Name: double-stranded rna oral delivery methods to induce rna interference in phloem and plant-sap-feeding hemipteran insects
Uploaded: 2018-05-04T14:00:00
Description: 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

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<...

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.

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	<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 ...

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