The authors gratefully acknowledge Donald Weber and Megan Herlihy (USDA, ARS Beltsville, MD) for providing BMSB and HB for experimentation and maintaining the colonies; and Maria T. Gonzalez, Salvador P. Lopez, (USDA, ARS, Fort Pierce, FL) and Jackie L. Metz (University of Florida, Fort Pierce, FL) for colony maintenance, sample preparation, and analyses.

1. BMSB Rearing
<ol>
	<li>Rear BMSB insects as per standard lab practice and previously described63.</li>
	<li>Raise ACP (D. citri) insects on Citrus macrophylla in a glasshouse (22 &#176;C) and natural light. Use adult ACP, at approximately 5-7 days post eclosion.</li>
</ol>
2. Selection of Gene Regions and In Vitro Synthesis of dsRNA
<ol>
	<li>Select genes specific to BMSB from previously published transcriptome profiles32<...

The authors have nothing to disclose.

RNAi has proven to be an important tool for exploring gene biological function and regulation, with great potential to be utilized for management of insect pests19,68,69,70,71. The design and selection of an appropriate gene(s) for silencing in a given insect species and the method of delivery of the corresponding dsRNA(s) to the insect are both of utmost impo...

Hemipteran insects comprise some of the most economically significant pests of agriculturebecause of their ability to attain elevated population growth and spread diseases in plants. The BMSB, H. halys St&#229;l, is an invasive pest that was accidentally introduced in the Western hemisphere in Allentown, Pennsylvania from Asia (China, Taiwan, Korea, and Japan) with the first sighting reported in 19961. Since its introduction, BMSB has been detected in 43 states, with the highest populations in the Mid-Atlantic (DE, MD, PA, NJ, VA, and WV), as well as in Canada and Europe, and represents a potential threat to agriculture2. As a polyphagous pest, BMSB can instigate damage to approximately 300 identified plant hosts including high-value crops such as apples, grapes, ornamental plants, seed crops, soybeans, and corn. Damage is caused primarily due the mode of feeding known as lacerate and flush where the animal pierces the host crop with its needle-like stylet to gain access to the nutrients from the vascular tissues2,3. BMSB is also an indoor pest as they may find residence in living areas such as schools and houses during autumn through winter2. Chemicals and aeroallergens released by BMSB were reported to illicit allergic reaction in fruit crop workers. BMSB may also contribute to allergic disease leading to contact dermatitis, conjunctivitis, and rhinitis in sensitive individuals4,5. Another hemipteran insect, the ACP, D. citri Kuwayama (Hemiptera: Liviidae), is a serious pest of citrus fruits, and transmits the phloem-limited bacteria (Candidatus Liberibacter asiaticus) causing Huanglongbing (HLB), better known as citrus greening disease6,7. HLB was first reported from Southern China and has spread to 40 different Asian, African, Oceanian, South and North American countries7. Citrus greening is a worldwide problem with threatening economic and financial losses due to citrus fruit loss; hence, management of ACP is considered of utmost importance to prevent and control HLB.
Measures for effective control of these insect pests usually requires the application of chemical pesticides that are relatively short lived. Chemical insecticide control strategies often lack safe environmental management strategies or have decreased susceptibility due to pesticide resistance in pest populations8,9. Hence, the biological control of pests with molecular biopesticides is a potential alternative, but its use globally remains modest, and various species of parasitoids (e.g., Trisolcus japonicus) may also be effective as natural biological controls. RNAi is a potential emerging technology for managing invasive insect pests with molecular biopesticides10. RNAi is a well described gene regulatory mechanism that facilitates the effective posttranscriptional gene silencing of endogenous as well as invading dsRNAs in a sequence-specific manner, that eventually leads to the regulation of gene expression at the mRNA level11,12. Briefly, when exogenous dsRNA is internalized into a cell it is processed into siRNAs by a member of the bidentate nuclease RNase III superfamily, called Dicer, which is evolutionarily conserved in worms, flies, plants, fungi, and mammals13,14,15. These 21-25 nucleotide siRNA duplexes are then unwound and integrated in the RNA-induced silencing complex (RISC) as guide RNAs. This RISC-RNA complex allows Watson-Crick base pairing to the complementary target mRNA; this eventually leads to cleavage by the Argonaute protein, a multi domain protein containing an RNase H-like domain, which degrades the corresponding mRNA and reduces protein translation, thereby leading to posttranscriptional gene silencing16,17,18.
RNAi for pest management requires the introduction of dsRNA in vivo to silence the gene of interest, thereby activating the siRNA pathway. Various methods that have been used for dsRNA delivery to insects and insect cells to induce systemic RNAi include feeding10,19, soaking20,21, microinjection22, carriers such as liposomes23, and other techniques24. RNAi was first demonstrated in Caenorhabditis elegans to silence unc-22 gene expression by Fire and Mello25, followed by knockdown in expression of the frizzled genes in Drosophila melanogaster26. Initial functional studies utilized microinjection to deliver dsRNA in insects, such as Apis mellifera22,27, Acyrthosiphon pisum28, Blattella germanica29, H. halys30, and lepidopteran insects (reviewed by Terenius et al.31). Microinjection is advantageous to deliver an accurate and precise dose to the site of interest in the insect. Albeit such septic punctures may elicit expression of immune related genes due to trauma32, hence, ruling out its practicality in agricultural biopesticides development.
Another method of delivering dsRNA in vivo is by soaking, which involves ingestion or absorption of dsRNA by suspension of animals or cells generally in extracellular medium containing dsRNA. Soaking has been used to efficiently induce RNAi in Drosophila S2 tissue culture cells to inhibit Downstream-of-Raf1 (DSOR1) mitogen-activated protein kinase kinase (MAPKK)20, as well as in C. elegans to silence the pos-1 gene33. However, dsRNA delivered using soaking is less efficient to induce RNAi compared to microinjection20. RNAi mediated silencing in a chewing insect was first shown in the Western corn rootworm (WCR) (Diabrotica virgifera virgifera) by infusing the dsRNA into an artificial agar diet10. Earlier reports have summarized methods to deliver dsRNA infused in natural diets specific to arthropods34. These delivery methods were further determined to be comparably effective to artificial means of delivery; such as the case of the tsetse fly (Glossina morsitans morsitans), where equal knockdown of an immune-related gene was observed when dsRNA was delivered either through blood meal or microinjected35. Similarly, delivery of dsRNA through droplets in light brown apple moth (Epiphyas postvittana)36, diamondback moth (Plutella xylostella) larvae37, as well as honey bees38,39 induced efficient RNAi. Most effective RNAi experiments in hemipteran have utilized injection of dsRNA40 because oral delivery of dsRNA in hemipteran insects is arduous since it must be delivered through the host plant&#39;s vascular tissues. Effective RNAi was also observed in ACP and glassy-winged sharpshooter leafhopper (GWSS), Homalodisca vitripennis: dsRNA was delivered through citrus and grapevines that had absorbed dsRNA into the vascular tissues through root drench, foliar sprays, trunk injections, or absorption by cuttings41,42,43,44,45,46. This also resulted in the first patent for dsRNA against the ACP (2016, US 20170211082 A1). Delivery of siRNA and dsRNA using carriers such as nanoparticles and liposomes imparts stability, and increases in delivered dsRNA efficacy are rapidly emerging23,47,48,49,50. A new class of nanoparticle-based delivery vehicles for nucleic acids for in vitro and in vivo that was summarized specifically for therapeutic applications may impart immense potential as suitable delivery vectors51. Nanoparticles as a delivery vehicle for dsRNA may have disadvantages including solubility, hydrophobicity, or limited bioaccumulation52, but a suitable polymer aiding delivery may compensate these disadvantages. Development and use of self-delivering nucleotides are also emerging called &#39;antisense oligonucleotides&#39;, which are single stranded RNA/DNA duplexes46.
Vitellogenesis in arthropods is a key process controlling reproduction and regulated by juvenile hormone (JH) or ecdysone, which are the key inducers of Vg synthesis by the body fat; the Vg is eventually taken up by the developing oocyte via Vg receptor mediated endocytosis53. Vg is a group of polypeptides synthesized extraovarially, which is essential for development of the major egg yolk protein, vitellin54,55, and therefore, it is important in reproduction and aging56. Vg has been successfully silenced in nematodes57 as well as in honey bee (Apis mellifera) where RNAi mediated depletion of Vg was observed in adults and eggs22. RNAi mediated posttranscriptional gene silencing of Vg was tested because it was thought its depletion would lead to an observable phenotypic effect such as reduced fertility and fecundity, to potentially aid in BMSB control. The JHAMT gene that encodes the S-adenosyl-L-methionine (SAM)-dependent JH acid O-methyltransferase, catalyzes the final step of the JH biosynthesis pathway58. In this pathway farnesyl pyrophosphate (FPP) is sequentially transformed from farnesol, to farnesoic acid followed by conversion of methyl farnesoate to JH by JHAMT. This pathway is conserved in insects and arthropods specifically for metamorphosis, a process that is developmentally regulated by hormones59,60,61. In B. mori, JHAMT gene expression and the JH biosynthetic activity in the Corpora allata suggest that the transcriptional suppression of the JHAMT gene is crucial for the termination of JH biosynthesis58. Therefore, the JHAMT and Vg genes were selected for targeted depletion using RNAi. RNAi was also tested in citrus trees for control of ACP and GWSS. Citrus trees were treated with dsRNA through root drench, stem tap (trunk injections), as well as foliar sprays with dsRNAs against insect specific arginine kinase (AK) transcripts42,44. The topical application of dsRNA was detected all over the canopy of citrus trees, indicating efficient delivery through the plants vascular tissues, and resulted in increased mortality in ACP and GWSS41,42,45.
In the current study, we have identified a natural diet delivery method for treatments such as dsRNA. This newly developed technique was subsequently used for silencing the JHAMT and Vg mRNA using gene specific dsRNAs in BMSB nymphs as demonstrated earlier62. These new delivery protocols demonstrated here supersede conventional RNA delivery systems that use topical sprays or microinjections. Vegetables and fruits, stem tap, soil drenching, and clay absorbents in may be used for delivery of dsRNA, which is critical to the continued development of biopesticide pest and pathogen management.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BMSB (H. halys) insects&#160;</td>
			<td style="width:176px;">USDA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACP (D. citri) insects&#160;</td>
			<td style="width:176px;">USDA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">organic green beans</td>
			<td style="width:176px;">N/A</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrus plants</td>
			<td style="width:176px;">USDA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium hypochlorite solution</td>
			<td>J.T. Baker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">green food coloring&#160;</td>
			<td>McCormick &#38; Co., Inc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Forma chambers&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magenta vessel (Culture)</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primers&#160;</td>
			<td style="width:176px;">IDT DNA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SensiMix SYBR</td>
			<td style="width:176px;">Bioline</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR ABI 7500</td>
			<td>Applied Biosystems&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spray bottle</td>
			<td style="width:176px;">N/A</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td>American Can Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TaKaRa Ex Taq</td>
			<td style="width:176px;">Clontech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAquick</td>
			<td style="width:176px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

double-stranded rna oral delivery methods to induce rna interference in phloem and plant-sap-feeding hemipteran insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

Vegetable mediated dsRNA delivery through feeding in BMSB 4th instar nymphs was tested for the development of molecular biopesticides using RNAi for invasive insect pests. BMSBs feed using their needle-like stylets by a mechanism known as lacerate and flush, which causes considerable damage to crops. Slender organic green beans, P. vulgaris L., were used to test if nutrients or dsRNA could be delivered in vivo to BMSB through feeding3. ...

Watch this Scientific Journal Video about Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects at JoVE.com

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

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

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.

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13.6K visualizações.  United States Department of Agriculture. 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 ...