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 aeg....

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 gree....

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

List of materials used in this article

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