Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes

https://doi.org/10.1016/j.ijbiomac.2016.01.030Get rights and content

Abstract

RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control.

Introduction

Mosquitoes are the most dangerous insects to human life because they transmit deadly pathogens like Plasmodium, Chikungunya virus, yellow fever virus, dengue virus, Japanese encephalitis virus and West Nile virus [1], [2]. Worldwide 3500 species of mosquitoes have been identified and grouped under 41 genera. Only 100 species are reported as vectors of human and other vertebrate diseases. The most commonly distributed genera are Aedes, Anopheles and Culex. Mosquitoes are controlled by chemical pesticides. But chemical pesticides fail to control mosquitoes due to the development of pesticide resistance in mosquitoes [3]. Alternate methods of mosquito control include biopesticides, biological control and sterilization techniques. Recently researchers are interested in applying gene silencing technique in mosquito control. RNAi technique was reported as a promising one for gene knockdown in eukaryotes [4].

RNAi is triggered by gene silencing mechanism that is initiated with the introduction of dsRNA into a cell [5]. Specifically designed RNAi molecules can target mRNAs and initiate their degradation. Several pioneering studies have demonstrated great possibilities for controlling agriculturally important insect pests [6], [7], mosquitoes [8], trypanosomes [9] and tick-borne pathogens [10], [11]. Some papers had reported that dsRNA or siRNA synthesized by in vitro methods potentially cleaved the targeted mRNA and silenced the gene of interest [12], [13]. Although RNAi is a conserved mechanism in eukaryotes including fungi, plants, insects and mammals, there have been great challenges for successful use of RNAi in some organisms or some developmental stages of an organism [14]. Lack of an effective delivery method for dsRNA or siRNA and the instability of the nucleic acids during and/or after the delivery are the major difficulties in gene silencing studies.

To increase the stability of dsRNA and to enhance their cellular uptake, polymeric nanoparticles have been used for nucleic acid delivery in RNAi-based gene therapeutics. One of the most commonly used polymers is chitosan which is used to generate nanoparticles for delivery of nucleic acids [15]. Chitosan is an efficient carrier of nucleic acids, which has gained tremendous interest as gene delivery system. Chitosan can attach with nucleic acids and form nanopolyplexes via ionic interactions. The nanopolyplexes are low toxic, biodegradable and biocompatible materials [16], [17]. The nanopolyplexes formed by cationic chitosan and anionic nucleic acid are used to optimize stability of the electrostatic interactions. Several pioneering studies have demonstrated that chitosan could carry and deliver dsRNA both in in vivo and in vitro systems [18]. The nanoparticle-mediated RNA interference was used to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae) [19].

Flying ability is an important evolutionary transition. It requires combination of morphological, physiological, and behavioral features [20]. It was reported that there were three key regulatory genes involved in insect wing development such as wingless (wg), vestigial (vg) and apterous (ap); this was studied in two basal insects [21]. Proper development of the wing in insect involves the coordinate action of several genes. One of the central genes involved in this process is vestigial (vg). The vg product is required in a variety of tissues during morphogenesis and is also required during late larval development (the third larval instar) primarily for formation of specific regions of the wing and haltere imaginal discs [22], [23], [24]. It is a target of the decapentaplegic (dpp) and Notch (N)/wingless (wg) pathways and patterned vg expression occurs through the entire wing field. The objective of the present study was to develop an effective method for the delivery of dsRNA using chitosan nanoparticles to target the vg gene in Aedes aegypti.

Section snippets

In silico analysis of vg gene

The sequence of Ae. aegypti wing development gene was retrieved from GenBank (accession no: XM_001656561.1). This sequence was used to generate a protein in multiple alignment with orthologs using Clustal W (http://www.ebi.ac.uk/Tools/msa/clustalw2). The conserved regions of deduced amino acids sequence were predicted by molecular modeling using T08 Sam server (https://compbio.soe.ucsc.edu/SAM_T08/T08-query.html). Phylogenetic analysis was done by the neighbor-joining method using MEGA version

Results and discussion

Mosquitoes are the principal vectors of many vector borne diseases affecting human beings and animals. In order to control the spread of these diseases, strategies to stop mosquitoes have become essential. The spread of these viral diseases can be kept under control if the wing development of the mosquitoes is impeded. This can be done by targeting the gene responsible for flight of mosquitoes through RNAi. Therefore, in this study the vg gene was targeted since it is responsible for wing

Conflict of interest

The authors have no conflict of interest.

Acknowledgement

The authors are grateful to Entomology Research Institute, Loyola College, Chennai. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group project No. RGP-VPP-213 for financial assistance.

References (44)

  • K.A. Howard et al.

    RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system

    Mol. Ther.

    (2006)
  • D.J. Gary et al.

    Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery

    J. Control. Release

    (2007)
  • A.A. James

    Mosquito molecular genetics: the hands that feed bite back

    Science

    (1992)
  • M.W. Service

    Management of vectors

  • J.F. Charles et al.

    Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena

    Mem. Inst. Oswaldo Cruz

    (2000)
  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • J.A. Baum et al.

    Control of coleopteran insect pests through RNA interference

    Nat. Biotechnol.

    (2007)
  • Y.B. Mao et al.

    Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol

    Nat. Biotechnol.

    (2007)
  • D.W. Severson et al.

    Mosquito genomics: progress and challenges

    Annu. Rev. Entomol.

    (2012)
  • T.J. Schuijt et al.

    Identification and characterization of Ixodesscapularis antigens that elicit tick immunity using yeast surface display

    PLoS One

    (2011)
  • G. Huang et al.

    Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene

    PNSA

    (2006)
  • S.C. Miller et al.

    Larval RNAi in Drosophila?

    Dev. Genes Evol.

    (2008)
  • Cited by (81)

    • Chitosan nanocarriers mediated dsRNA delivery in gene silencing for Helicoverpa armigera biocontrol

      2023, Pesticide Biochemistry and Physiology
      Citation Excerpt :

      Jayachandran et al. (2013) reported 40% mortality by daily oral feeding of 125 ng dsRNA continuously from neonate to pre-pupal stages, targeting trypsin like serine protease gene. Delivery of nanocomplexes of chitosan polymer bound to siRNA caused gene silencing in African malaria mosquito Anopheles gambiae, yellow fever mosquito Aedes aegypti and S. frugiperda (Zhang et al., 2010; Dhandapani et al., 2019; Kumar et al., 2016; Gurusamy et al., 2020). These nanocomplexes resulted in low insect mortalities (30–47%) at relatively high dsRNA concentrations (1–10 μg).

    View all citing articles on Scopus
    View full text