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

Specific gene silencing by RNA interference (RNAi) involving exogenous double stranded RNA (dsRNA) delivery has potential in Helicoverpa armigera control, a resistant insect pest. Here, ionotropically synthesized cationic chitosan nanoparticles (CNPs, 95 nm size, +36 mV charge) showed efficient dsRNA loading (95 %) and effective protection from insect gut nucleases and pH degradation. The CNPs were tagged with fluorescence and found to be stable on leaf surface (24 h) and were internalized by columnar insect gut cells. A single dose of CNPs:dsRNA complex (containing 0.1 µg dsRNA) ingested by H. armigera larvae via artificial/leaf feed effectively silenced lipase and chitinase target genes (2-2.7 fold downregulation) and suppressed their respective enzyme activities (2-5.3 fold). RNAi caused reduced pupation (5-fold) and impaired moth emergence. RNAi effects correlated significantly with 100% insect mortality (PCA 0.97-0.99). Furthermore, specific dsRNA did not affect non-target insects Spodoptera litura and Drosophila melanogaster. Developed CNPs:dsRNA complexes towards RNAi targets can serve as a safe, targeted insecticide for sustainable crop protection.

New Frontiers in Pest Control: Chitosan Nanoparticles-Shielded dsRNA as an Effective Topical RNAi Spray for Gram Podborer Biocontrol.

Chickpea pod borer, Helicoverpa armigera, displays resistance to chemical insecticides and transgenics. The potential nontransformative RNAi approach of specific gene silencing by mRNA breakdown through exogenous double-stranded (dsRNA) delivery to Helicoverpa faces problems of degradation by nucleases and insect gut pH. We demonstrate that chitosan nanoparticles (CNPs) effectively mediate specific dsRNA delivery against Helicoverpa armigerajuvenile hormone methyltransferase (JHAMT) and acetylcholine esterase (ACHE) target genes. Ionotropically synthesized cationic CNPs (100 nm size, +32 mV charge) loaded dsRNA efficiently and protected it effectively from degradation by nucleases and insect gut pH. Tagging CNPs with Calcofluor fluorescence illustrated its efficient uptake in columnar insect gut cells. The potential of CNPs-mediated dsRNA delivery was elucidated with effective silencing of green fluorescent protein transformed Sf9 cells. Furthermore, CNPs-dsRNA complexes were stable for 5 d on leaf surfaces, and their ingestion with leaf effectively silenced H. armigeraJHAMT and ACHE genes to suppress related enzyme activities and caused 100% insect mortality. Further, in planta bioassay with CNPs-dsRNA spray confirmed the RNAi induced insect mortality. Moreover, CNPs-dsRNA fed nontarget insects Spodoptera litura and Drosophila melanogaster were unaffected, and no toxicity was observed for CNPs in cell line studies. Remarkably, only two low dose (0.028 g/ha) topical CNPs-ache-dsRNA sprays on chickpea displayed reduced pod damage with high yields on par with chemical control in the field, which was followed by CNPs-jhamt-dsRNA nanoformulation. These studies can pave the way for the development of topical application of CNPs-dsRNA spray as a safe, specific, innovative insecticide for sustainable crop protection.