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 ³²P-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 ³²P-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.     

Inhibitor of apoptosis is an effective target gene for RNAi-mediated control of Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double-stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB-specific genes to identify targets for RNAi-mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat-killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.     

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. ³²P-labeled dsRNA injected into SGSB was processed into siRNA, but fed ³²P-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.     

RNA interference in the cat flea,Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2

Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2?days and sustained up to, at least, 7?days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3?h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.     

Transgenic soybean plants expressing Spb18S dsRNA exhibit enhanced resistance to the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae)

The soybean pod borer [SPB; Leguminivora glycinivorella (Mats.) Obraztsov] is a major soybean pest in northeastern Asia. A useful method for addressing this problem is the generation of transgenic plants producing double‐stranded RNA (dsRNA) that target essential insect genes. In this study, we confirmed that 18S ribosomal RNA is critical for SPB development. Downregulated Spb18S expression induced by dsRNA injection increased larval mortality rates and resulted in early pupation. We also assessed whether Spb18S is silenced in SPB larvae fed on transgenic soybean expressing Spb18S dsRNA. Transgenic plants downregulated Spb18S expression levels and second‐instar larval survival rates. Moreover, such plants were less damaged by SPB larvae than control plants under field conditions.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

Developmental control of Helicoverpa armigera by ingestion of bacteria expressing dsRNA targeting an arginine kinase gene

Cotton bollworm (Helicoverpa armigera) is a polyphagous pest that causes agricultural and commercial losses in many parts of the world. These losses are compounded by insecticide abuse, which leads to insecticide resistance as well as environmental and food pollution. RNA interference (RNAi) is a powerful tool used in gene functional research and RNAi-based pest control. In this study, arginine kinase (AK) of cotton bollworm was selected as the target gene, as it plays a critical role in cellular energy metabolism in invertebrates. Two fragments of the H. armigera AK gene (HarmAK) were cloned into the L4440 vector to express double-stranded RNA (dsRNA) in Escherichia coli (HT115). The effects of different factors on dsRNA stability and the effect of silencing HarmAK on cotton bollworm were subsequently investigated. Both AK gene and protein expression levels were significantly inhibited in larvae, and the peak cumulative mortality rate of 44.44% was recorded on day 5, after 2^nd instar larvae were exposed to the artificial diet coated with the engineered bacteria. The two dsRNAs (dsAK1 and dsAK2) also caused drastic reductions in body weight (38.43% and 17.37%, respectively), body length (26.73% and 11.23%, respectively) and pupation rate (48.89% and 42.95%, respectively) compared to the control on day 5. The development and morphology of the larvae, pupae and adults that fed on the dsAK1 and dsAK2 bacteria were significantly impaired, while the control was not. Thus, AK is a potential target gene for RNAi-mediated cotton bollworm control.     

Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci

In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well-formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double-strand RNA (dsRNA)-expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA-expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting “naked” dsRNA or dsRNA-expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding “naked” dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont-mediated dsRNA delivery, which we believe is the most convenient approach for large-scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.