Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells.

There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.

Protamine-Lipid-dsRNA Nanoparticles Improve RNAi Efficiency in the Fall Armyworm, Spodoptera frugiperda.

Developing safe and effective double-stranded RNA (dsRNA) delivery systems remains a major challenge for gene silencing, especially in lepidopteran insects. This study evaluated the protamine sulfate (PS)/lipid/dsRNA nanoparticle (NP) delivery system for RNA interference (RNAi) in cells and larvae of the fall armyworm (FAW), Spodoptera frugiperda, a major worldwide pest. A highly efficient gene delivery formulation was prepared using a cationic biopolymer, PS, and a cationic lipid, Cellfectin (CF), complexed with dsRNA. The NPs were prepared by a two-step self-assembly method. The formation of NPs was revealed by dynamic light scattering and transmission electron microscopy. The formation of CF/dsRNA/PS NPs was spherical in shape and size, ranging from 20 to 100 nm with a positive charge (+23.3 mV). Interestingly, prepared CF/dsRNA/PS NPs could protect dsRNA (95%) from nuclease degradation and thus significantly improve the stability of dsRNA. Formulations prepared by combining EGFP DNA with CF/PS increased transfection efficiency in Sf9 cells compared to PS/EGFP and CF/EGFP NPs. Also, the PS/CF/dsRNA NPs enhanced the endosomal escape for the intracellular delivery of dsRNA. The gene knockdown efficiency was assessed in Sf9 Luciferase (Luc) stable cells after a 72 h incubation with CF/dsRNA/PS, PS/dsRNA, CF/dsRNA, or naked dsRNA. Knockdown of the Luc gene was detected in CF/dsRNA/PS (76%) and PS/dsRNA (42.4%) not CF/dsRNA (19.5%) and naked dsRNA (10.3%) in Sf9 Luc cells. Moreover, CF/dsIAP/PS (25 µg of dsRNA targeting the inhibitor of apoptosis, IAP, gene of FAW) NPs showed knockdown of the IAP gene (39.5%) and mortality (55%) in FAW larvae. These results highlight the potential application of PS/lipid/dsRNA NPs for RNA-mediated control of insect pests.

Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in Caenorhabditis elegans requires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1 in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed CeSid1 in two Spodoptera frugiperda cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of CeSid1 enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic S. frugiperda expressing CeSid1 were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to CeSid1 expression showed tissue specificity. Compared to RNAi efficiency in wild-type S. frugiperda, CeSid1 expressing transgenic S. frugiperda showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The in vitro cell-type specific and in vivo tissue-specific enhancement of RNAi efficiency by CeSid1 in S. frugiperda provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

Development of Catechin, Poly-l-lysine, and Double-Stranded RNA Nanoparticles.

Developing strategies to optimize double-stranded RNA (dsRNA) delivery remains a significant challenge in improving RNA interference (RNAi) in insects. Nanoformulations may provide an avenue for the safe and effective delivery of dsRNA. We investigated nanoparticle-mediated gene silencing using biodegradable polymers, poly-l-lysine (PLL), and polyphenol (-)-epigallocatechin gallate (EGCG) for dsRNA delivery into Spodoptera frugiperda (Sf9) cells. Negatively charged cores were formed by EGCG and dsRNA complexes, and PLL was used to encapsulate the cores. The nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and energy-dispersive spectrometry (EDS) analysis. The stability of the nanoparticles was assessed by incubating them in nuclease-containing Sf9 cell conditioned media. The effectiveness of the nanoparticles was investigated in Sf9 cells stably expressing the luciferase gene. The results revealed that the nanoparticles formed were small and spherical. The PLL/EGCG/dsRNA nanoparticles exhibited better stability compared to that of PLL/dsRNA or naked dsRNA. Nanoparticles prepared with dsRNA targeting the luciferase gene induced an efficient knockdown (66.7%) of the target gene. In Sf9 cells, nanoparticles prepared with Cy3- or CyPHer-5E-labeled dsRNA showed higher cellular uptake and endosomal escape, respectively, than the naked dsRNA. The improvement in uptake and cytosolic delivery may have helped to increase the knockdown efficiency. In Sf9 cells, the nanoparticles prepared with dsRNA targeting the inhibitor of apoptosis gene induced apoptosis by knocking down its expression. In conclusion, we demonstrate that PLL/EGCG/dsRNA nanoparticles are stable, highly efficient, and effective in dsRNA delivery and knockdown of the target gene.

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.

Polymer-Coated Hydroxyapatite Nanocarrier for Double-Stranded RNA Delivery.

Conventional synthetic insecticides have limited success due to insect resistance and negative effects on off-target biota and the environment. Although RNA interference (RNAi) is a tool that is becoming more widely utilized in pest control products, naked dsRNA has limited success in most taxa. Nanocarriers have shown promising results in enhancing the efficacy of this tool. In this study, we used a layer-by-layer electrostatic assembly where we synthesized poly(acrylic acid) (PAA)-coated hydroxyapatite (HA) nanoparticles (PAA-HA NPs) as inorganic nanocarriers, which were then coated with a layer of a cationic poly(amino acid), 10 kDa poly-l-arginine (PLR10), to allow for binding of a layer of negatively charged dsRNA. Binding of PLR10-PAA-HA NPs to dsRNA was found to increase as the mass ratio of NPs to dsRNA increased. In vitro studies with transgenic SF9 cells (from Spodoptera frugiperda) expressing the firefly luciferase gene showed a significant gene silencing (35% decrease) at a 5:1 NP-to-dsRNA ratio, while naked dsRNA was ineffective at gene silencing. There was a significant concentration-response relationship in knockdown; however, cytotoxicity was observed at higher concentrations. Confocal microscopy studies showed that dsRNA from PLR10-PAA-HA NPs was not localized within endosomes, while naked dsRNA appeared to be entrapped within the endosomes. Overall, polymer-functionalized HA nanocarriers enabled dsRNA to elicit gene knockdown in cells, whereas naked dsRNA was not effective in causing gene knockdown.

RNAi in Spodoptera frugiperda Sf9 Cells via Nanomaterial Mediated Delivery of dsRNA: A Comparison of Poly-l-arginine Polyplexes and Poly-l-arginine-Functionalized Au Nanoparticles.

This work focused on the delivery of dsRNA either complexed with poly-l-arginine (PLR-polyplex) or loaded onto poly-l-arginine functionalized 20 nm Au nanoparticles (PLR/Au NPs). RNA interference (RNAi) of a luciferase gene expressed in Spodopteria frugiperda Sf9 stable cell line (Sf9_LUC) was used as a model system. The binding affinity of dsRNA with two modes of functionalization of Au NPs was investigated: the electrostatic binding of PLR on citrate stabilized Au NPs (e-PLR/Au NPs) via the layer-by-layer method or the covalent-linking reaction of the polymer with NHS groups on the Au NPs surface (c-PLR/Au NPs) with excess groups quenched with either hydroxylamine (c-PLR/Au/Hyd NPs) or bovine serum albumin (c-PLR/Au/BSA NPs). The formation of PLR-polyplex particles resulting from the complexation of PLR and dsRNA were revealed by transmission electron microscope (TEM), scanning transmission electron microscope (STEM), and elemental mapping by energy dispersive X-ray spectroscopy (EDS). Luciferase gene knockdown was evaluated after exposure of Sf9 cells for 72 h to 600 ng of dsRNA. Gene knockdown (GK) was found to be more efficient by exposing Sf9 cells to nanoscaled (size <100 nm) PLR-polyplex (58% GK), in contrast to microscaled (size >1 µm) PLR-polyplex (20% GK) or covalent PLR/Au/Hyd (7% GK) particles. The replacement of hydroxylamine by bovine serum albumin as ligand has significantly enhanced the efficiency of GK (31% GK). Investigation of endosomal escape, a key physiological barrier for dsRNA delivery, with CypHer5E labeled dsRNA showed the good ability for the dsRNA conjugated to the different nanosystems to enter the cells compared to the unconjugated one. This study provides valuable information concerning the required properties of materials used for dsRNA delivery in lepidopteran cells such as nanoparticle size, stability in the cell culture media, the polymer molecular weight and binding strength to the nanoparticle, and the nature of ligands on the surface.

Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of 32 P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-ß catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.

Double-stranded RNAs targeting inhibitor of apoptosis gene show no significant cross-species activity.

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double-stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species-specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.

Transport of orally delivered dsRNA in southern green stink bug, Nezara viridula.

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24-72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. 32 P-labeled dsRNA injected into SGSB was processed into siRNA, but fed 32 P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi-dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi-dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated 32 P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated 32 P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.

Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito, Aedes aegypti.

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.

Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects.

Genome-wide transcriptomic and proteomic analyses of bollworm-infested developing cotton bolls revealed the genes and pathways involved in the insect pest defence mechanism.

Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% of differentially expressed proteins were found to be involved in signalling followed by redox regulation. Further analysis showed that defence-related stress hormones and their lipid precursors, transcription factors, signalling molecules, etc. were stimulated, whereas the growth-related counterparts were suppressed during bollworm infestation. Around 26% of the significantly up-regulated proteins were defence molecules, while >50% of the significantly down-regulated were related to photosynthesis and growth. Interestingly, the biosynthesis genes for synergistically regulated jasmonate, ethylene and suppressors of the antagonistic factor salicylate were found to be up-regulated, suggesting a choice among stress-responsive phytohormone regulation. Manual curation of the enzymes and TFs highlighted the components of retrograde signalling pathways. Our data suggest that a selective regulatory mechanism directs the reallocation of metabolic resources favouring defence over growth under bollworm infestation and these insights could be exploited to develop bollworm-resistant cotton varieties.