in clean culture vessels (polypropylene). Place three starved 4^th instar BMSB nymphs in the culture vessels.
11. Treat three animals per culture vessel each containing three green beans with green food coloring or dsRNA solution. Maintain the insects at 25 °C and 72% relative humidity, under a 16L:8D photoperiod in an incubator.
12. Allow the insects to feed on the green beans (immersed and absorbed with dsRNA) for 5 days but replenish with fresh diets of dsRNA treatment green beads after 3 days.

4. Real-time Quantitative (qPCR) Analysis of Gene Expression Following RNAi Mediated Silencing in BMSB

1. Measure the effect of RNAi on the levels of transcript expression by qPCR.
2. Isolate the total RNA from the dsRNA treated animals and synthesize the cDNA⁶².
3. Setup the qPCR reactions using a real-time PCR system and the primers listed in Table 1. Use the following qPCR cycling condition: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, along with dissociation step including 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s.
4. Determine the qPCR standards: use the serial dilution of cDNA prepared from total RNA isolated from a normal animal as a reference standard for the quantification.
5. Use BMSB 18s RNA as an internal standard to correct for differences in RNA recovery from tissues³².

5. Foliar Spray Application in Large Potted Citrus Trees and Seedlings

Note: Plants of the citrus cultivar 'Carrizo' citrange (Citrus sinensis XPoncirus trifoliata, Rutaceae), were maintained in a glasshouse under natural light and temperature, grown in 1.2 L containers. The plants were constantly pruned to promote growth of new foliar shoots, called 'flush'. ACP prefers to feed and oviposition on the new growth of citrus⁶⁴.

1. Select plants or seedlings and do not water them for 2-3 days prior to use to let the soil dry out to damp but not completely dry.
2. Using a hand pump spray bottle apply a 200 mL of dsRNA solution (0.5 mg/mL) to the lower canopy (Figure 4A).
   NOTE: Prepare the above mentioned dsRNA solutions in DNase/RNase free water.
3. Post-spray application, allow the applied dsRNA solution to be completely absorbed by the leaves.
   NOTE: Citrus trees absorbed the applied dsRNA, and then leaves from either new growth or from branches that were covered prior to application, were extracted; the dsRNA was detectable using qPCR and showed systemic movement into the tree top canopy leaves in 3-4 h^44,45,46.
4. Sample the new growth from previously topped trees after 25-40 days by collecting approximately 10 leaves from the tips of four branches. Extract the total RNA and analyze it by reverse transcription PCR (RT-PCR) and qPCR for the presence of the applied dsRNA trigger using the primers listed in Table 1 and previously described methods⁴⁶.
   NOTE: The sample collection, total RNA isolation, RT-PCR, and qPCR were performed as previously described⁴⁶.
5. Similarly, topically spray 10 mL dsRNA to the lower region of a seedling or small potted tree foliage.
   NOTE: Hemipteran insects (ACP and GWSS) were given feeding access normally at 24 h, post treatments on new growth (leaves) which had not been directly sprayed, or which grew weeks later, or whole plants. This produced insects which tested positive for the dsRNA at 3, 6, and 10 days, post feeding.

6. Soil/Root Drench Application in Large or Small Potted Citrus Trees and Seedlings

1. Select plants or seedlings and do not water them for 2-3 days prior to use to let the soil dry out to damp but not completely dry (this creates air space to hold the liquid solution to be applied).
2. Add 1 L of dsRNA solution (0.2 mg/mL) to the soil of large potted plants (approximately 2.5 m) and add 1 L of water (chaser) after 1 h.
3. Apply 100 mL of dsRNA solution (1.33 mg/mL) to the soil of 1 m tall potted trees in partially dry soils.
4. For small seedlings, apply 10 mL of dsRNA solution (1 mg/mL) to the soil in the cones or to the bare roots (Figure 4B, C).
5. Allow the plants that receive the dsRNA solution applied as a soil drench to soak for 30 min. Then apply plain water only treatment to aid absorption by roots (20 mL for plants in yellow containers, or 100 mL if larger plant pots are > 1 gallon).
   NOTE: Topically applied dsRNA to foliage resulted in detection at most distal tips of branches within 3-6 h post treatments, showing systemic movement through trees. New growth branches tested positive for dsRNA at 60-90 days post treatments. Cuttings are provided to insects (ACP and GWSS) in a dsRNA feeding bioassay⁴⁴.

7. Stem Tap (Tree Trunk Injection) Application in Large Potted Citrus Trees and Seedlings

1. Select citrus seedlings, new, or approximately 3.5 years old plants for injecting dsRNA using the stem tap (trunk injections) method.
2. Drill holes in the citrus plants using a drill and a 10 mm drill bit, taking care not to exceed 2 cm, or about half the diameter of the stem.
3. Wrap the copper tip of each injector 4-6 times with a 0.6 cm (¼ inch) wide strip of sealing film to prevent leakage near the tip.
4. Fill the tree trunk injectors with 6 mL (1.7 mg/mL) of dsRNA solution diluted in DNase/RNase free water (denoted here as colored solution).
5. Inject the solution into the trunk of the tree and leave the injector in the trunk for 6-10 h to allow absorption of the dsRNA solution. Allow the insects to feed on the cuttings from treated trees at 3, 10, and 30 days post treatment.
   NOTE: The dsRNA injected using the trunk injection method persisted in the trees for a period of 30-60 days^41,42,44. Validation for RNAi was performed by qPCR using the primers listed in Table 1.

8. dsRNA Treated Clay Granules for Delivery to Insects Through Soil

1. Pour out the clay absorbent into a 50-mL conical tube to the 35 mL mark (approximately 30 g of clay absorbent) on the tube.
2. Pour 20 mL of dsRNA solution diluted in DNase/RNase free water (100 µg/mL) into the tube to wet all the absorbent. Cap the conical tube, tip the tube to help remove air, and cap the tube.
3. Place the tube upright and let it stand for 1-2 min for the clay particles to absorb the solution.
4. Add enough dsRNA-soaked clay into the soil mix to fill a 1 gallon pot.
5. Mix and turn the soil by hand to thoroughly mix the dsRNA-soaked clay into the soil.
6. Use this soil to repot seedlings selected for the dsRNA treatment.
7. Water the soil with 200 mL of plain water without dsRNA. After 30 min to 1 h, follow with 100 mL of plain water. After 24 h, put the plant on a normal watering schedule.
8. Test 4-6 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.
   NOTE: Plants or cuttings from these treated plants are fed to insects any time after 24 h post treatment and have been able to delivery RNAi for up to a year to insects (unpublished data).