This study was supported by grants from Taiwan (Ministry of Science and Technology, MOST 100-2923-B-002-002-MY3 and 106-2313-B-002-011-MY3 to H.J.L.), the Czech Republic (Grant agency of South Bohemia University, GAJU grant 065/2017/P to Y.H.L), and Spain (Spanish Ministry of Economy and Competitiveness, grants CGL2012-36251 and CGL2015-64727-P to X.B., and the Catalan Government, grant 2014 SGR 619 to X.B.); it also received financial support from the European Fund for Economic and Regional Development (FEDER funds to X.B.).

1. Synthesis and Preparation of dsRNA
<ol>
	<li>Identify the dsRNA target sites in the 3&#39; untranslated region of the target genes. The dsTub is used for targeting the &#945;-tubulin (tub) gene (GenBank accession number: KX228233), and dsEGFP as a negative dsRNA control is designed from the sequence of enhanced green fluorescence protein (EGFP; GenBank accession number: LC311024).</li>
	<li>Perform standard PCR amplification to synthesize the dsRNA templates with gene-specific primers containing the T7 promoter sequence (5&#39;-TAATACGACTCACTATAGGG-3&#39;). The PCR amplification conditions and primer information used are those reported by Lin et al. (2017)12 (see Table of Materials).</li>
	<li>Proceed with dsRNA preparation using a T7 Transcription Kit (see Table of Materials), following the manufacturer&#39;s instructions. 
	Note: To yield high quantity dsRNA, mix two reactions in one tube (in total 40 &#181;L), and incubate at 37 &#176;C overnight.</li>
	<li>Add 2 &#181;L of DNase (see Table of Materials) into dsRNA samples for 15 min at 37 &#176;C to digest DNA templates.</li>
	<li>Add 200 &#181;L extraction reagent (see Table of Materials) and 40 &#181;L chloroform, then vortex.</li>
	<li>Centrifuge for 10 min at 13,000 x g at 4 &#176;C, then collect supernatant (150 &#181;L) in a new microcentrifuge tube.</li>
	<li>Precipitate dsRNA by adding 150 &#181;L isopropanol and incubating for 15 min on ice.</li>
	<li>Centrifuge for 15 min at 13,000 x g at 4 &#176;C, then remove supernatant.</li>
	<li>Add 200 &#181;L of the 70% ethanol to wash dsRNA pellets. Centrifuge for 10 min at 13,000 x g at 4 &#176;C.</li>
	<li>Remove the ethanol and repeat the ethanol wash steps.</li>
	<li>Remove the ethanol and dry dsRNA pellet by centrifugal vacuum concentrators for 3 min, then resuspend dsRNA with RNase-free water.</li>
	<li>Determine the quantity of the purified dsRNA using a micro-volume UV-Vis spectrophotometer (see Table of Materials) according to the manufacturer&#39;s instructions and adjust to the desired concentration (e.g., 0.25 &#181;g/&#181;L for preparation of dsRNA lipoplexes).</li>
	<li>Store the dsRNA samples at -20 &#176;C for up to 3 months.</li>
</ol>
2. Preparation of dsRNA Lipoplexes
<ol>
	<li>Dilute 4 &#181;L of the transfection reagent (see Table of Materials) by adding 4 &#181;L of 5% glucose solution. Dilute 4 &#181;L of the dsRNA (1 &#181;g) by adding 4 &#181;L of 5% glucose solution.</li>
	<li>Add the diluted liposome reagent solution immediately into the diluted dsRNA solution all at once. Vortex briefly to mix them, then incubate for 15 min at 25 &#176;C. 
	Note: Do not mix the solutions in the reverse order.</li>
	<li>The dsRNA lipoplexes are ready for use. Use within 1 hour of preparation, and discard after 1 hour. 
	Note: According to the manufacturer&#39;s instructions, the lipoplexes should be used as soon as possible.</li>
</ol>
3. Collection of Extracellular Enzymes from Hemolymph and Midgut Juice
<ol>
	<li>Insect saline buffer (10x&#160;stock)
	<ol>
		<li>Mix 900 mL double distilled water with 109.3 g NaCl, 15.7 g KCl, 6.3 g CaCl2, and 8.3 g MgCl2&#183;6H2O.</li>
		<li>Adjust pH to 6.8, and fill up to 1,000 mL.</li>
	</ol>
	</li>
	<li>Hemolymph collection
	<ol>
		<li>Anesthetize the cockroaches on ice until they do not move for 3 min.</li>
		<li>Hold the cockroach with two fingers (thumb and index finger), and turn the ventral side of the cockroach up.</li>
		<li>Use the dissecting scissor to cut off the tip of a coxa of a hind leg off. 
		Note: Cut the connected intersection between the coxa and trochanter. The excision of the other part of coxa was less efficient to collect hemolymph.</li>
		<li>Squeeze the abdomen gently with middle finger, and collect the hemolymph bleeding from the incision with a 10 &#181;L micropipette.</li>
		<li>Pool the hemolymph from several cockroaches (5 individuals) in a microcentrifuge tube. 
		Note: Keep the microcentrifuge tubes on ice to prevent melanization. The average volume of hemolymph obtained from a male cockroach is 2 - 3 &#181;L.</li>
		<li>Spin down the hemolymph briefly with a benchtop centrifuge for 10 s.</li>
		<li>Transfer desired volume of hemolymph (i.e., 10 &#181;L), and mix it with 50 &#181;L of 1x&#160;insect saline buffer.</li>
		<li>Centrifuge for 10 min at 1,000 x g at 4 &#176;C to spin down hemocytes. Transfer the supernatant to a clean microcentrifuge tube.</li>
		<li>Quantify the total protein concentration using a micro-volume UV-Vis spectrophotometer by measuring A28015.</li>
		<li>Adjust each sample to have the same protein concentration (6 mg/&#181;L of total protein).</li>
	</ol>
	</li>
	<li>Midgut juice collection
	<ol>
		<li>Anesthetize the cockroaches on ice. Fix the cockroaches ventral side up on the dissection plate with cold 1x&#160;insect saline buffer using insect pins.</li>
		<li>Dissect the abdomen with fine tweezers. Remove the entire gut (including the front, mid, and hind gut) to a fresh dish with 1x&#160;insect saline buffer.</li>
		<li>Remove the front and hind gut, and quickly transfer the midgut to a microcentrifuge tube that contains 100 &#181;L insect saline buffer.</li>
		<li>Pool the midguts from six cockroaches in the same tube, and vortex for 10 s.</li>
		<li>Centrifuge for 10 min at 1,000 x g at 4 &#176;C to spin down the hemocytes and gut tissues. Transfer the supernatant to a clean microcentrifuge tube.</li>
		<li>Quantify the total protein concentration using a micro-volume UV-Vis spectrophotometer by measuring A28015.</li>
		<li>Adjust each sample to have the same protein concentration (6 mg/&#181;L of total protein).</li>
	</ol>
	</li>
</ol>
4. dsRNA Degradation Assay
<ol>
	<li>Freshly prepare 4 &#181;L of the naked dsRNA or dsRNA lipoplexes (1 &#181;g).</li>
	<li>Mix dsRNA solutions with 10 &#181;L of insect saline buffer (control), extracted enzymes from hemolymph, or midgut juice.</li>
	<li>Add 2 &#181;L of either EGTA (20 mM) or RNase-free water to separate samples as controls for inhibition of enzyme activities.</li>
	<li>Incubate for 1 hour or longer (6, 12, or 24 hours) at 25 &#176;C.</li>
	<li>Add 200 &#181;L extraction reagent and 40 &#181;L chloroform, then vortex.</li>
	<li>Centrifuge for 10 min at 13,000 x g at 4 &#176;C, then collect supernatant (150 &#181;L) in a new microcentrifuge tube.</li>
	<li>Precipitate dsRNA by adding 150 &#181;L isopropanol and incubating for 15 min on ice.</li>
	<li>Centrifuge for 15 min at 13,000 x g at 4 &#176;C, then remove supernatant.</li>
	<li>Add 200 &#181;L of the 70% ethanol to wash dsRNA pellets. Centrifuge for 10 min at 13,000 x g at 4 &#176;C.</li>
	<li>Remove the ethanol and repeat the ethanol wash steps.</li>
	<li>Remove the ethanol and dry dsRNA pellet by centrifugal vacuum concentrators for 3 min. Resuspend dsRNA with 10 &#181;L RNase-free water.</li>
	<li>Check the integrity of treated dsRNA via gel electrophoresis on a 1.5% agarose gel.</li>
</ol>
5. Oral Administration of dsRNA
<ol>
	<li>Maintain cockroaches on an ad libitum diet of dry food (dog chow). Deprive water supply from the cockroaches one day before dsRNA ingestion experiments.</li>
	<li>Hold a cockroach by grabbing its wings with the flexible forceps (Figure 1, step 5). 
	Note: Do not grab the body parts of cockroaches.</li>
	<li>Prepare the dsRNA lipoplexes, as well as naked dsRNA, for feeding, as described in section 2. 
	Note: The dsRNA lipoplexes should be prepared freshly before feeding.</li>
	<li>Feed 4 &#181;L of dsRNA lipoplexes or naked dsRNA (250 ng) with a micropipette.</li>
	<li>Slowly pipet the droplet of the dsRNA solution close to the mouthparts of cockroaches, and let the cockroaches ingest the droplet completely.</li>
	<li>Perform the ingestion experiments twice a day (1 h after lights on and 1 h before lights off) for 8 days or 16 days continuously. 
	Note: All cockroaches acquired water only through manual feeding with dsRNA solution or control reagents (e.g., nuclease free water, glucose solution, and liposome reagent) during ingestion experiments.</li>
	<li>Provide water bottles to the cockroaches after 16 days of ingestion of dsRNA experiments.</li>
</ol>
6. Assess Knockdown
<ol>
	<li>At chosen time points (2, 9, and 17 days after dsRNA ingestion assay), collect the dsEGFP- and dsTub-treated insects and dissect the chosen tissues (e.g., midgut).</li>
	<li>Perform quantitative real-time PCR (qRT-PCR). The qRT-PCR amplification conditions and primer information as reported by Lin et al. (2017)12.</li>
	<li>Assess the control and dsRNA-treated insects daily to check mortality and remove the cockroaches that are no longer moving.</li>
</ol>

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The authors have nothing to disclose.

This protocol presents a method for effective RNAi through oral delivery of dsRNA lipoplexes, involving protection against ribonuclease digestion in the midgut juice of the German cockroach. As shown in other studies in various insect species, the poor RNAi effect through oral delivery of dsRNA is mostly accounted for by the degradation of dsRNA8,9,10. This protocol produces liposomes that serve as protective vehicles in oral de...

RNAi has been demonstrated as an effective method to knockdown gene expression through a mechanism of a post-transcriptional silencing pathway triggered by dsRNA molecules in many eukaryotes1. Over the past decade of study, RNAi has become a useful tool to study the functions of genes from development to behavior by depleting the expression of specific genes via injection and/or feeding of dsRNA in various taxa of insects2,3. Due to the specificity and robustness of the depleting effect, the application of RNAi is currently being considered as a potential strategy for pest control management4,5. However, the efficiency of RNAi varies widely between insect species, depending on the different genes being targeted and the delivery methods. A growing body of evidence suggests that the instability of dsRNA, which is degraded by ribonucleases, is a critical factor in the limited efficacy of RNAi5,6. For instance, the low RNAi sensitivity in Manduca sexta has been explained by the fact that the dsRNA mixed with hemolymph was quickly degraded within 1 hour7. Similarly, the presence of alkaline nucleases in the midgut, which efficiently degrade ingested dsRNA, is strongly correlated with low RNAi efficiency in different insect orders8,9,10.
The oral delivery of dsRNA is particularly interesting for the application of RNAi in a pest control strategy, but a method to retard the degradation of dsRNA by the nucleases in the midgut has not yet been developed, which would have the potential to ensure effective RNAi through feeding. However, the unresponsiveness of RNAi to oral delivery of dsRNA has been reported by feeding large amount of dsRNA, e.g. 50 &#181;g in Bombyx mori, or continuously feeding for 8 days (8 &#181;g dsRNA in total) in the locust species. The German cockroach, Blattella germinica, is highly sensitive to RNAi by the injection of dsRNA11,12,13,14, but is not responsive to dsRNA through feeding. Recently, Lin et al. (2017) have demonstrated that the dsRNA encapsulated with liposome carriers results in successful RNAi to knockdown the &#945;-tubulin gene expression in the midgut and trigger significant mortality of the German cockroach15. As the degradation of dsRNA in the midgut is the limiting factor for oral RNAi, the liposome carriers serve as a vehicle to protect dsRNA from degradation, which is readily applicable in other insects with strong nuclease activities in the gut. Of note, the reason for choosing the particular transfection reagent (see Table of Materials) we used as liposome carrier in the current protocol is that it has been tested for insect cell line transfection with less toxicity, according to the manufacturer&#39;s instructions. According to the comparison of different liposome transfection systems in Gharavi et al. (2013)16, the efficiency of transfecting small interfering RNA (siRNA) is approximately the same between this and other commercially available systems that have been used for dsRNA delivery systems in other insects17,18.Furthermore, our feeding method is careful enough to ensure the proper amount of dsRNA is ingested by each cockroach, and that the results are robust and confirmed. In summary, the present protocol and results demonstrate that using dsRNA lipoplexes improves dsRNA stability and opens the door to the design of the strategy oral delivery of RNAi, which is a promising approach for pest control in the future.

<table><tbody><tr><td>GenJe Plus DNA in vitro Transfection reagent</td><td>SignaGen</td><td>SL100499</td><td>for lipoplexes preparation</td></tr><tr><td>Blend Taq plus</td><td>TOYOBO&amp;nbsp;</td><td>BTQ-201</td><td>for PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ABI&amp;nbsp;</td><td>4385612</td><td>for qPCR</td></tr><tr><td>FirstChoice RLM-RACE Kit</td><td>Invitrogen</td><td>AM1700</td><td>for 3&#039; UTR identification&amp;nbsp;</td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Invitrogen</td><td>AMB13345</td><td>for dsRNA synthesis</td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2239</td><td>remove DNA template from dsRNA</td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596018</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>RQ1 RNase-Free Dnase</td><td>Promega</td><td>M6101</td><td>remove DNA template from total RNA&amp;nbsp;</td></tr><tr><td>chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2432</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>I9516</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>24102</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Diethyl pyrocarbonate, DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>for RNase free water preparation</td></tr><tr><td>glucose solution</td><td>Sigma-Aldrich</td><td>G3285</td><td>for lipoplexes preparation</td></tr><tr><td>Sodium chloride, NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>insect saline buffer formula</td></tr><tr><td>Potassium chloride, KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>insect saline buffer formula</td></tr><tr><td>Calcium chloride, CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016</td><td>insect saline buffer formula</td></tr><tr><td>Magnesium chloride hexahydrate, MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670</td><td>insect saline buffer formula</td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E3889</td><td>enzyme inhibitor&amp;nbsp;</td></tr><tr><td>dissecting scissor</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>fine tweezers</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>flexible tweezer</td><td>F.S.T.</td><td></td><td>cockroach holding&amp;nbsp;</td></tr><tr><td>pipetman</td><td>RAININ</td><td>P10</td><td>sample preparation</td></tr><tr><td>microcentrifuge tube</td><td>Axygen</td><td>MCT175C, PCR02C</td><td>sample preparation</td></tr><tr><td>pipette tip&amp;nbsp;</td><td>Axygen</td><td></td><td>sample preparation</td></tr><tr><td>vortexter</td><td>Digisystem</td><td>vm1000</td><td>sample preparation</td></tr><tr><td>Minispin centrifuge</td><td>The Gruffin Group</td><td>GMC 206</td><td>for liquid spin down&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>ALC</td><td>PK121R</td><td>sample preparation</td></tr><tr><td>pH meter&amp;nbsp;</td><td>JENCO</td><td>6071</td><td>for pH adjust</td></tr><tr><td>micro-volume spectrophotometer</td><td>Quawell</td><td>Q3000</td><td>nucleic acid quantitative</td></tr><tr><td>PCR Thermal cycler</td><td>ABI&amp;nbsp;</td><td>2720</td><td>for template PCR or&amp;nbsp; dsRNA synthesis incubation&amp;nbsp;</td></tr><tr><td>quantitative real-time PCR</td><td>ABI&amp;nbsp;</td><td>StepOne plus</td><td>gene expression quantitative</td></tr><tr><td>Centrifugal Vacuum Concentrators</td><td>eppendorf</td><td>5301</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Multipette&amp;nbsp;</td><td>eppendorf</td><td>xstream</td><td>for real-time PCR sample loading&amp;nbsp;</td></tr><tr><td>Agarose I</td><td>amresco</td><td>0710</td><td>for nucleic acid electrophoresis</td></tr><tr><td>tub gene specfifc forward preimer</td><td>tri-I biotech</td><td></td><td>GGG ACA AGC CGG AGT GCA GA</td></tr><tr><td>tub gene specfifc reverse preimer</td><td>tri-I biotech</td><td></td><td>TCC TGC TCC TGT CTC GCT GA</td></tr><tr><td>dsTub template forward primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&amp;nbsp;</td></tr><tr><td>dsTub template reverse primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&amp;nbsp;</td></tr><tr><td>dsEGFP template forward preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td></tr><tr><td>dsEGFP template reverse preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td></tr><tr><td>tub qPCR forward primer</td><td>tri-I biotech</td><td></td><td>GGA CCG CAT CAG GAA ACT GGC</td></tr><tr><td>tub qPCR reverse preimer</td><td>tri-I biotech</td><td></td><td>CCA CAG ACA GCC TCT CCA TGA GC</td></tr><tr><td>ef1 qPCR forward primer</td><td>tri-I biotech</td><td></td><td>CGC TTG AGG AAA TCA AGA AGG A</td></tr><tr><td>ef1 qPCRreverse preimer</td><td>tri-I biotech</td><td></td><td>CCT GCA GAG GAA GAC GAA G</td></tr></tbody></table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

A simplified scheme of the protocol for the oral delivery of dsRNA is presented in Figure 1, where the key steps for preparation of dsRNA lipoplexes are shown.
In order to investigate the protection given by liposome carriers upon dsRNA degradation in the midgut juice of B. germanica, an ex vivo assay where dsTub lipoplexes were incubated with midgut juice was conducted, and the in...

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Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

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Research

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Biology

9.3K Views.  National Taiwan University. This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

Video: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Genetics

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.

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 ...

Environment

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

Developmental Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.

Author Spotlight: Advancing Gene Silencing Research in Silkworms with dsRNA Delivery Through Feeding Chitosan Nanoparticles

Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

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Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

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

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Applications of RNA Interference in American Cockroach

RNAi Interference by dsRNA Injection into Drosophila Embryos

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery