Name: applications of rna interference in american cockroach
Uploaded: 2021-12-17T15:00:00.000+00:00
Duration: 6 min 32 s
Description: Watch this Scientific Journal Video about Applications of RNA Interference in American Cockroach at JoVE.com

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>
	<li>Use a sieve with 4 mm aperture to separate the hatched nymphs from oothecae.</li>
	<li>Keep the nymphs in cylindrical containers (12 cm in diameter and 10 cm in height) in the dark at 28 &#176;C, 70% humidity. Brush the inside edge of the containers with vaseline to prevent cockroaches from escaping. Provide rat food, water, and shelter (egg trays). Pay attention to the activity of the nymphs and regularly clean up the feces and debris.</li>
</ol>
2. Selection of the nymphs in proper instar
<ol>
	<li>Use a glass tube to pick out the freshly molted P. americana (white in body color). Keep them in new containers and wait for the correct stage for treatment. 
	&#8203;NOTE: After ~19 days under the above feeding conditions, the nymphs in the 3rd instars will be available for injection. The color of freshly molted cockroaches is white, and the instars are clarified by molting times.</li>
</ol>
3. Preparation of the target fragment with T7 promoters
<ol>
	<li>Using the cDNA of P. americana as a template, design paired primers to perform PCR to obtain 300-800 bp DNA fragment of the target gene9. Then clone the PCR fragment into a pTOPO vector (see Table of Materials) for sequencing.</li>
	<li>Using the target fragment DNA as a template, synthesize one new pair of primers with T7 promoter sequence (5&#39;-GGATCCTAATACGACTCACTATAGG-3&#39;) at the 5&#39; terminals and perform another round of PCR to obtain the target fragment with T7 promoters on each side9.</li>
</ol>
4. Transcription and synthesis of dsRNA in vitro
<ol>
	<li>Add 10 &#181;L of T7 2X Buffer, 2 &#181;L of the Enzyme Mix, T7 Express (see Table of Materials), and 2 &#181;g of PCR product (obtained in step 3.2) in the reaction system and add up the total volume to 20 &#181;L with ddH2O. Gently mix upside down manually and incubate at 37 &#176;C for 30 min and 70 &#176;C for 10 min, and then slowly cool to room temperature.</li>
	<li>Dilute RNase A solution (4 &#181;g/&#181;L) with ddH2O at a ratio of 1:200. Then add 1 &#181;L of RQ1 RNase-Free DNase (1 U/&#181;L) and 1 &#181;L of diluted RNase A solution to the system (see Table of Materials). 
	NOTE: Now, the volume of a single system is 22 &#181;L.</li>
	<li>Incubate at 37 &#176;C for 30 min. Then add 10% of the total volume (when performing N reactions simultaneously, the total volume is N x 22 &#181;L) of Sodium acetate and three volumes of the total volume of isopropanol.</li>
	<li>Gently mix upside down manually, place on ice for 5 min, and centrifuge at 13,000 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant with a pipette, wash the residue with 75% ethanol (with 25% diethyl pyrocarbonate (DEPC) treated water), and then centrifuge at 13,000 x g for 5 min at 4 &#176;C.</li>
	<li>Remove supernatant again. Air-dry the pellet for 15 min at room temperature. Use ~100 &#181;L of DEPC water to dissolve dsRNA, then dilute the dsRNA to the final 2 &#181;g/&#181;L. 
	&#8203;NOTE: The dsRNA could be stored at -80 &#176;C for 6 months.</li>
</ol>
5. Loading dsRNA solution into the syringe
<ol>
	<li>Set up the program in the micro-injection pump (see Table of Materials) in advance to ensure that the volume of each injection is consistent. Before using the syringe, clean the syringe by filling it with DEPC water 8-10 times.</li>
	<li>Install the 10 &#181;L Syringe on the micro-injection pump, start the pump, and fill the syringe with 10 &#181;L of dsRNA solution (prepared at step 4.6). Inject 1 &#181;L of the dsRNA into a 3rd instar nymph (see step 2) and ensure that it does not leak out to the body.</li>
</ol>
6. Injecting dsRNA to P. americana
<ol>
	<li>Anesthetize the cockroaches with an increased concentration of CO2 in a container until the cockroaches do not move anymore, and then proceed immediately with the injection.</li>
	<li>Gently pick up the cockroach with tweezers, and deliver the cockroach toward the needle with hand. Next, insert the needle via the gap between two abdominal somites horizontally against the epidermis. About&#160;insert depth, the shallower, the better.</li>
	<li>Then, inject the dsRNA solution into the cockroach. Finally, pull out the needle tip. Ensure that the needle tip is as close as possible to the epidermis to avoid damaging internal organs. 
	NOTE: The injection should be made under a dissection microscope.</li>
	<li>Put the injected cockroaches into clean bioassay containers. Wait for about 10-20 min to let them recover from the CO2 effects. Label the containers with the date of injection, type and dose of dsRNA, and age of the P. americana.</li>
	<li>Place the injected cockroaches in a dark environment at 28 &#176;C, 70% humidity, provide water, feed, and shelter, and observe possible changes in the phenotype of the cockroaches regularly.</li>
</ol>
7. Knockdown confirmation and phenotypic analysis
<ol>
	<li>Evaluate the efficiency of RNAi using any available molecular biology techniques such as quantitative Real-Time PCR (qRT-PCR) and Western blotting. For detailed qRT-PCR and Western blotting procedures, see References1,12.</li>
	<li>To observe and analyze RNAi-related phenotypes, use the microscope suitable for living animals. 
	NOTE: RNAi may affect the morphology, behavior, molting, limb regeneration, and other physiological activities of cockroaches. The specific frequency of phenotype is calculated according to specific conditions.</li>
</ol>

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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><tbody><tr><td>701 N 10 &amp;micro;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 &amp;mu;g/&amp;mu;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 ...

applications-of-rna-interference-in-american-cockroach

Research

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Developmental Biology

2.4K Views.  South China Normal University. The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Video: Applications of RNA Interference in American Cockroach

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 ...

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

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 ...

Biology

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

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 ...

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) ...

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.

Applications of RNA Interference in American Cockroach

Summary

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Chapters in this video

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

Electroporation-mediated RNA Interference Method in Odonata

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNAi Trigger Delivery into Anopheles gambiae Pupae

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

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

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura