Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1. Mosquito Species and Rearing
<ol>
	<li>Maintain A. gambiae G3 and A. aegypti&#160;Liverpool IB12 strains (used in the representative studies below) or other strains of interest according to standard lab practice or as described previously23,24.</li>
</ol>
2. dsRNA and siRNA Design and Production
<ol>
	<li>Design primers to construct long dsRNA templates specific to the gene of interest. Use the E-RNAi tool25. Produce dsRNA according to the method of choice or as previously described19.
	<ol>
		<li>For a negative control, synthesize dsRNA19 corresponding to the sequence of green fluorescent protein (GFP), &#946;-galactosidase (&#946;-gal), or some other gene not expressed in mosquitoes. If so desired, dsRNA19 utilized to generate the representative results summarized below can be used as a positive control. Store dsRNA dissolved in RNAse-free water at -80 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Alternatively, design gene-specific siRNA according to standard lab practice or as described previously10. Scramble the sequence of a knockdown siRNA to design negative control siRNA that does not correspond to any mosquito gene. Purchase the custom siRNAs, which are available through a number of reputable vendors.
	<ol>
		<li>If so desired, siRNAs20,21utilized to obtain the representative results summarized below can be used as positive controls. Store siRNA dissolved in RNAse-free water at -80 &#176;C until needed.</li>
	</ol>
	</li>
</ol>
3. Preparing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Gather pre-made RT 100 mM sodium sulfate (100 mM Na2SO4 in deionized H2O) and 0.1 M sodium acetate (0.1&#160;M NaC2H3O2&#8211;0.1&#160;M acetic acid, pH 4.5 in deionized H2O) buffers.</li>
	<li>Dissolve chitosan (&#8805;75% deacetylated) in 0.1 M sodium acetate buffer to make 0.02% (w/v) working solution and keep the solution at RT before use.</li>
	<li>Dissolve dsRNA or siRNA in 50 &#181;l deionized H2O and add it to the 100 mM sodium sulfate buffer to make a 100 &#181;l solution of 32 &#181;g of dsRNA/siRNA per 100 &#181;l of 50 mM sodium sulfate.</li>
	<li>Add 100 &#181;l of chitosan solution to the dsRNA/siRNA solution and then heat the mixture in a water bath at 55 &#176;C for 1&#160;min. Set up a control by adding 100 &#181;l of 50 mM sodium sulfate to 100 &#181;l of chitosan solution and follow the same procedure.</li>
	<li>Mix the solutions immediately for 30 sec by high-speed vortexing at RT to facilitate the formation of nanoparticles.</li>
	<li>Centrifuge the mixture at 13,000 x g for 10 min at RT, after which time a pellet should be visible. Transfer the supernatant to a new 1.5 ml tube. Air-dry the pellet for &#8776;10 min at RT before using it to prepare mosquito food.</li>
	<li>Measure the concentration of dsRNA/siRNA in the supernatant by using the supernatant from the control (see 3.5) as a blank to calculate the total amount of dsRNA/siRNA that remained in the supernatant. Use the difference between starting amounts of dsRNA/siRNA and amounts remaining in the supernatants to calculate the percentage of dsRNA/siRNA entrapped in the nanoparticles. This loading efficiency is normally over 90%.</li>
	<li>Repeat the same procedure if more nanoparticles are needed. Use the dried nanoparticles immediately. The impact of cold storage of particles prior to use has not been evaluated.</li>
</ol>
4. Preparing Mosquito Food containing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Prepare 1 ml of a 2% agarose solution (w/v) in deionized water, melt the agarose, and keep melted agarose solution in a 55 &#730;C water bath before use.</li>
	<li>Mix fish food flakes (47% crude protein, min. crude fat 10%, max. crude fiber 3%) and dry yeast at a ratio of 2:1 (w/w). Grind the mixture to small particles with a mortar and pestle (passable through No. 50 USA standard test sieve). Use the ground food, which should be brownish in color, either immediately or store it in a sealed container at 4 &#730;C for several weeks.</li>
	<li>In a 1.5 ml tube, mix 6 mg of ground food with the dried nanoparticles from Section 3.7 with a toothpick.</li>
	<li>Add 30&#160;&#181;l of 2% pre-melted agarose gel solution to the food-nanoparticle mixture; stir immediately and thoroughly by using a toothpick or pipet tip.</li>
	<li>Use the gel containing food and nanoparticles to feed the mosquito larvae immediately. Alternatively, once the gel is completely solidified at RT, store the gel at 4 &#176;C and use the next day, or at -80 &#176;C for later use.</li>
</ol>
5. Feeding Mosquito Larvae with Food Containing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Feeding A. gambiae mosquito larvae:
	<ol>
		<li>Remove a single gel pellet from the 1.5 ml tube using a toothpick and cut it into 6 equal slices using a clean razor blade or tooth pick.</li>
		<li>Transfer 20 third instar larvae to a 500 ml Petri dish containing 100 ml of deionized water.</li>
		<li>Feed the mosquito larvae by adding one slice of the gel pellet (finely chopped into smaller pieces) per Petri dish once a day for four days. Be sure to observe larvae feeding on the pellet, which should be significantly reduced in size or completely absent by the next day. After time, mosquitoes will develop into late fourth instar larvae.</li>
		<li>Record any visible phenotypic changes during the experiment. Examine the transcript levels and other phenotypic changes as discussed in section 6 at the end of the four day period.</li>
	</ol>
	</li>
	<li>Feeding A. aegypti mosquito larvae:
	<ol>
		<li>Cut the gel pellet into 6 equal slices using a clean razor blade or toothpick.</li>
		<li>Place 50 age-synchronized 24 hr after egg hatching first instar larvae into a petri dish in &#8776;40 ml deionized water.</li>
		<li>Feed larvae one slice per petri dish for 4 hr/day, then transfer larvae back to the regular larval diet of 2:1 ground fish food flakes and dry yeast for the rest of the day. Repeat procedure daily throughout the four larval instars.</li>
		<li>Examine the transcript levels and other phenotypic changes as discussed in section 6 at the desired developmental time points.</li>
	</ol>
	</li>
</ol>
6. Confirmation of Gene Knockdown
<ol>
	<li>Relative transcript quantification by qRT-PCR in A. aegypti and A. gambiae larvae.
	<ol>
		<li>Perform and analyze qRT-PCR assays with three biological replicates on at least 10 pooled control vs. experimental larvae as described11,19,20 , or according to standard lab procedure. Representative results are included below.</li>
		<li>For analysis of a particular tissue type/body part (i.e., brain or antenna), perform qRT-PCR as described in 6.1.1 following dissection to recover the tissue of interest (for example, see Mysore et al.21).</li>
	</ol>
	</li>
	<li>Confirmation of knockdown by in situ hybridization
	<ol>
		<li>Synthesize digoxygenin-labeled antisense and sense control riboprobes according to standard lab practice or as described26.&#160;</li>
		<li>Execute in situ hybridization per the Haugen et al.27 protocol for confirmation of knockdown as described previously11,20 and discussed in the representative results section below.</li>
	</ol>
	</li>
	<li>Confirmation of knockdown by immunohistochemistry
	<ol>
		<li>If antibodies against the protein product of the gene targeted are available, perform immunohistochemistry as previously described28, which facilitates identification of individuals with the greatest levels of knockdown for phenotype analysis20 as exemplified in the representative results section below.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The chitosan/interfering RNA nanoparticle methodology described herein has been used to effectively target genes during larval development in A. gambiae (Figures&#160;2, 3) and A. aegypti (Figures 4, 5,&#160;6, Tables 1, 2). Chitosan nanoparticles can be prepared with either long dsRNA or siRNA, both of which have been used successfully in mosquitoes as evidenced by the representative results described herein. Synthesis of dsRNA is less...

Blood feeding vector mosquitoes of the Anopheline and Aedine genera transmit disease-causing agents responsible for several of the worst scourges of humankind. An estimated 3.4 billion people are at risk for contracting malaria, which is responsible for over one-half million deaths annually worldwide. Malaria results from infection by Plasmodiumsp. parasites, which are transmitted to people through the bites of infected mosquitoes of the Anopheles genus, including the principal African vector Anopheles gambiae (<a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a>, 2014)1. Aedes aegypti is the primary mosquito vector for dengue virus, which causes dengue fever, a nonspecific febrile illness that is the most widespread and significant arboviral disease in the world. Dengue virus is presently a threat to &#62;2.5 billion people in the tropics, with a yearly incidence of approximately 50 million cases resulting in ~24,000 deaths annually (<a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a>, 2014)2. Despite the devastating global impact of mosquito-borne illnesses on human health, effective means of preventing and treating these diseases are lacking. Mosquito control is presently the best method of disease prevention.
The potential for controlling arthropod-borne diseases by the genetic manipulation of vector insects has been recognized for over four decades3. Transgenic strains of A. aegypti engineered to have a repressible female-specific flightless phenotype have recently made the potential for using transgenic vector control strategies a reality4-6. These advancements have challenged researchers to identify novel genetic targets for vector control and additional means of manipulating gene function in vector mosquitoes. Alteration of gene expression during development, as was the case in female-flightless mosquitoes4, may promote the elucidation of novel vector control strategies. However, largely due to technical challenges, the functions of very few genes have been characterized during development of A. gambiae, A. aegypti, or other mosquito species.
Since its discovery in C. elegans7, RNA interference (RNAi), which is conserved in animals, plants, and microorganisms, has been extensively used for functional genetic studies in a wide variety of organisms, including insects8,9. The RNAi pathway is initiated by Dicer, which cleaves long dsRNA into short 20-25 nucleotide-long siRNAs that function as sequence-specific interfering RNA. siRNAs silence genes that are complementary in sequence by promoting transcript turnover, cleavage, and disruption of translation9. Long dsRNA molecules (typically 300-600 bp) or custom siRNAs targeting a particular sequence can be used in the research laboratory for silencing any gene of interest. By managing when interfering RNA is delivered, researchers can control the time at which gene silencing initiates. This advantage is useful as it can be used to overcome challenges such as developmental lethality or sterility, which can hinder the production and maintenance of strains bearing heritable mutations, an expensive and labor-intensive process that is not yet available in all insect species. Although the degree of gene silencing by RNAi can vary from gene to gene, tissue to tissue, and animal to animal, RNAi is widely used for functional analysis of genes in mosquitoes and other insects8,9.
Three interfering RNA delivery strategies have been used in mosquitoes: microinjection, soaking/topical application, and ingestion. For a detailed history and comparison of the use of these three techniques in insects, please refer to Yu et al8. We have successfully used microinjection10 as a means of delivering siRNAs to target developmental genes in A. aegypti embryos, larvae, and pupae11-14. However, this labor-intensive delivery strategy requires both a microinjection setup and a skilled hand. Moreover, microinjection is stressful to the organism, a confounding factor, particularly when behavioral phenotypes will be assessed. Finally, microinjection delivery cannot be extended to the field for vector control. As an alternative, soaking the organism in interfering RNA solution has also become a popular means of inducing gene silencing, as it is convenient and requires little equipment or labor. Soaking has primarily been applied in insect cell line studies8, but in a recent study, knockdown was achieved in A. aegypti larvae immersed in a solution of dsRNA15, which the animals appeared to be ingesting. However, for studies involving analysis of multiple experimental groups or phenotypes, soaking is rather costly. Lopez-Martinez et al.16 described rehydration driven RNAi, a novel approach for interfering RNA delivery that involves dehydration in saline solution and rehydration with a single drop of water containing interfering RNA. This approach does cut the costs associated with whole animal immersion, but is more expensive than microinjection and may be limited in its application to species that can tolerate high osmotic pressures. Moreover, it is difficult to envision how immersion or dehydration/rehydration immersion methodology could be adapted for vector control in the field. For these reasons, for post-embryonic studies, delivery of interfering RNA with ingested food is a viable alternative strategy.
Although ingestion-based strategies do not work in all insect species, perhaps most notably Drosophila melanogaster, oral delivery of interfering RNA mixed with food has promoted gene silencing in a variety of insects8,17, including A. aegypti adults18. We described chitosan nanoparticle-mediated RNAi in A. gambiae larvae19 and have successfully applied this approach for reduction of gene expression in A. aegypti larvae20,21. Here, methodology for this RNAi procedure, which involves entrapping of interfering RNA by the polymer chitosan, is detailed. Chitosan/interfering RNA nanoparticles are formed by self-assembly of polycations with interfering RNA through the electrostatic forces between positive charges of the amino groups in chitosan and negative charges carried by the phosphate groups on the backbone of interfering RNA19. The procedure described is compatible with both long dsRNA molecules (hereafter referred to as dsRNA) or double stranded siRNA (hereafter referred to as siRNA). Following synthesis, chitosan/interfering RNA nanoparticles are mixed with larval food and delivered to larvae through oral ingestion. This methodology is relatively inexpensive, requires little equipment and labor19, and facilitates high-throughput analysis of multiple phenotypes, including analysis of behaviors20,21. This methodology, which can be adapted for gene silencing studies in other insects, including other disease vectors and insect agricultural pests, could potentially be used for gene silencing in a variety of other animal species. Moreover, chitosan, an inexpensive, non-toxic and biodegradable polymer22, could potentially be utilized in the field for species-specific mosquito control.

<table><tbody><tr><td>0.02 ml pipetteman</td><td>Rainin</td><td>PR20</td><td>for resuspension of interfering RNA</td></tr><tr><td>0.2 ml pipetteman</td><td>Rainin</td><td>PR200</td><td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td></tr><tr><td>0.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-120</td><td>for aliquoting resuspended interfering RNA</td></tr><tr><td>1 ml pipetteman</td><td>Rainin</td><td>PR1000</td><td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td></tr><tr><td>1.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-046</td><td>for preparation of interfering RNA/nanoparticles</td></tr><tr><td>1M Sodium Acetate, pH 4.5</td><td>TEKnova</td><td>S0299</td><td>to prepare sodium acetate buffer</td></tr><tr><td>Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td><td>BDH</td><td>VWR BDH3092-500MLP</td><td>to prepare sodium acetate buffer</td></tr><tr><td>Agarose Genetic Technology Grade</td><td>MP Biomedicals LLC.</td><td>800668</td><td>to coat prepared interfering RNA/nanoparticles</td></tr><tr><td>Centrifuge</td><td>Eppendorf AG</td><td>5415D</td><td>to pellet interfering RNA/nanoparticles</td></tr><tr><td>Chitosan, from shrimp shells</td><td>Sigma-Aldrich</td><td>C3646-25G</td><td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td></tr><tr><td>Dry Yeast</td><td>Universal Food Corp</td><td>NA</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>E-RNAi tool</td><td>German Cancer Research Center</td><td>NA</td><td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td></tr><tr><td>goldfish food</td><td>Wardley</td><td>Goldfish FLAKE FOOD</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>Heated water bath</td><td>Thermo Scientific</td><td>51221048</td><td>to heat the interfering RNA/nanoparticles at 55&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ice</td><td>not applicable</td><td>not applicable</td><td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>02-591-44</td><td>for storage of ice used during the procedure</td></tr><tr><td>liver powder</td><td>MP Biomedicals LLC.</td><td>900396</td><td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td></tr><tr><td>Microwave oven</td><td>A variety of vendors</td><td>not applicable</td><td>to prepare 2% agarose solution</td></tr><tr><td>petridish (100 x 15 mm)</td><td>Fischer Scientific</td><td>875713</td><td>interfering RNA/nanoparticle feeding chamber for larvae</td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>S220</td><td>for preparation of buffers</td></tr><tr><td>Razor blade</td><td>Fischer Scientific</td><td>12-640</td><td>to divide the interfering RNA/nanoparticle pellet for feedings</td></tr><tr><td>siRNA</td><td>Thermo Scientific/Dharmacon</td><td>custom</td><td>for preparation of siRNA/nanoparticles</td></tr><tr><td>Sodium Sulfate, Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>BDH/distributed by VWR</td><td>VWR BDH0302-500G</td><td>to prepare 50 mM sodium sulfate solution in which the interfering RNA will be resuspended</td></tr><tr><td>Thermometer</td><td>VWR</td><td>61066-046</td><td>to measure the water bath temperature</td></tr><tr><td>Tooth picks</td><td>VWR</td><td>470146-908</td><td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td></tr><tr><td>Ultralow freezer</td><td>A variety of vendors</td><td>not applicable</td><td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Vortex mixer</td><td>Fischer Scientific</td><td>02-216-108</td><td>for preparation of chitosan/interfering RNA</td></tr><tr><td>Weight paper</td><td>Fischer Scientific</td><td>NC9798735</td><td>to divide the interfering RNA/nanoparticle pellet for feedings</td></tr></tbody></table>

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.

A. gambiae: 
Chitosan/dsRNA nanoparticles are formed due to the electrostatic interaction between amino groups of chitosan and the phosphate groups of dsRNA. The efficiency of dsRNA incorporation into nanoparticles is usually above 90% as measured by depletion of dsRNA from the solution. Atomic force microscopy images show that chitosan-dsRNA particle size averages 140 nm in diameter, ranging from 100-200 nm (Figure 1).
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Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

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 ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.3K Views.  Kansas State University.  Hier beschrijven we een werkwijze voor het remmen van genfunctie in ziektedragers muggen door het gebruik van chitosan / interfererende RNA nanodeeltjes die worden opgenomen door larven. 

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

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

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 ...

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Developmental Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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) ...

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

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

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

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

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

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

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

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