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, ...

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

<ol>
	<li>Xia, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+draft+sequence+for+the+genome+of+the+domesticated+silkworm+(bombyx+mori).">A draft sequence for the genome of the domesticated silkworm (bombyx mori).</a> Science. 306 (5703), 1937-1940 (2004).</li><li>Kawamoto, M., Kiuchi, T., Katsuma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silkbase:+An+integrated+transcriptomic+and+genomic+database+for+bombyx+mori+and+related+species.">Silkbase: An integrated transcriptomic and genomic database for bombyx mori and related species.</a> Database (Oxford). 2022, (2022).</li><li>Yokoi, K., Tsubota, T., Jouraku, A., Sezutsu, H., Bono, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+transcriptome+data+in+silkworm+bombyx+mori.">Reference transcriptome data in silkworm bombyx mori.</a> Insects. 12 (6), (2021).</li><li>Mita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+construction+of+an+est+database+for+bombyx+mori+and+its+application.">The construction of an est database for bombyx mori and its application.</a> Proc Natl Acad Sci U S A. 100 (24), 14121-14126 (2003).</li><li>Zhou, Q. Z., Zhang, B., Yu, Q. Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmncrnadb:+A+comprehensive+database+of+non-coding+rnas+in+the+silkworm,+bombyx+mori.">Bmncrnadb: A comprehensive database of non-coding rnas in the silkworm, bombyx mori.</a> BMC Bioinformatics. 17 (1), 370 (2016).</li><li>Prasad, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silksatdb:+A+microsatellite+database+of+the+silkworm,+bombyx+mori.">Silksatdb: A microsatellite database of the silkworm, bombyx mori.</a> Nucleic Acids Res. 33 (Database issue), D403-D406 (2005).</li><li>Ding, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rna-based+antiviral+immunity.">Rna-based antiviral immunity.</a> Nat Rev Immunol. 10 (9), 632-644 (2010).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+dsRNA+for+RNAi+in+insects:+An+overview+and+future+directions.">Delivery of dsRNA for RNAi in insects: An overview and future directions.</a> Insect Sci. 20 (1), 4-14 (2013).</li><li>Liang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+the+immune+gene+bmpgrp-l4+in+the+midgut+affects+the+growth+of+silkworm+(bombyx+mori)+larvae.">Silencing of the immune gene bmpgrp-l4 in the midgut affects the growth of silkworm (bombyx mori) larvae.</a> Insect Mol Biol. 32 (4), 340-351 (2023).</li><li>Tian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+a+lepidopteran+pest+Spodoptera+exigua+by+ingestion+of+bacteria+expressing+dsrna+of+a+non-midgut+gene.">Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsrna of a non-midgut gene.</a> PLoS One. 4 (7), e6225 (2009).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+rna-mediated+interference+through+ingested+double-stranded+rna+in+Caenorhabditis+elegans.">Effectiveness of specific rna-mediated interference through ingested double-stranded rna in Caenorhabditis elegans.</a> Genome Biol. 2 (1), RESEARCH0002 (2001).</li><li>Arimatsu, Y., Kotani, E., Sugimura, Y., Furusawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+cdna+encoding+extracellular+dsrnase+and+its+expression+in+the+silkworm+Bombyx+Mori.">Molecular characterization of a cdna encoding extracellular dsrnase and its expression in the silkworm Bombyx Mori.</a> Insect Biochem Mol Biol. 37 (2), 176-183 (2007).</li><li>Liu, J., Swevers, L., Iatrou, K., Huvenne, H., Smagghe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bombyx+mori+DNA/rna+non-specific+nuclease:+Expression+of+isoforms+in+insect+culture+cells,+subcellular+localization+and+functional+assays.">Bombyx mori DNA/rna non-specific nuclease: Expression of isoforms in insect culture cells, subcellular localization and functional assays.</a> J Insect Physiol. 58 (8), 1166-1176 (2012).</li><li>Christiaens, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+rnai+efficacy+in+Spodoptera+exigua+via+the+formulation+of+dsrna+with+guanylated+polymers.">Increased rnai efficacy in Spodoptera exigua via the formulation of dsrna with guanylated polymers.</a> Front Physiol. 9, 316 (2018).</li><li>Dass, C. R., Choong, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+chitosan+formulations+in+cancer+therapy.">The use of chitosan formulations in cancer therapy.</a> J Microencapsul. 25 (4), 275-279 (2008).</li><li>Zhang, X., Zhang, J., Zhu, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/double-stranded+RNA+nanoparticle-mediated+RNA+interference+to+silence+chitin+synthase+genes+through+larval+feeding+in+the+African+malaria+mosquito+(Anopheles+gambiae).">Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae).</a> Insect Mol Biol. 19 (5), 683-693 (2010).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/interfering+rna+nanoparticle+mediated+gene+silencing+in+disease+vector+mosquito+larvae.">Chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae.</a> J Vis Exp. (97), e52523 (2015).</li><li>Sandal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-shielded+dsrna+delivery+for+enhancing+rnai+efficiency+in+cotton+spotted+bollworm+Earias+vittella+(lepidoptera:+Nolidae).">Nanoparticle-shielded dsrna delivery for enhancing rnai efficiency in cotton spotted bollworm Earias vittella (lepidoptera: Nolidae).</a> Int J Mol Sci. 24 (11), 9161 (2023).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/dsrna+polyplex+nanoparticles+advance+environmental+rna+interference+efficiency+through+activating+clathrin-dependent+endocytosis.">Chitosan/dsrna polyplex nanoparticles advance environmental rna interference efficiency through activating clathrin-dependent endocytosis.</a> Int J Biol Macromol. 253 (Pt 4), 127021 (2023).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+function+of+bombyx+toll9-2+in+the+silkworm+(Bombyx+mori)+larval+midgut:+Activation+by+escherichia+coli/lipopolysaccharide+and+regulation+of+growth.">Immunological function of bombyx toll9-2 in the silkworm (Bombyx mori) larval midgut: Activation by escherichia coli/lipopolysaccharide and regulation of growth.</a> Arch Insect Biochem Physiol. 116 (4), e22130 (2024).</li><li>Cheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silkworm+larvae+growth+and+properties+of+silk+fibres+after+direct+feeding+of+copper+or+silver+nanoparticles.">Characterization of silkworm larvae growth and properties of silk fibres after direct feeding of copper or silver nanoparticles.</a> Mater Design. 129, 125-134 (2017).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+efficacy+of+rnai+mediated+by+various+nanoparticles+in+the+rice+striped+stem+borer+(Chilo+suppressalis).">Comparison of efficacy of rnai mediated by various nanoparticles in the rice striped stem borer (Chilo suppressalis).</a> Pestic Biochem Physiol. 165, 104467 (2020).</li><li>Gurusamy, D., Mogilicherla, K., Palli, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+nanoparticles+help+double-stranded+rna+escape+from+endosomes+and+improve+rna+interference+in+the+fall+armyworm,+Spodoptera+frugiperda.">Chitosan nanoparticles help double-stranded rna escape from endosomes and improve rna interference in the fall armyworm, Spodoptera frugiperda.</a> Arch Insect Biochem Physiol. 104 (4), e21677 (2020).</li><li>Qi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+ros+scavenging+and+fluorescent+gold+nanoparticles+to+deliver+sirna+to+improve+plant+resistance+to+Pseudomonas+syringae.">Rational design of ros scavenging and fluorescent gold nanoparticles to deliver sirna to improve plant resistance to Pseudomonas syringae.</a> J Nanobiotechnol. 22 (1), 446 (2024).</li><li>Laisney, J., Loczenski Rose, V., Watters, K., Donohue, K. V., Unrine, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+rna+in+the+neotropical+brown+stink+bug,+Euschistus+heros,+using+a+composite+nanomaterial.">Delivery of short hairpin rna in the neotropical brown stink bug, Euschistus heros, using a composite nanomaterial.</a> Pestic Biochem Physiol. 177, 104906 (2021).</li><li>Terenius, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rna+interference+in+Lepidoptera:+An+overview+of+successful+and+unsuccessful+studies+and+implications+for+experimental+design.">Rna interference in Lepidoptera: An overview of successful and unsuccessful studies and implications for experimental design.</a> J Insect Physiol. 57 (2), 231-245 (2011).</li><li>Kolge, H., Kadam, K., Ghormade, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+nanocarriers+mediated+dsrna+delivery+in+gene+silencing+for+Helicoverpa+armigera+biocontrol.">Chitosan nanocarriers mediated dsrna delivery in gene silencing for Helicoverpa armigera biocontrol.</a> Pestic Biochem Physiol. 189, 105292 (2023).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>P13473G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>100 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>Q12115G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>2.5 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>P20777G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>20 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>H19229E</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>200 &#956;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&#160;</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&#160;</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>&#160;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&#160;</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&#160;</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.

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

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

The scope of this research is to develop a methodology to feed the larvae of the silkworm Bombyx mori with dsRNA, resulting in gene silencing effect. This will help in gene regulation studies and high throughput gene screening. Chitosan is an inexpensive, non-toxic and biodegradable polymer.Chitosan/dsRNA nanoparticles are found to be effective in causing the RNAi effect by feeding the mosquitoes. In the silkworm, RNAi is achieved only by injection of dsRNA so far. However, enough authenticated devices are required for injection.This technique is also time consuming. Feeding is a natural behavior in insects. Feeding the chitosan dsRNA nanoparticles to silkworm to cause the silencing effect has never been reported yet.Additionally, this technique is labor saving, cost effective and easy to perform.

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...

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 ...

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JoVE Journal

Biology

Preparation of the Chitosan/dsRNA Nanoparticles for Feeding-Based RNA Interference in Silkworms

To begin mix 0.1 molar sodium acetate and 0.1 molar acetic acid in deionized water at pH 4.5. Then prepare 100 millimolar sodium sulfate buffer in deionized water at room temperature. Weigh the commercialized chitosan to prepare a 0.02%weight by volume solution and dissolve it in the prepared 100 millimolar sodium acetate buffer.Then dissolve 20 micrograms of double stranded RNA or dsRNA in 50 microliters of nuclease free water. Add this solution to 50 microliters of the sodium sulfate buffer to make a 100 microliter dsRNA solution. Next, add 100 microliters of the prepared chitosan solution to 100 microliters each of the dsRNA solution and 50 millimolar sodium sulfate.After mixing, heat the solutions at 55 degrees Celsius for one minute. Immediately vortex the mixture at high speed for 30 seconds to facilitate nanoparticle formation. Centrifuge the mixture at 13, 000 G at room temperature for 10 minutes to obtain a white pellet.Transfer the supernatant to a fresh 1.5 milliliter tube and let the pellet air dry at room temperature for 10 minutes. Using a micro volume spectrophotometer, determine the concentration of dsRNA in the supernatant. To calculate the percentage of dsRNA encapsulated, divide the amount remaining in the supernatant by the initial amount of dsRNA.

Chitosan/dsRNA Nanoparticle Feeding for Reverse Gene Functional Studies in Silkworm Larvae

To begin, prepare the chitosan double stranded RNA nanoparticles for feeding the silkworm larvae. Then pick freshly molted fifth instar silkworm larvae of similar size for the feeding experiment. Place each larvae individually into a well of a six-well plate.Cover the plate and starve the larvae for 24 hours. Next, rinse fresh mulberry leaves with deionized water and dry them using clean kitchen paper. Cut the fully dried leaves into one centimeter by one centimeter leaf discs.Dissolve the chitosan dsRNA nanoparticles in nuclease-free water at a concentration of 500 nanograms per microliter before use. Coat 10 microliters of chitosan dsRNA nanoparticles, control chitosan nanoparticles, and naked dsRNA separately on the surface of each mulberry leaf disc. Let the solutions air dry on the leaf discs at room temperature for five minutes.Next, offer one nanoparticle-coated or dsRNA-coated leaf disc to each larvae every day. Once the coated leaf disc is fully consumed by the larvae, each day, provide fresh mulberry leaves. On day six, place the larvae on ice until they are immobile.To dissect the larvae, cut off the thorax with scissors on a clean Petri dish and use tweezers to pull out the midgut. After removing the contents. wash the midgut in a Petri dish with nuclease-free water.Freeze the midgut at minus 70 degrees Celsius in a 1.5 milliliter tube. Feeding chitosan dsRNA nanoparticles in silkworms resulted in a 79%reduction in BmToll9-2 transcript levels compared to control treatments. Silkworm larvae treated with BmToll9-2 silencing chitosan dsRNA nanoparticles were visibly smaller than the control larvae.The cocoons from BmToll9-2 silenced larvae were also significantly smaller than those from the control group.

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

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

Summary