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.</li>
	<li>Ensure the regions of interest selected vary between 200 to 500 base pairs.</li>
	<li>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.
	<ol>
		<li>PCR reaction: In a 0.25 mL PCR tube, combine 5 &#181;L of 10X PCR Buffer, 4 &#181;L of dNTP Mixture (2.5 mM each), 2 &#181;L of DNA template (50 ng/&#181;L), 2.5 &#181;L each of Primers 1 and 2 (10 &#181;M), 0.25 &#181;L of DNA polymerase (5 U/&#181;L), and DNase/RNase free water up to 50 &#181;L.</li>
		<li>PCR condition: Cycle the PCR reaction to amplify the region of interest at 95 &#176;C for 3 min followed by 30 cycles of 98 &#176;C for 10 s, 55 &#176;C for 30 s, 72&#176;C for 1 min. Incubate the reaction at 72 &#176;C for an additional 10 min. Purify the PCR reaction using a purification kit.</li>
	</ol>
	</li>
	<li>Amplify the obtained PCR fragments further with gene specific primers flanked with the T7 RNA polymerase promoter sequence (5&#39;-GAA TTA ATA CGA CTC ACT ATA GGG AGA-3&#39;) as mentioned earlier62.</li>
	<li>Use LacZ gene as a negative control (mock) for RNAi. 
	NOTE: LacZ is a gene that encodes &#946;-galactosidase amplified from Escherichiacoli genomic DNA (the primers used are listed in Table 1).</li>
	<li>Perform in vitro transcription to yield dsRNA as described earlier62.</li>
	<li>Dissolve and resuspend the resulting dsRNA in 150 &#181;L DNase/RNase free water, measure the concentration, and store at -80 &#176;C for future use.</li>
</ol>
3. Delivery of dsRNA Using Green Beans
<ol>
	<li>Select early 4th instar BMSB nymphs hatched from the same egg mass and starve them for 24 h prior to dsRNA feeding.</li>
	<li>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.</li>
	<li>Wash 3 times with ddH2O and allow to air dry.</li>
	<li>Trim the green beans from the calyx end to a total length of 7.5 cm using a clean razor blade.</li>
	<li>Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 &#181;L of control solution (a 1:10 dilution of green food coloring (ingredients: Water, Propylene Glycol, Fd&#38;C Yellow 5, Fd&#38;C Blue 1, and Propylparaben as preservative)).</li>
	<li>Make dilutions of the in vitro synthesized LacZ, JHAMT, or Vg dsRNAs by diluting 5 &#181;g or 20 &#181;g in 300 &#181;L of RNase/DNase free water to yield final concentrations of 0.017 &#181;g/&#181;L or 0.067 &#181;g/&#181;L, respectively.</li>
	<li>Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 &#181;L of dsRNA solution (from step 3.6).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place these tubes in clean culture vessels (polypropylene). Place three starved 4th instar BMSB nymphs in the culture vessels.</li>
	<li>Treat three animals per culture vessel each containing three green beans with green food coloring or dsRNA solution. Maintain the insects at 25 &#176;C and 72% relative humidity, under a 16L:8D photoperiod in an incubator.</li>
	<li>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.</li>
</ol>
4. Real-time Quantitative (qPCR) Analysis of Gene Expression Following RNAi Mediated Silencing in BMSB
<ol>
	<li>Measure the effect of RNAi on the levels of transcript expression by qPCR.</li>
	<li>Isolate the total RNA from the dsRNA treated animals and synthesize the cDNA62.</li>
	<li>Setup the qPCR reactions using a real-time PCR system and the primers listed in Table 1. Use the following qPCR cycling condition: 95 &#176;C for 10 min followed by 40 cycles of 95 &#176;C for 15 s, 60 &#176;C for 1 min, along with dissociation step including 95 &#176;C for 15 s, 60 &#176;C for 1 min, 95 &#176;C for 15 s, and 60 &#176;C for 15 s.</li>
	<li>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.</li>
	<li>Use BMSB 18s RNA as an internal standard to correct for differences in RNA recovery from tissues32.</li>
</ol>
5. Foliar Spray Application in Large Potted Citrus Trees and Seedlings
Note: Plants of the citrus cultivar &#39;Carrizo&#39; 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 &#39;flush&#39;. ACP prefers to feed and oviposition on the new growth of citrus64.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 h44,45,46.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Soil/Root Drench Application in Large or Small Potted Citrus Trees and Seedlings
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Apply 100 mL of dsRNA solution (1.33 mg/mL) to the soil of 1 m tall potted trees in partially dry soils.</li>
	<li>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).</li>
	<li>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 &#62; 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.</li>
</ol>
7. Stem Tap (Tree Trunk Injection) Application in Large Potted Citrus Trees and Seedlings
<ol>
	<li>Select citrus seedlings, new, or approximately 3.5 years old plants for injecting dsRNA using the stem tap (trunk injections) method.</li>
	<li>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.</li>
	<li>Wrap the copper tip of each injector 4-6 times with a 0.6 cm (&#188; inch) wide strip of sealing film to prevent leakage near the tip.</li>
	<li>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).</li>
	<li>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 days41,42,44. Validation for RNAi was performed by qPCR using the primers listed in Table 1.</li>
</ol>
8. dsRNA Treated Clay Granules for Delivery to Insects Through Soil
<ol>
	<li>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.</li>
	<li>Pour 20 mL of dsRNA solution diluted in DNase/RNase free water (100 &#181;g/mL) into the tube to wet all the absorbent. Cap the conical tube, tip the tube to help remove air, and cap the tube.</li>
	<li>Place the tube upright and let it stand for 1-2 min for the clay particles to absorb the solution.</li>
	<li>Add enough dsRNA-soaked clay into the soil mix to fill a 1 gallon pot.</li>
	<li>Mix and turn the soil by hand to thoroughly mix the dsRNA-soaked clay into the soil.</li>
	<li>Use this soil to repot seedlings selected for the dsRNA treatment.</li>
	<li>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.</li>
	<li>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).</li>
</ol>

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 ...

double-stranded-rna-oral-delivery-methods-to-induce-rna-interference

Universidad San Sebastian

Research

JoVE Journal

Environment

13.7K Views.  United States Department of Agriculture. 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.

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

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

Data Processing Methods for 3D Seismic Imaging of Subsurface Volcanoes: Applications to the Tarim Flood Basalt

The morphology and structure of plumbing systems can provide key information on the eruption rate and style of basalt lava fields. The most powerful way to study subsurface geo-bodies is to use industrial 3D&#160;reflection seismological imaging. However, strategies to image subsurface volcanoes are very different from that of oil and gas reservoirs. In this study, we process seismic data cubes from the Northern Tarim Basin, China, to illustrate how to visualize sills through opacity rendering techniques and how to image the conduits by time-slicing. In the first case, we isolated probes by the seismic horizons marking the contacts between sills and encasing strata, applying opacity rendering techniques to extract sills from the seismic cube. The resulting detailed sill morphology shows that the flow direction is from the dome center to the rim. In the second seismic cube, we use time-slices to image the conduits, which corresponds to marked discontinuities within the encasing rocks. A set of time-slices obtained at different depths show that the Tarim flood basalts erupted from central volcanoes, fed by separate pipe-like conduits.

Three-dimensional (3D) reflection seismology is a powerful method for imaging subsurface volcanoes. By using industrial 3D seismological data from the Tarim Basin, we illustrate how to extract the sills and the conduits of subsurface volcanoes from seismic data cubes.

The morphology and structure of plumbing systems can provide key information on the eruption rate and style of basalt lava fields. The most powerful way ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...