This work was supported by the National Natural Science Foundation of China (Grant Nos. 32070500, 31620103917, 31330072, and 31572325 to C.R., Sh.L.), by the Natural Science Foundation of Guangdong Province (Grant No. 2021B1515020044 and 2020A1515011267 to C.R.), by the Department of Science and Technology in Guangdong Province (Grant Nos. 2019B090905003 and 2019A0102006), by the Department of Science and Technology in Guangzhou (Grant No. 202102020110), by the Shenzhen Science and Technology Program (Grant No. KQTD20180411143628272 to Sh.L.).

The line of P. americana was initially provided by Dr. Huiling Hao. This species has been maintained with inbreeding for 30 years9.
1. Hatching and feeding of P. americana
<ol>
	<li>Collect fresh oothecae (immediately post egg-laying) of P. americana and incubate in the dark incubator at 25 &#176;C and 60% humidity for ~25 days. Then increase the temperature and humidity&#160;to 30 &#176;C and 75% 3 days before hatching.</li...

<ol>
	<li>Miller, S. C., Miyata, K., Brown, S. J., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+systemic+RNA+interference+in+the+red+flour+beetle+Tribolium+castaneum:+Parameters+affecting+the+efficiency+of+RNAi.">Dissecting systemic RNA interference in the red flour beetle Tribolium castaneum: Parameters affecting the efficiency of RNAi.</a> PloS One. 7 (10), 47431 (2012).</li><li>Fire, A., 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=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li><li>Ambesajir, A., Kaushik, A., Kaushik, J. J., Petros, S. T. <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:+A+futuristic+tool+and+its+therapeutic+applications.">RNA interference: A futuristic tool and its therapeutic applications.</a> Saudi Journal of Biological Sciences. 19 (4), 395-403 (2012).</li><li>Younis, A., Siddique, M. I., Kim, C. K., Lim, K. B. <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+(RNAi)+induced+gene+silencing:+A+promising+approach+of+hi-tech+plant+breeding.">RNA interference (RNAi) induced gene silencing: A promising approach of hi-tech plant breeding.</a> International Journal of Biological Sciences. 10 (10), 1150-1158 (2014).</li><li>Bell&#233;s, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Drosophila:+RNAi+in+vivo+and+functional+genomics+in+insects.">Beyond Drosophila: RNAi in vivo and functional genomics in insects.</a> Annual Review of Entomology. 55, 111-128 (2010).</li><li>French, A. S., Meisner, S., Liu, H., Weckstr&#246;m, M., Torkkeli, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+and+RNA+interference+of+cockroach+phototransduction+indicate+three+opsins+and+suggest+a+major+role+for+TRPL+channels.">Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels.</a> Frontiers in Physiology. 6, 207 (2015).</li><li>Hennenfent, A., Liu, H., Torkkeli, P. H., French, A. S. <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+supports+a+role+for+Nanchung-Inactive+in+mechanotransduction+by+the+cockroach,+Periplaneta+americana,+tactile+spine.">RNA interference supports a role for Nanchung-Inactive in mechanotransduction by the cockroach, Periplaneta americana, tactile spine.</a> Invertebrate Neuroscience: IN. 20 (1), 1 (2020).</li><li>Immonen, E. V., 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=EAG+channels+expressed+in+microvillar+photoreceptors+are+unsuited+to+diurnal+vision.">EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision.</a> The Journal of Physiology. 595 (16), 5465-5479 (2017).</li><li>Li, 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=The+genomic+and+functional+landscapes+of+developmental+plasticity+in+the+American+cockroach.">The genomic and functional landscapes of developmental plasticity in the American cockroach.</a> Nature Communications. 9 (1), 1008 (2018).</li><li>Zhao, Z., 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=Grainy+head+signaling+regulates+epithelium+development+and+ecdysis+in+Blattella+germanica.">Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.</a> Insect Science. 28 (2), 485-494 (2021).</li><li>Lozano, J., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+repressive+function+of+Kr&#252;ppel+homolog+1+on+insect+metamorphosis+in+hemimetabolous+and+holometabolous+species.">Conserved repressive function of Kr&#252;ppel homolog 1 on insect metamorphosis in hemimetabolous and holometabolous species.</a> Scientific Reports. 1, 163 (2011).</li><li>Philip, B. N., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+knockdown+analysis+by+double-stranded+RNA+injection.">Gene knockdown analysis by double-stranded RNA injection.</a> Methods in Molecular Biology (Clifton, N. J). 772, 471-497 (2011).</li><li>Zheng, 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=CRISPR+interference-based+specific+and+efficient+gene+inactivation+in+the+brain.">CRISPR interference-based specific and efficient gene inactivation in the brain.</a> Nature Neuroscience. 21 (3), 447-454 (2018).</li><li>Garbutt, J. S., Bell&#233;s, X., Richards, E. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+double-stranded+RNA+in+insect+hemolymph+as+a+potential+determiner+of+RNA+interference+success:+Evidence+from+Manduca+sexta+and+Blattella+germanica.">Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica.</a> Journal of Insect Physiology. 59 (2), 171-178 (2013).</li><li>Parrish, S., Fleenor, J., Xu, S., Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+a+dsRNA+trigger:+differential+requirement+for+the+two+trigger+strands+in+RNA+interference.">Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference.</a> Molecular Cell. 6 (5), 1077-1087 (2000).</li><li>Lemonds, T. R., Liu, J., Popadi&#263;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+the+melanin+pathway+to+overall+body+pigmentation+during+ontogenesis+of+Periplaneta+americana.">The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana.</a> Insect Science. 23 (4), 513-519 (2016).</li><li>Jackson, A. L., Linsley, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+amidst+the+silence:+Off-target+effects+of+siRNAs.">Noise amidst the silence: Off-target effects of siRNAs.</a> Trends in Genetics: TIG. 20 (11), 521-524 (2004).</li><li>Patel, M., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DISE-inducing+shRNAs+by+monitoring+cellular+responses.">Identification of DISE-inducing shRNAs by monitoring cellular responses.</a> Cell Cycle (Georgetown, Tex). 17 (4), 506-514 (2018).</li><li>Vent&#243;s-Alfonso, A., Ylla, G., Monta&#241;es, J. C., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNMT1+promotes+genome+methylation+and+early+embryo+development+in+cockroaches.">DNMT1 promotes genome methylation and early embryo development in cockroaches.</a> iScience. 23 (12), 101778 (2020).</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=Variation+in+RNAi+efficacy+among+insect+species+is+attributable+to+dsRNA+degradation+in+vivo.">Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo.</a> Insect Biochemistry and Molecular Biology. 77, 1-9 (2016).</li><li>Bi, F., Liu, N., Small Fan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interfering+RNA:+A+new+tool+for+gene+therapy.">interfering RNA: A new tool for gene therapy.</a> Current Gene Therapy. 3 (5), 411-417 (2003).</li></ol>

The authors declare that they have no conflicts of interest.

This report described a methodological step-by-step RNAi strategy in P. americana; of note, it also can be applied to other cockroaches (Blattella germanica, for example) and many other insects with minor changes. However, the gene silencing efficiency of RNAi is not always high enough, with an obvious disadvantage compared with the gene knockout strategy13. The following residual effect of gene-level may interfere with the real phenotypes. To ensure the RNAi treatment is success...

RNA interference (RNAi), an evolutionarily conserved mechanism, gradually becomes an essential reverse-genetic tool to inhibit gene expression in many organisms1, since Andrew Fire and Craig Mello2 developed the double-stranded RNA (dsRNA) mediated gene silence strategy. dsRNA is cleaved into fragments of 21-23 nucleotides, small interfering RNAs (siRNAs), by the enzyme Dicer in cells to activate the RNAi pathway. Then siRNAs are incorporated into the RNA-induced silencing complex (RISC), which couples to the target mRNA, causes mRNA cleavage, and finally results in the loss of gene function3,4,5. Among the insect species, many systemic RNAi experiments have so far been reported in lots of insect orders, such as Orthoptera, Isoptera, Hemiptera, Coleoptera, Neuroptera, Diptera, Hymenoptera, Lepidoptera, and Blattodea5,6,7,8.
Cockroaches (Blattaria) are an essential insect family in developmental and metamorphic studies with their rapid growth cycles, strong adaptability to the environment, and high developmental plasticity9. Before discovering that RNAi was compatible with cockroaches, previous research only focused on cockroach prevention and control due to a scarcity of genetic manipulation techniques in cockroaches. The cockroach ootheca&#39;s unique structure made it challenging to perform embryo injection-based gene knockout with the CRISPR-Cas9 system. Besides, most tissues in cockroaches (such as P. americana) show robust systemic RNAi response, allowing for the rapid generation of dysfunctional phenotypes by injecting one or more targeting dsRNAs9,10,11. These features made RNAi an indispensable technique in gene functional research in P. americana.
Even though the use of RNAi in functional gene research in P. americana has been reported, no detailed or step-by-step description was available. This report provides one step-by-step operational guideline for RNAi in P. americana, useful for gene function study in other cockroaches. Furthermore, this guide is not limited to Blattodea and can be applied to many other insects with minor modifications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>701 N 10 &#181;L Syr (26s/51/2)</td>
			<td>Hamilton</td>
			<td>PN:80300</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ningbo Jiangnan Instrument Factory</td>
			<td>RXZ-380A-LED</td>
			<td>For cockroaches hatching and feeding</td>
		</tr>
		<tr>
			<td>Micro-injection pump</td>
			<td>Alcott Biotechnology</td>
			<td>ALC-IP600</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>pTOPO-Blunt Cloning Kit</td>
			<td>Aidlab Biotechnology</td>
			<td>CV16</td>
			<td>For Gene clonging</td>
		</tr>
		<tr>
			<td>quantitative Real-Time PCR Systems</td>
			<td>Bio-Rad</td>
			<td>CFX Connect</td>
			<td>For qRT-PCR analysis</td>
		</tr>
		<tr>
			<td>T7 RiboMAX Express RNAi System</td>
			<td>Promega</td>
			<td>P1700</td>
			<td>For dsRNA synthesis, which contains Rnase A Solution (4 &#956;g/&#956;L), Sodium Acetate, 3.0M (pH 5.2), Enzyme Mix, T7 Express, Nuclease-Free water, Express T7 2x Buffer, RQ1 RNase-Free DNase</td>
		</tr>
		<tr>
			<td>Thermal Cyclers</td>
			<td>Bio-Rad</td>
			<td>S1000</td>
			<td>For DNA amplification</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows a successful injection. The microinjection syringe with a micro diameter needle should be horizontally placed on the booster (Figure 1A). The needle is inserted via the gap between two abdominal somites horizontally against the epidermis (Figure 1B). Ensure that the liquid goes into the P. americana abdomen. The too steep angle of the needle will damage the internal organs (Figur...

Watch this Scientific Journal Video about Applications of RNA Interference in American Cockroach at JoVE.com

Applications of RNA Interference in American Cockroach

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 ...

The protocol provides a simple way to study American cockroaches. A common sanitary pest and a beneficial insect for traditional Chinese medicine. It will help to explore the molecular mechanisms of multiple life activities of American cockroaches by knocking down specific genes.The main advantage of this technology is that it can knock down genes in American cockroach or other similar insects that are not suitable for gene editing. This technology can be used to explore various fields such as the genetic and epigenetic regulation, development process of tissues, regeneration of appendages, mating behavior, and other interesting sides of American cockroach. This technology is easy to operate and versatile.We suggest to keep the animals healthy during DSRA injection treatment. I believe novices can easily master this technique after several practice sessions. To begin, separate the hatched nymphs from oothecae with a four millimeter aperture sieve.Pick out the white freshly malted P.americana with a glass tube. Place the P.americana in new containers and wait for the correct stage for treatment. In the reaction system, add 10 microliters of T7 2X buffer, 2 microliters of the T7 Express Enzyme Mix and two micrograms of PCR product.Finally, make up the total volume to 20 microliters with double distilled water. Gently mix upside down manually and incubate at 37 degrees Celsius for 30 minutes, then 70 degrees Celsius for 10 minutes. Dilute 4 micrograms per microliter RNase A solution with DEPC water at a ratio of 1 to 200.Then add 1 microliter of one unit per microliter RQ1 RNase Free Dnase and 1 microliter of diluted RNase A solution to the system. Incubate at 37 degrees Celsius for 30 minutes. Then add 10%of the total volume of sodium acetate and three volumes of the total volume of isopropanol.Gently mix upside down manually, then place on ice for five minutes and centrifuge at 13, 000 g for 10 minutes at 4 degrees Celsius. Remove the supernatant with a pipette. Next, wash the residue with 75%ethanol, with 25 percent DEPC treated water, then centrifuge at 13, 000 g for 5 minutes at 4 degrees Celsius.Remove supernatant again, air dry the pellet for 15 minutes at room temperature. Then dissolve the double stranded RNA with about 100 microliters of DEPC water and dilute it to the final concentration of 2 micrograms per microliter. Set up the program in the microinjection pump to ensure that the volume of each injection is consistent.Then clean the syringe by filling it with DEPC water 8 to 10 times. Install the 10 microliter syringe on the micro injection pump. Then start the pump and fill the syringe with 10 microliters of double stranded RNA solution.Anesthetize the cockroaches with carbon dioxide, then gently pick them up with tweezers and deliver the cockroach toward the needle by hand. Next, insert the needle via the gap between two abdominal somites horizontally against the epidermis. Inject the double stranded RNA solution into the cockroach ensuring that the needle tip is as close as possible to the epidermis to avoid damaging internal organs.Finally, pull out the needle tip. Put the injected cockroaches into clean bioassay containers. Wait for about 10 to 20 minutes to let them recover from the effects of carbon dioxide.Label the containers with the date of injection, type and dose of double stranded RNA and age of the P.americana. Ddc or DOPA decarboxylase and Dpp or decapentaplegic genes were selected as two examples to observe the phenotype of the malted cockroaches after double stranded RNA injection and dsMocK which has no target in the animals was taken as a negative control. The RNAi efficiency was detected by qRT PCR.The genes were significantly knocked down with a P value of less than 0.01 for dsDdc and less than 0.05 for dsDpp. P.americana with knockdown Ddc genes had the white epidermis due to blocked melanization. In the experiment with dsDpp injections, the RNAi disrupted limb regeneration.The insects should be healthy and injected without harm. This technology provides a simple way to explore gene functions for insects that cannot be gene edited such as American cockroach.

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2.2K visualizações.  South China Normal University. O protocolo fornece uma maneira simples de estudar baratas americanas. Uma praga sanitária comum e um inseto benéfico para a medicina tradicional chinesa. Ajudará a explorar os mecanismos moleculares de múltiplas atividades de vida de baratas americanas derrubando genes específicos.A principal vantagem desta tecnologia é que ela pode derrubar genes em baratas americanas ou outros insetos similares que não são adequados para a edição de genes. Essa tecnologia pode ser usada pa...