Suppressing tawny crazy ant (Nylanderia fulva) by RNAi technology

A bstract The tawny crazy ant(Nylanderia fulva)is a new invasive pest in the United States.At present,its management mainly relies on the use of synthetic insecticides,which are generally ineffective at producing lasting control of the pest,necessitating alternative environmentally friendly measures.In this study,we evaluated the feasibility of gene silencing to control this ant species.Six housekeeping genes encoding actin(NfActin),coatomer subunit β (NfCOPP),arginine kinase(NfArgK),and V-type proton ATPase subunits A(NfvATPaseA),B(NfvATPaseB)and E(NfvATPaseE)were cloned.Phylogenetic analysis revealed high sequence similarity to homologs from other ant species,particularly the Florida carpenter ant(Camponotus floridanus).To silence these genes,vector L4440 was used to generate six specific RNAi constructs for bacterial expression.Heat-inactivated,dsRNA-expressing Escherichia coli were incorporated into artificial diet.Worker ants exhibited reduced endogenous gene expression after feeding on such diet for 9 d.However,only ingestion of dsRNAs of NfCOPfi(a gene involved in protein trafficking)and NfArgK(a cellular energy reserve regulatory gene in invertebrates)caused modest but significantly higher ant mortality than the control.These results suggest that bacterially expressed dsRNA can be orally delivered to ant cells as a mean to target its vulnerabilities.Improved efficacy is necessary for the RNAi-based approach to be useful in tawny crazy ant management.     

Systemic RNAi in western corn rootworm,Diabrotica virgifera virgifera,does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.     

Orally delivered dsRNA induces knockdown of target genes and mortality in the Asian long-horned beetle,Anoplophora glabripennis

The Asian long-horned beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries, including the United States. Methods available to manage or eradicate this pest are extremely limited, but RNA interference (RNAi) technology is a potentially effective method to control ALB. In this study, we used sucrose feeding bioassay for oral delivery of double-strand RNA (dsRNA) to ALB larvae. ³²P-labeled dsRNA orally delivered to ALB larvae using the sucrose droplet feeding method was processed to small interfering RNA. Feeding neonate larvae with dsRNA targeting genes coding for the inhibitor of apoptosis (IAP), vacuolar sorting protein SNF7 (SNF7), and snakeskin (SSK) induced knockdown of target genes and mortality. Feeding 2 µg of dsRNA per day for 3 days did not induce a significant decrease in the expression of target genes or mortality. However, feeding 5 or 10 µg of dsRNA per day for 3 days induced a significant decrease in the expression of target genes and 50–90% mortality. Interestingly, feeding 2.5 µg each of dsIAP plus dsSNF7, dsIAP plus dsSSK, or dsSNF7 plus dsSSK per day for 3 days induced a significant decrease in the expression of both target genes and approximately 80% mortality. Our findings demonstrate that orally delivered dsRNA induces target gene knockdown and mortality in ALB neonate larvae and RNAi technology may have the potential for effective ALB control.     

Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.     

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.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

The OST‐complex as target for RNAi‐based pest control in Nilaparvata lugens

RNAi‐based pest control strategies are emerging as environment friendly and species‐specific alternatives for the use of conventional pesticides. Because N‐glycosylation is important for many biological processes, such as growth and development, the early steps of protein N‐glycosylation are promising targets for an RNAi‐based pest control strategy. Through injection of dsRNAs, the expression of the catalytic subunits of the oligosaccharyl transferase complex was efficiently silenced in nymphs of the notorious rice pest insect Nilaparvata lugens. Silencing of both STT3 isoforms resulted in a high mortality of the N. lugens nymphs. However, our data reveals the occurrence of a functional redundancy between the two isoforms when silencing only one of the isoforms. These observations confirm the potential to use the early genes in the N‐glycosylation pathway as targets for an RNAi‐based pest control strategy. In addition, the existence of a functional redundancy between the two STT3 isoforms presents a factor which one must take into account when designing RNAi‐based approaches.     

New insights into an RNAi approach for plant defence against piercing‐sucking and stem‐borer insect pests

Insect double‐stranded (ds)RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. Arabidopsis Mob1A is required for organ growth and reproduction. When Arabidopsis roots were soaked in dsMob1A, the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty‐four hours after rice roots were immersed in fluorescent‐labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of Ago and Dicer in rice and planthoppers were induced by dsEYFP. When rice roots were soaked in dsActin, their growth was also significantly suppressed. When planthoppers or Asian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing‐sucking and stem‐borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.     

Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.     

Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

RNA interference of carboxyesterases causes nymph mortality in the Asian citrus psyllid,Diaphorina citri

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest of citrus. In addition, D. citri is the vector of Huanglongbing, a destructive disease in citrus, also known as citrus greening disease caused by Candidatus Liberibacter asiaticus. Huanglongbing causes huge losses for citrus industries. Insecticide application for D. citri is the major strategy to prevent disease spread. The heavy use of insecticides causes development of insecticide resistance. We used RNA interference (RNAi) to silence genes implicated in pesticide resistance in order to increase the susceptibility. The activity of dsRNA to reduce the expression of carboxyesterases including esterases FE4 (EstFE4) and acetylcholinesterases (AChe) in D. citri was investigated. The dsRNA was applied topically to the fourth and fifth instars of nymphs. We targeted several EstFE4 and AChe genes using dsRNA against a consensus sequence for each of them. Five concentrations (25, 50, 75, 100, 125 ng/μl) from both dsRNAs were used. The treatments with the dsRNA caused concentration dependent nymph mortality. The highest gene expression levels of both AChe and EstFE4 were found in the fourth and fifth nymphal instars. Gene expression analysis showed that AChe genes were downregulated in emerged adults from dsRNA‐AChe‐treated nymphs compared to controls. However, EstFE4 genes were not affected. In the same manner, treatment with dsRNA‐EstFE4 reduced expression level of EstFE4 genes in emerged adults from treated nymphs, but did not affect the expression of AChe genes. In the era of environmentally friendly control strategies, RNAi is a new promising venue to reduce pesticide applications.