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

Summary

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.

Abstract

Vector mosquitoes inflict more human suffering than any other organism—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.

Introduction

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 (http://www.who.int/topics/malaria/en/, 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 >2.5 billion people in the tropics, with a yearly incidence of approximately 50 million cases resulting in ~24,000 deaths annually (http://www.cdc.gov/dengue/, 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 reality^4-6. 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 insects^8,9. 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 insects^8,9.

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 pupae^11-14. 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 insects^8,17, 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 larvae^20,21. 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 behaviors^20,21. 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.

Protocol

1. Mosquito Species and Rearing

1. Maintain A. gambiae G3 and A. aegypti Liverpool IB12 strains (used in the representative studies below) or other strains of interest according to standard lab practice or as described previously^23,24.

2. dsRNA and siRNA Design and Production

1. 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.
   1. For a negative control, synthesize dsRNA¹⁹ corresponding to the sequence of green fluorescent protein (GFP), β-galactosidase (β-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 °C until needed.
2. 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.
   1. If so desired, siRNAs^20,21utilized to obtain the representative results summarized below can be used as positive controls. Store siRNA dissolved in RNAse-free water at -80 °C until needed.

3. Preparing Chitosan/interfering RNA Nanoparticles

1. Gather pre-made RT 100 mM sodium sulfate (100 mM Na₂SO₄ in deionized H₂O) and 0.1 M sodium acetate (0.1 M NaC₂H₃O₂–0.1 M acetic acid, pH 4.5 in deionized H₂O) buffers.
2. Dissolve chitosan (≥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.
3. Dissolve dsRNA or siRNA in 50 µl deionized H₂O and add it to the 100 mM sodium sulfate buffer to make a 100 µl solution of 32 µg of dsRNA/siRNA per 100 µl of 50 mM sodium sulfate.
4. Add 100 µl of chitosan solution to the dsRNA/siRNA solution and then heat the mixture in a water bath at 55 °C for 1 min. Set up a control by adding 100 µl of 50 mM sodium sulfate to 100 µl of chitosan solution and follow the same procedure.
5. Mix the solutions immediately for 30 sec by high-speed vortexing at RT to facilitate the formation of nanoparticles.
6. 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 ≈10 min at RT before using it to prepare mosquito food.
7. 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%.
8. 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.

4. Preparing Mosquito Food containing Chitosan/interfering RNA Nanoparticles

1. Prepare 1 ml of a 2% agarose solution (w/v) in deionized water, melt the agarose, and keep melted agarose solution in a 55 ˚C water bath before use.
2. 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 ˚C for several weeks.
3. In a 1.5 ml tube, mix 6 mg of ground food with the dried nanoparticles from Section 3.7 with a toothpick.
4. Add 30 µl of 2% pre-melted agarose gel solution to the food-nanoparticle mixture; stir immediately and thoroughly by using a toothpick or pipet tip.
5. 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 °C and use the next day, or at -80 °C for later use.

5. Feeding Mosquito Larvae with Food Containing Chitosan/interfering RNA Nanoparticles

1. Feeding A. gambiae mosquito larvae:
   1. 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.
   2. Transfer 20 third instar larvae to a 500 ml Petri dish containing 100 ml of deionized water.
   3. 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.
   4. 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.
2. Feeding A. aegypti mosquito larvae:
   1. Cut the gel pellet into 6 equal slices using a clean razor blade or toothpick.
   2. Place 50 age-synchronized 24 hr after egg hatching first instar larvae into a petri dish in ≈40 ml deionized water.
   3. 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.
   4. Examine the transcript levels and other phenotypic changes as discussed in section 6 at the desired developmental time points.

6. Confirmation of Gene Knockdown

1. Relative transcript quantification by qRT-PCR in A. aegypti and A. gambiae larvae.
   1. Perform and analyze qRT-PCR assays with three biological replicates on at least 10 pooled control vs. experimental larvae as described^11,19,20 , or according to standard lab procedure. Representative results are included below.
   2. 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).
2. Confirmation of knockdown by in situ hybridization
   1. Synthesize digoxygenin-labeled antisense and sense control riboprobes according to standard lab practice or as described²⁶. 
   2. Execute in situ hybridization per the Haugen et al.²⁷ protocol for confirmation of knockdown as described previously^11,20 and discussed in the representative results section below.
3. Confirmation of knockdown by immunohistochemistry
   1. 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.

Results

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).

Discussion

The chitosan/interfering RNA nanoparticle methodology described herein has been used to effectively target genes during larval development in A. gambiae (Figures 2, 3) and A. aegypti (Figures 4, 5, 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...

Materials

Name	Company	Catalog Number	Comments
0.02 ml pipetteman	Rainin	PR20	for resuspension of interfering RNA
0.2 ml pipetteman	Rainin	PR200	for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles
0.5 ml graduated tube	Fischer Scientific	05-408-120	for aliquoting resuspended interfering RNA
1 ml pipetteman	Rainin	PR1000	for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles
1.5 ml graduated tube	Fischer Scientific	05-408-046	for preparation of interfering RNA/nanoparticles
1M Sodium Acetate, pH 4.5	TEKnova	S0299	to prepare sodium acetate buffer
Acetic Acid, AIRSTAR. ACS, USP, FCC Grade	BDH	VWR BDH3092-500MLP	to prepare sodium acetate buffer
Agarose Genetic Technology Grade	MP Biomedicals LLC.	800668	to coat prepared interfering RNA/nanoparticles
Centrifuge	Eppendorf AG	5415D	to pellet interfering RNA/nanoparticles
Chitosan, from shrimp shells	Sigma-Aldrich	C3646-25G	to combine with interfering RNA for prepararation of interfering RNA/nanoparticles
Dry Yeast	Universal Food Corp	NA	to prepare mosquito larval food with interfering RNA/nanoparticles
E-RNAi tool	German Cancer Research Center	NA	for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//
goldfish food	Wardley	Goldfish FLAKE FOOD	to prepare mosquito larval food with interfering RNA/nanoparticles
Heated water bath	Thermo Scientific	51221048	to heat the interfering RNA/nanoparticles at 55oC
Ice	not applicable	not applicable	for thawing interfering RNA and preparation of interfering RNA/nanoparticles
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Ice bucket	Fisher Scientific	02-591-44	for storage of ice used during the procedure
liver powder	MP Biomedicals LLC.	900396	to feed mosquito larvae post interfering RNA/nanoparticle treatment
Microwave oven	A variety of vendors	not applicable	to prepare 2% agarose solution
petridish (100 x 15 mm)	Fischer Scientific	875713	interfering RNA/nanoparticle feeding chamber for larvae
pH meter	Mettler Toledo	S220	for preparation of buffers
Razor blade	Fischer Scientific	12-640	to divide the interfering RNA/nanoparticle pellet for feedings
siRNA	Thermo Scientific/Dharmacon	custom	for preparation of siRNA/nanoparticles
Sodium Sulfate, Anhydrous (Na2SO4)	BDH/distributed by VWR	VWR BDH0302-500G	to prepare 50 mM sodium sulfate solution in which the interfering RNA will be resuspended
Thermometer	VWR	61066-046	to measure the water bath temperature
Tooth picks	VWR	470146-908	for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets
Ultralow freezer	A variety of vendors	not applicable	for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC
Vortex mixer	Fischer Scientific	02-216-108	for preparation of chitosan/interfering RNA
Weight paper	Fischer Scientific	NC9798735	to divide the interfering RNA/nanoparticle pellet for feedings