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 armigera juvenile 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. armigera JHAMT 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.
Kaas Plateau is located in Western Ghats of Maharashtra, India. The region is one of its kinds being a biodiversity hot spot declared by UNESCO representing rich biodiversity of Western Ghats in India. However, insect biodiversity of this region has not been studied in detail so far. Thus, the present study was aimed at identification of insect community based on mitochondrial gene, cytochrome c oxidase subunit I (MT-CO1), for quick and reliable identification. During a collection trip, several insect specimens were collected, which belonged to seven insect orders, viz., Lepidoptera, Coleoptera, Hemiptera, Hymenoptera, Diptera, Orthoptera and Thysanoptera. Based on their morphological characteristics, specimens collected were delineated in to various orders and families. This resulted in determination of possible 15 different insect species, of which 7 could be identified up to species level. Remaining 8 sequences were matched with existing GenBank database that was > 96%, therefore, were considered as putative species. One specimen could be identified up to genus level, viz., Cicindela sp. and one up to family level - Pentatomidae and six up to order level only, i.e., Coleoptera, Diptera, Lepidoptera, Orthoptera and Thysanoptera (2 specimens). The results suggested that 50% of the community could be identified to species level with MT-CO1 gene and at least about 8 specimens could possibly be new species for India. The insects thus collected from Kaas plateau were molecularly identified and at least 50% of collections were delineated to species level on the basis of their DNA barcodes for the first time.
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