RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within the same order. We identified homologs of key RNAi genes in the genomes of some of these insects and studied their domain architecture. These data suggest that dsRNA digestion by dsRNases and its processing to siRNAs in the cells are among the major factors contributing to differential RNAi efficiency reported among insects.

RNA interference (RNAi) is a method of gene silencing where dsRNA is digested into small interfering RNA (siRNA) in the presence of enzymes. These siRNAs then target homologous mRNA sequences aided by the RNA-induced silencing complex (RISC). The mechanism of dsRNA uptake has been well studied and established across many living organisms including insects. In insects, RNAi is a novel and potential tool to develop future pest management means targeting various classes of insects including dipterans, coleopterans, hemipterans, lepidopterans, hymenopterans and isopterans. However, the extent of RNAi in individual class varies due to underlying mechanisms. The present review focuses on three major insect classes viz hemipterans, lepidopterans and coleopterans and the rationale behind this lies in the fact that studies pertaining to RNAi has been extensively performed in these groups. Additionally, these classes harbour major agriculturally important pest species which require due attention. Interestingly, all the three classes exhibit varying levels of RNAi efficiencies with the coleopterans exhibiting maximum response, while hemipterans are relatively inefficient. Lepidopterans on the other hand, show minimum response to RNAi. This has been attributed to many facts and few important being endosomal escape, high activity dsRNA-specific nucleases, and highly alkaline gut environment which renders the dsRNA unstable. Various methods have been established to ensure safe delivery of dsRNA into the biological system of the insect. The most common method for dsRNA administration is supplementing the diet of insects via spraying onto leaves and other commonly eaten parts of the plant. This method is environment-friendly and superior to the hazardous effects of pesticides. Another method involves submergence of root systems in dsRNA solutions and subsequent uptake by the phloem. Additionally, more recent techniques are nanoparticle- and Agrobacterium-mediated delivery systems. However, due to the novelty of these biotechnological methods and recalcitrant nature of certain crops, further optimization is required. This review emphasizes on RNAi developments in agriculturally important insect species and the major hurdles for efficient RNAi in these groups. The review also discusses in detail the development of new techniques to enhance RNAi efficiency using liposomes and nanoparticles, transplastomics, microbial-mediated delivery and chemical methods.

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest.

Whitefly Bemisia tabaci is a global invasive pest that causes substantial losses to agricultural crops worldwide either by direct feeding or vectoring numerous plant viruses. Management with insecticides remains a big challenge due to its rapid resistance development potential as well as the impact of these chemicals on non-target organisms. Thus, in search of alternate and novel pest management strategies RNA interference (RNAi) has come up as potential future tool in this direction. The present study targets nine potential genes (Aquaporin (AQP), Calcitonin (CAL), CyclophilinB (CYCP), Knottin-1 (k-1), Heat shock proteins (Hsp20, Hsp40 and Hsp70), SWItch/Sucrose Non-fermentable (SNF7) and inhibitor of apoptosis (IAP) of whitefly that have been implicated to play a role in various vital physiological functions and virus transmission. The RNAi mediated knockdown efficiency of these genes has been improved through the conjugation of respective target gene dsRNA with CQD (carbon quantum dots) nanoparticles or simultaneous knockdown of dsRNA specific gut nucleases. The studies revealed that feeding of dsRNA (40 μg/ml sucrose diet) of the target gene(s) either conjugated with CQD or along with dsRNA against dsRNase (dsdsRNase) (40 μg/ml sucrose diet) enhanced the RNAi efficiency by 24–89% compared to whiteflies fed with naked dsRNA of the same target gene. The studies provide insights about the functional role of various genes in whitefly, which can possibly be exploited for the management of this pest in the future.

Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time. From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci. Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes (Dicer-2/Aubergine/Staufen) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci. The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci, which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest.