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

Abstract

SHORT ABSTRACT 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. LONG 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.

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

“…Current methods for inducing non-transgenic RNAi in mosquitoes involve direct injection of dsRNA into the adult hemocoel 12,13 or larval feeding of RNAi trigger-coated nanoparticles [14][15][16][17] or microalgae-based dsRNA molecules 18 . Targeting the adult mosquito, while extremely valuable, can exclude a large number of genes that function during earlier developmental periods.…”

Chitosan, Carbon Quantum Dot, and Silica Nanoparticle Mediated dsRNA Delivery for Gene Silencing in Aedes aegypti: A Comparative Analysis

“…[8][9][10][11][12][13][14][15][16] Three general strategies have been elucidated for RNAi in insects: microinjection, soaking and feeding of dsRNA. 17 dsRNA feeding is perhaps the most convenient and cost effective RNAi approach for insect control. Delivery of dsRNA by expressing them in plants as well as direct feeding are being developed.…”

“…26 A simple and cost effective method to conduct successful RNAi in mosquitoes has been developed. 17 Here, we have evaluated efficiency of NP mediated dsRNA delivery method to knockdown Ae. aegypti genes.…”

Developmental neurogenetics of sexual dimorphism in Aedes aegypti

“…The time at which gene silencing initiates can be managed through control of siRNA delivery. This advantage facilitates the study of embryonic lethal genes during post-embryonic stages of development; it also permits identification of the critical periods in which loss of gene function generates phenotypes of interest, information that may inform the design of control strategies (Clemons et al, 2010b; Zhang et al, 2015). …”

“…siRNA can also be delivered to A. aegypti larvae via chitosan nanoparticles (Mysore et al, 2013, 2014a,b) that are mixed with larval food and orally ingested by larvae, and which may promote the stability and cellular uptake of interfering RNA (Zhang et al, 2010). This technique, for which detailed methodology is available (Zhang et al, 2015), is relatively inexpensive, requires little equipment and labor, facilitates high-throughput analysis of multiple phenotypes including behavioral analyses (Zhang et al, 2010, 2015; Mysore et al, 2013, 2014a,b), and could likely be adapted for gene silencing studies in other insect species. Furthermore, chitosan, a non-toxic and biodegradable polymer (Dass and Choong, 2008), could potentially be utilized in the field.…”