This study was funded by the National Natural Science Foundation of China (31501898), the Science and Technology Program of Guangzhou (202102010465), the Guangzhou Higher Education Teaching Quality and Teaching Reform Project (2022JXGG057), and the Research project of the Open Online Course Steering Committee of Guangdong Provincial Universities (2022ZXKC381).

1. Silkworm species and rearing
<ol>
	<li>Rear at least 120 freshly hatched 1st instar silkworm larvae of B. mori P50 strain with fresh mulberry leaves at 25 &#177; 1 &#176;C, photoperiod 12 h Light:12 h Dark, and 75% &#177; 5% relative humidity.</li>
</ol>
2. Selection of silkworm larvae
<ol>
	<li>Pick day 1 of the 5th instar larvae for the RNAi experiment; the silkworm larvae grow fast after day 3 of the 5th instar, which is ideal for comparing the differences in the larval appearance. 
	NOTE: Alternatively, younger stages, such as the 3rd or 4th instar, could be used for the RNAi experiment. However, it requires constant dsRNA treatment until the 5th instar, which is relatively expensive and material costing.</li>
</ol>
3. Synthesis of dsRNA
<ol>
	<li>Identifying dsRNA target fragment: Select the coding sequence of a target gene and align it with other homologous genes to determine the conserved region. Design gene-specific primers in the non-conserved region for subsequent dsRNA target fragment amplification. For RNAi experiments in insects, the typical dsRNA target fragment is generally 400-600 bp. The minimum size could be 200 bp. 
	NOTE: To improve the efficiency and successful rate of RNAi experiments, some web-based tool can be used to design the dsRNA target, such as dsRNA Engineer (https://dsrna-engineer.cn/).</li>
	<li>Producing PCR product template: Add a T7 RNA polymerase promoter (5&#39;-TAATACGACTCACTATAGG-3&#39;) to the 5&#39;-end of either primer designed above. Perform standard PCR to amplify the dsRNA target fragment. Run a 1% agarose gel in 1x TAE to verify a single PCR product of the expected size. After that, purify the PCR product with a purification kit (Table of Materials) and use it for subsequent transcription. 
	NOTE: To store the target PCR product for long-term and obtain high yields, it is suggested to ligate the PCR product to a plasmid. The plasmid should be linearized before the PCR reaction.</li>
	<li>Generation of dsRNA: Use a T7 RNAi System (Table of Materials) to generate the dsRNA. Set up the reaction by adding the components at room temperature according to the manufacturer&#39;s instructions. Mix the reaction by pipetting gently and incubate at 37 &#176;C for 30 min. 
	NOTE: To maximize the yield, incubation at 37 &#176;C can be extended up to 2-6 h. For templates containing a secondary structure or GC-rich, incubation can be performed at 42 &#176;C instead to improve the yield of dsRNA.</li>
	<li>Annealing to form dsRNA: Incubate the reaction at 70 &#176;C for 10 min, then slowly cool to room temperature (~20 min). This will anneal the dsRNA.</li>
	<li>DNase and RNase treatment: Pipette 1 &#181;L of the supplied RNase solution to 199 &#181;L of nuclease-free water to make a freshly diluted RNase solution. Add 1 &#181;L of freshly diluted RNase solution and 1 &#181;L of the supplied RQ1 RNase-free DNase per 20 &#181;L reaction volume, mix gently, and incubate at 37 &#176;C for 30 min. The single-stranded RNA (ssRNA) and the template DNA in the reaction will be removed after this treatment.</li>
	<li>Alcohol precipitation: Add 1 volume of isopropanol or 2.5 volumes of 95% ethanol along with 0.1 volume of 3 M Sodium Acetate (pH 5.2) to the reaction. Mix the reaction by vortexing and incubate on ice for 5 min to form a cloudy mass. Spin at maximum speed in a centrifuge for 10 min to collect a white pellet at the bottom of the tube. Discard the supernatant and wash the pellet with 0.5 mL of cold 70% ethanol to remove the residual salt. Remove the ethanol after the wash. Allow the pellet to air dry at room temperature for 15 min. Resuspend the pellet with nuclease-free water in 2-5 times of the initial reaction volume. Store at -20 &#176;C or -70 &#176;C. 
	NOTE: The dsRNA pellet should not be overly dried, since this could make it difficult to fully resuspend. To ensure sufficient resuspension, a minimum of 2 volumes are needed.</li>
	<li>Quantity and quality check of dsRNA: Dilute the dsRNA in nuclease-free water at a ratio of 1:100 to 1:300. Determine the concentration of dsRNA using a microvolume spectrophotometer (Table of Materials) according to the manufacturer&#39;s instructions. To quantify dsRNA, multiply the concentration by the volume. To assess the quality of dsRNA, use agarose gel electrophoresis. Prepare a 1% agarose gel in 1x TAE and dilute the dsRNA at 1:50 with nuclease-free water. Use 5 &#181;L of diluted dsRNA per lane and stain the gel in 0.5 mg/mL ethidium bromide for at least 15 min for visualization. 
	NOTE: dsRNA migrates more slowly than dsDNA.</li>
</ol>
4. Preparation of the chitosan/dsRNA nanoparticles
<ol>
	<li>Make 100 mM sodium acetate (0.1 M NaC2H3O2 and 0.1 M acetic acid, pH 4.5 in deionized water) and 100 mM sodium sulfate (100 mM Na2SO4 in deionized water) buffer at room temperature.</li>
	<li>Dissolve commercialized chitosan (from shrimp shells, &#8805;75% deacetylated; Table of Materials) in 100 mM sodium acetate buffer to make a 0.02% (w/v) chitosan solution.</li>
	<li>Dissolve 20 &#181;g of dsRNA in 50 &#181;L of nuclease-free water and add it to 50 &#181;L of 100 mM sodium sulfate buffer to make a 100 &#181;L dsRNA solution.</li>
	<li>Add 100 &#181;L of chitosan solution to 100 &#181;L of dsRNA solution. Simultaneously, prepare a control by adding 100 &#181;L of chitosan solution to 100 &#181;L of 50 mM sodium sulfate. Mix and heat the mixtures at 55 &#176;C for 1 min.</li>
	<li>Vortex the mixture immediately with a high-speed vortex for 30 s to allow the formation of the nanoparticles.</li>
	<li>Centrifuge the mixture at 13,000 x g at room temperature for 10 min to obtain a white pellet. Transfer the supernatant to a fresh 1.5 mL tube. Let the pellet air-dry at room temperature for 10 min.</li>
	<li>Determine the concentration of dsRNA in the supernatant by using a microvolume spectrophotometer (Table of Materials). Use the supernatant from the control as a blank. Multiply the concentration by volume to calculate the total amount of dsRNA remaining in the supernatant. Calculate the percentage of dsRNA encapsulated in the nanoparticles by the amount remaining in the supernatant divided by the starting amount of dsRNA. 
	NOTE: Even though the nanoparticles could be kept for 4-37 &#176;C for at least 15 days before use14, it is suggested to use the nanoparticles as soon as possible.</li>
</ol>
5. Feeding the silkworm larvae with chitosan/dsRNA nanoparticles
<ol>
	<li>Pick freshly molted 5th instar silkworm larvae (i.e., Day 1 of the 5th instar) of the same size for the feeding experiment. Place the larvae individually into each well of a 6-well plate before closing the cover and starve for 24 h.</li>
	<li>Rinse fresh mulberry leaves with deionized water and dry them with clean kitchen paper. The mulberry leaves should be dry without water drops. Cut into 1 cm x 1 cm mulberry leaf discs.</li>
	<li>Dissolve chitosan/dsRNA nanoparticles into nuclease-free water at 500 ng/&#181;L prior to use. Dilute the control chitosan nanoparticles (prepared in 4.4) with same volume of nuclease-free water. Prepare naked dsRNA to 500 ng/&#181;L in nuclease-free water.</li>
	<li>Use the chitosan/dsRNA nanoparticles for RNAi knockdown experiment, the control chitosan nanoparticles as a blank/negative control. Use the naked dsRNA to compare the differences with chitosan/dsRNA nanoparticles.</li>
	<li>Coat 10 &#181;L of chitosan/dsRNA nanoparticles, chitosan, and naked dsRNA on the surface of each leaf disc, respectively. Air-dry the nanoparticle solution and dsRNA solution on the leaf discs at room temperature for 5 min.</li>
	<li>Feed each larva with one nanoparticle-coated or dsRNA-coated leaf disc per day. Provide nanoparticle-coated or dsRNA-coated leaf discs to the larvae continuously for 5 days. Fresh mulberry leaves can be provided after the coated leaf disc is fully eaten by the larvae each day.</li>
	<li>On day 6, anesthetize the larvae on ice until they do not move and dissect the larvae for sampling. Cut off the thorax with scissors on a clean Petri dish. Pull out the midgut with a tweezer. Remove the content in the midgut and wash the midgut in another Petri dish filled with nuclease-free water. Store the midgut in a 1.5 mL tube and keep at -70 &#176;C.</li>
</ol>
6. Confirmation of gene silencing
<ol>
	<li>Perform quantitative Real-time PCR (qRT-PCR) to calculate the relative transcript quantification. At least three biological replicates and three technical replicates for each biological replicate are required. At least two reference genes are recommended to normalize the relative transcript. A Bombyx Toll9-2 (BmToll9-2) gene is tested. Evaluate the gene silencing efficiency by comparing the mean value of the relative transcript to the control group and convert it to percentage. 
	NOTE: The qRT-PCR amplification condition, primer information, and relative transcript calculation can be found in our recent publication20.</li>
	<li>Analyze the RNAi phenotype to evaluate the gene silencing effect. Observe the size of the larvae and cocoons. Take photos daily to record appearances. 
	NOTE: RNAi can affect morphology, metamorphosis, physiology, and behavior. The observation of RNAi-related phenotypes depends on the genes targeted.</li>
</ol>

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

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The authors have nothing to disclose.

A proper stage is important for RNAi phenotype observation, depending on the genes targeted. Our preliminary results showed that Toll9-2 is involved in the growth of the silkworm. The size and weight of the silkworm larvae increase rapidly at the 5th instar21. Therefore, the 5th instar larvae are selected as the stage for the chitosan/dsRNA nanoparticles feeding experiment. It is also possible to select the 3rd or 4th instar larvae for feeding e...

The silkworm, Bombyx mori, is an insect domesticated more than 5000 years ago in China. Due to its ability to produce silk, the silkworm is an important economic insect in Chinese agriculture and sericulture. The silkworm is second only to the fruit fly as the model insect. As a model insect in Lepidoptera, the silkworm is easy to rear, with a large body size and plenty of mutants. Meanwhile, the silkworm is the first Lepidopteran insect with its complete genome sequenced1. A lot of databases providing information for genome2, transcriptome3, expressed sequence tag (EST)4, non-coding RNA5, and microsatellite6 are also available to the public. The above facts make the silkworm a perfect model for genetic research.
RNA interference (RNAi) is a cellular process in which double-stranded RNA (dsRNA) molecules bind and slice the complementary messenger RNA (mRNA), thereby achieving the silencing effect of the target gene. This mechanism is naturally present in bacteria to defend against the invasion of viruses7. Later, it was found that RNAi is conserved in animals, plants, and microbes. Due to its powerful sequence-specific silencing effect, RNAi is used in fundamental research to manipulate gene expression and study gene function. RNAi is achieved through the delivery of dsRNA into cells.
In insects, there are three common ways to deliver dsRNA, which are microinjection, feeding, and soaking8. At the moment, successful RNAi reports in the silkworms through naked dsRNA delivery are conducted by dsRNA injection9. The advantages of microinjection are the immediate delivery of dsRNA into the hemolymph and precise dsRNA amount control. However, certain disadvantages of microinjection also exist. For example, it is time-consuming, and it requires delicate devices. It is also important to optimize the injection needles, injection volume, and dsRNA amount. Therefore, an alternative way to deliver dsRNA to silkworms becomes necessary. Because an insect&#39;s exoskeleton is a water-tight barrier that is made of chitin, soaking insect larvae to achieve RNAi is rarely reported, which is not a good option for RNAi in insects. Feeding of dsRNA is labor-saving, cost-effective, and easy to perform10. This method is also applicable for high-throughput gene screening11. However, it is found that a DNA/RNA non-specific nuclease, namely BmdsRNase, is present in the midgut and midgut juice of the silkworm larvae12. This nuclease is shown to digest dsRNA, preferably13. Therefore, feeding naked dsRNA to the silkworm to silence the gene expression seems to be difficult.
Recently, nanoparticle-shielded dsRNA is proved to be a good alternative to increase the RNAi efficiency by feeding14. Chitosan is an inexpensive, nontoxic, and biodegradable polymer, which can be prepared by deacetylation of chitin, a naturally occurring and the second most abundant biopolymer after cellulose15. Because the amino group in the chitosan is positively charged and the phosphate group on the backbone of the dsRNA is negatively charged, the chitosan/dsRNA nanoparticles could be formed by self-assembly of polycations16. Chitosan/dsRNA nanoparticles are effective in achieving RNAi through larval feeding in mosquitos Aedes aegypti and Anopheles gambiae17, cotton spotted bollworm Earias vittella18 and carmine spider mite Tetranychus cinnabarinus19.
In order to develop a methodology for dsRNA delivery by feeding in silkworms to gain successful RNAi efficiency, this report focuses on describing step-by-step procedures on how to prepare the chitosan/dsRNA nanoparticles and feed the nanoparticles to the silkworm larvae. This methodology is relatively inexpensive, labor-saving and easy to follow, which can be adapted for gene silencing studies in other insects. We aim to provide an easier protocol for the Lepidopteran dsRNA delivery method with higher RNAi efficiency.

<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>Sangon</td><td>F601620</td><td>for dsRNA or nanoparticles reaction</td></tr><tr><td>10 &amp;mu;l pipette</td><td>Eppendorf</td><td>P13473G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>100 &amp;mu;l pipette</td><td>Eppendorf</td><td>Q12115G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>2.5 &amp;mu;l pipette</td><td>Eppendorf</td><td>P20777G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>20 &amp;mu;l pipette</td><td>Eppendorf</td><td>H19229E</td><td>to aspirate or resuspend liquid</td></tr><tr><td>200 &amp;mu;l pipette</td><td>Eppendorf</td><td>H20588E</td><td>to aspirate or resuspend liquid</td></tr><tr><td>6-well Clear TC-treated Multiple Well Plates</td><td>Costar</td><td>3516</td><td>for silkworm rearing individually</td></tr><tr><td>Acetic acid</td><td>Aladdin</td><td>A116165</td><td>to make TAE</td></tr><tr><td>Agarose M</td><td>BBI Life Sciences</td><td>A610013</td><td>for agarose gel electrophosis</td></tr><tr><td>Analytical balance</td><td>Sartorius</td><td>BSA224S</td><td>to weight ingredients</td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Sartorius</td><td>Centrisart A-14C</td><td>to centrifuge to form dsRNA or nanoparticles</td></tr><tr><td>Chitosan</td><td>Sigma-Aldrich</td><td>C3646</td><td>to combine with dsRNA for preparation of nanoparticles</td></tr><tr><td>EDTA</td><td>Sangon</td><td>A500895</td><td>to make TAE</td></tr><tr><td>Ethanol</td><td>Aladdin</td><td>E130059</td><td>to make TAE, or for dsRNA precipitation</td></tr><tr><td>Freezer</td><td>Siemens</td><td>iQ300</td><td>to store dsRNA or nanoparticles</td></tr><tr><td>GoTaq Green Master Mix</td><td>Promega</td><td>M712</td><td>for PCR reaction</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>for qRT-PCR reaction</td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Aladdin</td><td>I112011</td><td>for dsRNA precipitation</td></tr><tr><td>NanoDrop Microvolume UV-Vis Spectrophotometer</td><td>ThermoFisher</td><td>One</td><td>to determine the concentration of dsRNA</td></tr><tr><td>ph meter</td><td>Sartorius</td><td>&amp;nbsp;PB-10</td><td>to prepare buffers</td></tr><tr><td>SanPrep Column PCR Product Purification Kit</td><td>Sangon</td><td>B518141</td><td>for PCR product purification</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>to make 100 mM sodium acetate buffer</td></tr><tr><td>Sodium sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>239313</td><td>to make 100 mM sodium sulfate buffer</td></tr><tr><td>T7 RiboMAX Express RNAi System</td><td>Promega</td><td>P1700</td><td>for dsRNA synthesis</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>C</td><td>for dsRNA generation or nanoparticles heating</td></tr><tr><td>Tris</td><td>Sangon</td><td>A501492</td><td>to make TAE</td></tr><tr><td>Vortex&amp;nbsp;</td><td>IKA</td><td>Vortex 3</td><td>to prepare chitosan/dsRNA nanoparticles</td></tr></tbody></table>

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.

To evaluate the RNAi efficiency, an immune gene targeting BmToll9-2 was chosen for analysis. BmToll9-2 gene is well characterized in the lab, and gene silencing by dsRNA injection results in lighter and smaller larvae in our recent publication20. To confirm the RNAi efficacy by ingestion through chitosan/dsRNA nanoparticles, chitosan nanoparticles were used as a control, and naked dsRNA was compared at the same time.
Compared with the control, chitosan...

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.

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 ...

larval-rna-interference-silkworm-bombyx-mori-through-chitosandsrna

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JoVE Journal

Biology

1.7K Views.  Guangzhou University. Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

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 ...

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.

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 ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

Advancing RNAi Techniques in Trichogramma dendrolimi for Gene Function Analysis

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

Author Spotlight: Investigating Wolbachia-Induced Thelytokous Parthenogenesis and Genetic Toolkit Development Through RNA Interference

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

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

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Chapters in this video

Dissection of Larval CNS in Drosophila Melanogaster

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

Drosophila Larval NMJ Immunohistochemistry

Visualizing RNA Localization in Xenopus Oocytes

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

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

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

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura