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Recent advances in RNA interference research in insects: Implications for future insect pest management strategies
Crop Protection 45 (2013) 36e40
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Review
Recent advances in RNA interference research in insects: Implications for future insect pest management strategies Liuqi Gu, Douglas C. Knipple* Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Received in revised form 2 October 2012 Accepted 3 October 2012
The manipulation of the posttranscriptional gene silencing phenomenon known as RNA interference (RNAi), demonstrated more than a decade ago in the genetic model systems Caenorhabditis elegans Maupas (Fire et al., 1998) and Drosophila melanogaster Meigen (Kennerdell and Carthew, 1998), has provided a powerful reverse genetic tool for the elucidation of gene function. Since its discovery, myriad reports have been published describing efforts to apply RNAi approaches in insect species lacking well developed genetics or characterized genomes. Here we review recent progress in this area, focusing in particular on several recent landmark studies that demonstrate the potential practical value of this gene silencing technique for the development of new tools for the management of insect pests of agriculture. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: RNA interference Systemic RNAi High throughput screening Microbial insecticide Transgenic crop plants
1. Mechanism of RNAi The introduction of exogenous double-stranded RNA (dsRNA) into the cells of diverse eukaryotic organisms has been shown to induce rapid and sustained degradation of mRNAs containing sequences complementary to the dsRNA (Mello and Conte, 2004). This evolutionarily conserved post-transcriptional gene silencing mechanism is hypothesized to represent an active organismal response against viral infection and mobilized transposable elements, as well as playing a role in developmentally regulated translational suppression (Ding, 2010). The RNAi pathway in the cell is initiated by an RNase III enzyme called Dicer, which processes dsRNAs into short (21e25 nucleotide) small interfering RNAs (siRNAs) (Elbashir et al., 2001). These siRNAs become incorporated into a protein complex known as the RNA induced silencing complex (RISC). Once formed, the RISC is guided to a specific mRNA that is complementary to one of the strands of the siRNA causing its degradation. Argonaute protein is the major component in the RISC and mediates target recognition and cleavage (Hammond et al., 2001).
* Corresponding author. Department of Entomology, Cornell University, New York State Agricultural Experiment Station, 630 W. North Street, Geneva, New York 14456-1371, USA. Tel.: þ1 315 787 2363; fax: þ1 315 787 2326. E-mail address: [email protected] (D.C. Knipple). 0261-2194/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2012.10.004
Three types of RNAi response can be defined according to Whangbo and Hunter (2008): cell autonomous, environmental and systemic, with the latter two also referred together as non-cell autonomous RNAi. In cell autonomous RNAi the silencing effect is encompassed within the cells where dsRNA is constitutively expressed or exogenously introduced whereas in environmental RNAi silencing signal is directly picked up by cells from the immediate environment, such as gut or hemocoel. If the silencing signal spreads to neighboring cells from an epicenter of cells, then systemic RNAi is triggered. Notably, silencing signal can be takenup and passed-on even among cells where the target genes are absent (Winston et al., 2007). With regard to the effort to apply RNAi to pest management, the focus has been on non-cell autonomous RNAi. Two types of dsRNA uptake mechanisms have been identified. In Caenorhabditis elegans Maupas, the best characterized animal for RNAi, two transmembrane proteins involved in the dsRNA uptake in non-cell autonomous RNAi were identified. SID-1 (Systemic RNAi Defective) is essential and sufficient to mediate systemic spreading of RNAi signal while SID-2 is gut-specific and mainly facilitates environmental RNAi in cooperation with SID-1 (Feinberg and Hunter, 2003; McEwan et al., 2012; Winston et al., 2002, 2007). The second dsRNA uptake mechanism involves a receptormediated endocytosis pathway specific for environmental RNAi. It was first discovered in Drosophila S2 cells and later shown to also play a role in worms indicating its evolutionary conservation (Jose and Hunter, 2007; Saleh et al., 2006; Ulvila et al., 2006).
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2. Variable sensitivity of different insect species to systemic RNAi While C. elegans demonstrated a very strong RNAi response, among the thirty or so insect species in which the RNAi phenomenon has been investigated thus far, sensitivity to systemic RNAi has been found to vary considerably, with successful suppression of gene expression presumed to depend on intrinsic properties of species, as well as the genes and tissues being targeted (reviewed in Bellés (2010)). In several less derived insect species, systemic RNAi responses are quite robust, even persisting into subsequent generations via germ line transmission (Bucher et al., 2002; Liu and Kaufman, 2004a, b; Lynch and Desplan, 2006; Mito et al., 2008; Ronco et al., 2008). In contrast, some of the more derived dipteran and lepidopteran species that have been examined appear to be refractory to systemic RNAi. Responses to injected dsRNA in the Lepidoptera have been found to be particularly variable (reviewed in Terenius et al. (2011)). Among several proposed contributing factors in the susceptibility of insect species to RNAi, the stability of dsRNA after entering into the insect has been highlighted by a few recent studies. DNA/ RNAse non-specific activity distinct from that of dicer has been reported in several lepidopteran species (Allen and Walker, 2012; Arimatsu et al., 2007; Garbutt et al., 2012; Liu et al., 2012). These extracellular enzymes are secreted into various tissues and digest dsRNA. This at least partially explains the observation that in Drosophila melanogaster Meigan and lepidopterans, hemocytes are in general much easier to target for RNAi than other tissues, since dsRNAs are usually directly injected into hemolymph. More intriguingly, one study showed that for an RNAi-insensitive insect, Manduca sexta Linnaeus, exogenous dsRNA was subject to rapid degradation in hemolymph whilst for Blattella germanica Linnaeus, a phylogenetically more basal species known to be highly susceptible to RNAi, dsRNA persisted much longer (Garbutt et al., 2012). In C. elegans, RNA-dependent RNA polymerase (RdRP) amplifies the primary siRNA forming secondary dsRNAs that feed back into the front end of the RNAi pathway (Sijen et al., 2001). However, core RNAi machineries for siRNA production are not involved in systemic spreading of RNAi, and siRNA amplification is not necessary for the systemic RNAi effect (Tomoyasu et al., 2008). In general, the core RNAi machineries are conserved among all insects species examined, while RdRP homologs have never been identified, even in those showing robust systemic RNAi (Tomoyasu et al., 2008). Nonetheless, RdRP-like activity via alternative enzymes has been reported in Drosophila cells (Lipardi et al., 2005). SID-1 is a dsRNA-selective dsRNA-gated channel (Shih and Hunter, 2011) and its role in dsRNA uptake is the key to systemic spreading of RNAi in C. elegans. In insects, the presence of SID-1-like (SIL) proteins appears to vary phylogenetically, e.g., being notably absent in Dipterans (Tomoyasu et al., 2008). However, several studies cast doubts on their roles in dsRNA uptake. First, sensitivity to RNAi is not always associated with the presence of sil. For instance, the silkmoth Bombyx mori Linnaeus possesses three sid-1 orthologs but is not susceptible to experimental RNAi. However, when ectopically expressed in Bombyx cells, C. elegans SID-1 could aid dsRNA uptake thereby greatly enhance the cells’ sensitivity to RNAi (Kobayashi et al., 2012). Second, for those species with robust systemic RNAi that also possess sils, the sils are actually dispensable with regard to the RNAi effect (Luo et al., 2012; Tomoyasu et al., 2008). Further, insect sils appear to share more similarity in sequence with C. elegans tag-130, which is not involved in RNAi (Tomoyasu et al., 2008). Therefore, insects amenable to systemic RNAi must possess alternative mechanism(s) for the systemic spreading of RNAi signal.
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3. Potential of RNAi-based approaches for crop protection In insects other than D. melanogaster, research on RNAi has largely focused on the non-cell autonomous (environmental and systemic) RNAi response. Until recently, most investigations of RNAi in insects have involved delivery of in vitro synthesized dsRNAs into embryos or the hemocoel by microinjection. This method of dsRNA delivery has provided a powerful reverse genetic tool for investigating gene function in species lacking well developed genetics as well as a means to evaluate the relative sensitivity of a given species to systemic RNAi. However, microinjection is obviously not a useful means to deliver dsRNA for pest control. The potential utility of RNAi for insect pest control was suggested by two studies published in 2006 demonstrating that RNAi can be elicited in insects by oral administration of dsRNA (Araujo et al., 2006; Turner et al., 2006). In the former study, second instar nymphs of the triatomine bug Rhodnius prolixus Stål were fed dsRNAs targeting transcripts encoding the anticoagulant hemebinding protein nitrophorin 2, resulting in significantly reduced levels of anticoagulant activity in salivary glands. In the latter study third instar larvae of the light brown apple moth Epiphyas postvittana Walker were fed dsRNAs targeting transcripts encoding a larval gut enzyme and a pheromone binding protein (PBP) in adult antennae, resulting in reduced levels of both transcripts in the tissues in which they are normally expressed. The fact that PBP transcript levels were significantly reduced in adult moths demonstrated that the ingested dsRNA was not only taken up by larval midgut cells, but also was transported to cells in the eye/antennal disc, where it persisted for at least 18 days from third larval instar through adult eclosion. These two studies demonstrated that administration of dsRNA via oral route can also induce systemic RNAi. Subsequently, several studies have been published that corroborate the general utility of direct feeding of in vitro synthesized dsRNA to elicit RNAi in a variety of pest species covering a broad spectrum of different orders. Investigations in the mosquito Aedes aegypti Linnaeus provided the first demonstration that RNAi can be induced in insects by topical application of dsRNA (Pridgeon et al., 2008). In this study, expression of an inhibitor of apoptosis protein 1 gene (AaeIAP1) was suppressed by applying dsRNA diluted in acetone to the dorsal thorax of adult females producing significant mortality. Subsequently, the topical application of dsRNA was also demonstrated in the Asian corn borer Ostrinia furnacalis Guenée (Wang et al., 2011). In this study, RNAi was induced by spraying an aqueous solution of dsRNA directly onto larvae leading to developmental stunting or death. It was further shown that eggs soaked in dsRNA solutions had significantly decreased rates of hatching relative to control treatments and that fluorescently labeled dsRNA delivered to eggs persisted in larvae to reach gut, hemocytes and silk fiber. The demonstration that topical application of dsRNA could induce RNAi was quite unexpected, since it previously had been thought that oral administration was the only possible way to deliver dsRNAs to target tissues, other than injection, as the insect midgut is not protected by chitin. Assuming that the chitinous exoskeleton of the insect does, in fact, present an impervious barrier to exogenous dsRNA delivery, the induction of RNAi by topical application of dsRNA reported here could be explained by passage to interior tissues via the tracheal system. In most RNAi studies of nonmodel insects, RNAi reagents are produced through in vitro enzymatic reverse transcription or chemical synthesis. However, this is impractical for field application for pest control because of its high cost. An alternative way of inducing RNAi is to express the dsRNA in vivo via vector constructs harboring segments of target gene sequence. Recently, three such systems, mediated respectively by bacteria (Li et al., 2011; Tian
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et al., 2009; Zhu et al., 2011), host plant (Baum et al., 2007; Kumar et al., 2012; Mao et al., 2007; Pitino et al., 2011; Zha et al., 2011) and host plant virus (Kumar et al., 2012), have been successfully implemented to target the expression of insect genes. These studies employing transgene mediated RNAi represent significant progress toward developing RNAi approaches for pest management. In the first system, dsRNAs of the targeted insect genes are expressed from a plasmid with T7 promoters in inverted orientation flanking the inserted partial cDNA sequence of the target gene in an Escherichia coli Migula strain. Thus, dsRNA is produced in the bacterial cells by a process similar to in vitro synthesis. The ingestion of such bacteria expressing dsRNA has been shown to produce robust RNAi responses at both transcriptional and phenotypic levels in Spodoptera frugiperda J. E. Smith (Tian et al., 2009), Bactrocera dorsalis Hendel (Li et al., 2011), and Leptinotarsa decemlineata Say (Zhu et al., 2011). Notably, all three of these investigations showed gene silencing effects induced in tissues beyond the gut, i.e., systemic RNAi. These dsRNA expressing bacteria could potentially serve as novel biological insecticides. However, multiple applications might still be required in order to achieve effective control of insect pests. Thus, the idea of developing transgenic plants capable of inducing RNAi in insect pests has drawn considerable attention in recent years. In this system, the host plants are transformed via Agrobacterium tumefaciens Smith & Townsend with vectors carrying inverted repeats of target insect gene sequences, which when transcribed form hairpin RNAs (hpRNAs) that are functionally equivalent to linear dsRNAs. So far, this approach has been shown to effectively induce RNAi resulting in mortality in the western corn rootworm Diabrotica virgifera LeConte (Baum et al., 2007), the cotton bollworm Helicoverpa armigera Hübner (Mao et al., 2007), the tobacco hornworm M. sexta (Kumar et al., 2012) and two phloem sap feeders, the brown planthopper Nilaparvata lugens Stål (Zha et al., 2011) and the green peach aphid Myzus persicae Sulzer (Pitino et al., 2011). Notably, all five studies focused on direct silencing of gut specific genes, i.e., environmental RNAi, although the latter study also showed that the expression of a gene expressed in salivary gland but not in gut was also effectively suppressed, suggesting systemic RNAi. Although these in planta expressed insect hpRNAs were able to reduce transcript levels of targeted genes to a certain extent, the level of induction of lethal phenotypes they produced were generally lower than those obtained in the bacteria based system. The most dramatic outcome was observed in transgenic corn plants expressing hpRNA that targeted the A subunit of V-ATPase, an integral membrane proton pump expressed in the D. virgifera midgut, resulting in significant mortality and concomitant reduction in feeding damage by this pest (Baum et al., 2007). It has been observed that hpRNA expressed in planta are at least partially processed into siRNA by plant Dicer before being ingested by insects (Pitino et al., 2011; Zha et al., 2011). In both these studies, siRNAs were detected in the phloem sap of the transgenic plants. Intriguingly, knocking out the host plant dicer genes resulted in substantially higher levels of long hpRNA and exhibited more profound silencing effect in the targeted insect compared to wild-type plants, indicating that originally longer hpRNA promotes more efficient RNAi in insect than siRNA readily processed by the host plant (Kumar et al., 2012; Mao et al., 2007). Although the development of pest resistant RNAi transgenic plants seems quite promising, the production of stable transgenic lines is a time-consuming and laborious process. In plants, virusinduced gene silencing (VIGS) provides a more simple and efficient alternative to knockdown of endogenous gene expression. In VIGS, a recombinant virus is used as the silencing vector to target a host plant gene by way of triggering the antiviral RNA-silencing
pathway with little induction of viral disease symptoms in the host plant. Quite a few plant viruses have been used to develop VIGS vectors in a wide range of host plant species (Bachan and Dinesh-Kumar, 2012). A recent study provides the first instance of using VIGS to silence genes in an herbivore of the host plant (Kumar et al., 2012). In this system, Nicotiana attenuata Torr. ex S. Watson was inoculated with tobacco rattle virus as the silencing vector to target three midgut expressed cytochrome P450 (CYP) genes in M. sexta. For comparison, they also carried out plant mediated RNAi and demonstrated that the VIGS based approach gives comparable silencing effect in term of specificity and robustness. Whereas VIGS provides a more rapid process than constructing RNAi transgenic plants, its silencing effect is transient since genetic material does not integrate into the plant genome. Thus, VIGS enables high throughput screening of potential targets in insect pests, as well as permitting the investigation of a multitude of ecological questions at the molecular level (Bachan and Dinesh-Kumar, 2012). 4. High selectivity of RNAi permits species-specific gene silencing The high selectivity of RNAi is conferred by the nucleotide sequence identity of the dsRNA to its target sequence. This selectivity was elegantly demonstrated in a study in which dsRNA was made to target the expression of the E subunit of the V-ATPase midgut-expressed proton pump in D. melanogaster, M. sexta, Tribolium castaneum Herbst, and Acyrthosiphon pisum Harris (Whyard et al., 2009). Feeding of each unique dsRNA to the four species resulted in the selective killing of only the species from which the sequence of the dsRNA was matched to its target. This investigation further demonstrated that, by designing dsRNA targeting the variable 30 untranslated regions of g-tubulin genes of four Drosophila species, even orthologous genes of closely related species with high sequence identity in their coding regions can be selectively silenced by RNAi. High intraspecific specificity of RNAi was also shown using dsRNAs designed to silence three CYP genes in M. sexta (Kumar et al., 2012). In this study no off-target effect was observed even in genes sharing the highest sequence similarity with the targets. These investigations compellingly demonstrate that the RNAi response can be exploited to devise species-specific insect pest control strategies through careful target sequence selection and design of dsRNA. 5. Streamlining of high throughput target selection and screening As noted above, the variable effectiveness of dsRNAs to inhibit target gene expression in insects has been attributed not only to the relative sensitivity of a given species to systemic RNAi, but also to intrinsic properties of specific genes and gene products as well as the tissues in which they are expressed. Until recently, most experimental work attempting to identify genes of insect pest species that might be suitable candidates for future RNAi-based control strategies has involved injection of dsRNA targeting the expression of individual genes of known function. This approach is labor intensive and inefficient, insofar as it requires preexisting genomic or cDNA libraries that are not available for most nonmodel organisms, and the vast majority of potential targets are not considered. A recent landmark paper establishes methodological advances that address the above limitations (Wang et al., 2011). In this investigation, RNAseq, Illumina’s second-generation sequencing technology, was used in combination with 30 digital gene expression tag (DGE-tag) technology to characterize the expression profiles of several tens of thousands of unique tagged sequences at
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each of four developmental stages (embryonic, larval, pupal and adult) of the Asian corn borer O. furnacalis. This methodological approach is not biased toward genes of known function and is highly comprehensive. In this investigation about 1000 developmental stage-specific unique tagged sequences corresponding to expressed genes were identified at each developmental stage and their relative levels of expression measured. Remarkably, of ten abundantly expressed, larval stage-specific sequences tested for the ability of their corresponding dsRNAs to induce an RNAi response, nine produced high levels of mortality and developmental stunting following spraying of dsRNA onto newly hatched O. furnacalis larvae. This work establishes a paradigm for efficiently identifying suitable targets in pest insects for RNAi-based pest control, by combining high throughput genome-wide searching for candidate target genes and screening for optimal ones with bioassays. As a consequence of the greatly reduced cost of second generation sequencing, more transcriptomes are becoming available for a broad spectrum of species at different developmental stages and in different tissues. Although currently good quality full genome sequences are available for only a few pests of agricultural significance (such as the red flour beetle T. castaneum and the pea aphid A. pisum), in 2011 the i5k initiative was launched with the objective of sequencing the genomes of 5000 insect and related arthropod species over the following 5 years (http://arthropodgenomes.org/ wiki/i5K). This transformative project is intended to cover all insect species known to be important to worldwide agriculture, food safety, medicine, and energy production. Comprehensive genomic information will not only facilitate the selection of the most desirable targets, but will also ensure the specificity and maximal effectiveness of RNAi reagents. For example, the open source software NEXT-RNAi facilitates the automated design of dsRNAs to maximize silencing efficiency and minimize off-target effects (Horn et al., 2010). Meanwhile, genomic information will permit cross-referencing among ecologically interacting species such as predators and natural enemies in order to avoid off-target effects. Furthermore, the above mentioned methods of RNAi administration, including topical application of dsRNA, bacteria or plant virus based RNAi systems, are all amenable to streamlined high throughput screening. For RNAi screening in plants, transient transformation based on agro-infiltration of leaf discs can also be used to evaluate the system before investing the time to construct a stable transgenic line (Pitino et al., 2011). The efficient construction of transgenic RNAi plant lines has been facilitated by the development of hpRNA-expressing vectors, such as the widely used GATEWAY system including the pHELLSGATE and pIPK vectors (Helliwell and Waterhouse, 2005; Waterhouse and Helliwell, 2003; Wielopolska et al., 2005). More recently, a newly developed approach, pRNAi-GG, allows the building of an hpRNA expression construct from a single PCR product of the gene of interest by one-tube restriction-ligation and one-step transformation, further improving the cloning efficiency (Yan et al., 2012). 6. Concluding remarks The results of the recent research summarized in this review point to the tremendous potential of using RNAi approaches to develop novel management tools for the control of insect pests of agriculture. Because the core RNAi machinery is present in all insects, it is theoretically possible to devise RNAi-based management strategies for virtually any pest species by disrupting the expression of essential genes. Importantly, it appears that even for those insect species lacking a systemic RNAi response, genes expressed in the midgut are susceptible to silencing by ingested dsRNA. Future research and discovery efforts aimed at developing
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novel RNAi-based crop protection strategies should focus on identifying additional gene targets in this tissue, particularly for species lacking systemic RNAi. For insect pest species with systemic RNAi, the recent advances in high throughput screening approaches (Wang et al., 2011) permit the efficient identification of a wide variety of potential candidate target genes, the expression of which can be disrupted by RNAi to produce diverse physiological, developmental, reproductive and homeostatic effects. The enormous inventory of genes with various functions and expression profiles that can be targeted in species with systemic RNAi makes it feasible to explore the usefulness of RNAi-induced phenotypic effects other than direct mortality and developmental stunting, such as increased susceptibility to insecticides (Mao et al., 2007), disruption of host seeking behavior (Zhao et al., 2011) and infertility (Pitino et al., 2011), potentially enabling the development of multi-dimensional management strategies. A desirable feature of RNAi approaches for crop protection is the exquisite selectivity of RNAi based on the sequence identity of the dsRNA with the sequence of its target transcript. This selectivity can be exploited to devise RNAi-based pest management strategies that have no effect on non-target species, thus permitting their integration into existing integrated pest management programs. Optimization of pest management strategies based on RNAi must take into consideration potential pitfalls and limitations, most notably, the ability of a pest species to develop resistance to an RNAi-based control agent. It has been suggested that the ability of a dsRNA to produce a useful phenotypic effect could be overcome by sequence polymorphisms in the target gene of a pest population (Gordon and Waterhouse, 2007). It is therefore important to evaluate the extent of sequence polymorphism in specific target genes in pest populations and to design dsRNAs that act on large stretches of target gene sequence before investing in the development and deployment of dsRNA agents targeting their expression. It is also possible that another biochemical pathway or a paralogous gene with partially overlapping function could compensate for the loss of function of an RNAi-induced phenotype (Price and Gatehouse, 2008). The potential to develop this type of resistance can be minimized by careful design of dsRNAs targeting the expression of well understood target genes. It has also been reported that continuous feeding of dsRNA over several days induced up-regulation of some targeted genes in B. dorsalis (Li et al., 2011). Although the expression of other genes examined in the latter study were effectively suppressed by their corresponding dsRNAs, it would be desirable to conduct further investigations to elucidate the mechanism underlying the observed over-expression to determine whether it reflects intrinsic properties of these particular genes or a more general compensatory response. Other strategies to minimize the possibility of resistance development and to enhance the efficacy of RNAi-based control strategies include deployment of combinations of dsRNAs targeting different genes and the use of more than one delivery method, such as a combination of a microbial insecticide and transgenic host plants expressing insect pest dsRNAs. Although our understanding of RNAi in insects is still limited, with many knowledge gaps, recent advances suggest the exceptional promise this field holds for developing a new generation of management tools for the control of agricultural pests. References Allen, M.L., Walker, W.B., 2012. Saliva of Lygus lineolaris digests double stranded ribonucleic acids. J. Insect Physiol. 58, 391e396. Araujo, R.N., Santos, A., Pinto, F.S., Gontijo, N.F., Lehane, M.J., Pereira, M.H., 2006. RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochem. Mol. Biol. 36, 683e693.
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Terenius, O., Papanicolaou, A., Garbutt, J.S., Eleftherianos, I., Huvenne, H., Kanginakudru, S., Albrechtsen, M., An, C., Aymeric, J.L., Barthel, A., Bebas, P., Bitra, K., Bravo, A., Chevalier, F., Collinge, D.P., Crava, C.M., de Maagd, R.A., Duvic, B., Erlandson, M., Faye, I., Felföldi, G., Fujiwara, H., Futahashi, R., Gandhe, A.S., Gatehouse, H.S., Gatehouse, L.N., Giebultowicz, J.M., Gómez, I., Grimmelikhuijzen, C.J., Groot, A.T., Hauser, F., Heckel, D.G., Hegedus, D.D., Hrycaj, S., Huang, L., Hull, J.J., Iatrou, K., Iga, M., Kanost, M.R., Kotwica, J., Li, C., Li, J., Liu, J., Lundmark, M., Matsumoto, S., Meyering-Vos, M., Millichap, P.J., Monteiro, A., Mrinal, N., Niimi, T., Nowara, D., Ohnishi, A., Oostra, V., Ozaki, K., Papakonstantinou, M., Popadic, A., Rajam, M.V., Saenko, S., Simpson, R.M., Soberón, M., Strand, M.R., Tomita, S., Toprak, U., Wang, P., Wee, C.W., Whyard, S., Zhang, W., Nagaraju, J., ffrench-Constant, R.H., Herrero, S., Gordon, K., Swevers, L., Smagghe, G., 2011. RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J. Insect Physiol. 57, 231e245. Tian, H., Peng, H., Yao, Q., Chen, H., Xie, Q., Tang, B., Zhang, W., 2009. Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene. PLoS ONE 4, e6225. Tomoyasu, Y., Miller, S.C., Tomita, S., Schoppmeier, M., Grossmann, D., Bucher, G., 2008. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium. Genome Biol. 9, R10. Turner, C.T., Davy, M.W., MacDiarmid, R.M., Plummer, K.M., Birch, N.P., Newcomb, R.D., 2006. RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. Insect Mol. Biol. 15, 383e391. Ulvila, J., Parikka, M., Kleino, A., Sormunen, R., Ezekowitz, R.A., Kocks, C., Ramet, M., 2006. Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281, 14370e14375. Wang, Y., Zhang, H., Li, H., Miao, X., 2011. Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS ONE 6, e18644. Waterhouse, P.M., Helliwell, C.A., 2003. Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. Genet. 4, 29e38. Whangbo, J.S., Hunter, C.P., 2008. Environmental RNA interference. Trends Genet. 24, 297e305. Whyard, S., Singh, A.D., Wong, S., 2009. Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochem. Mol. Biol. 39, 824e832. Wielopolska, A., Townley, H., Moore, I., Waterhouse, P., Helliwell, C., 2005. A highthroughput inducible RNAi vector for plants. Plant Biotechnol. J. 3, 583e590. Winston, W.M., Molodowitch, C., Hunter, C.P., 2002. Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295, 2456e2459. Winston, W.M., Sutherlin, M., Wright, A.J., Feinberg, E.H., Hunter, C.P., 2007. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc. Natl. Acad. Sci. U. S. A. 104, 10565e10570. Yan, P., Shen, W., Gao, X., Li, X., Zhou, P., Duan, J., 2012. High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants. PLoS ONE 7, e38186. Zha, W., Peng, X., Chen, R., Du, B., Zhu, L., He, G., 2011. Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS ONE 6, e20504. Zhao, Y.Y., Liu, F., Yang, G., You, M.S., 2011. PsOr1, a potential target for RNA interference-based pest management. Insect Mol. Biol. 20, 97e104. Zhu, F., Xu, J., Palli, R., Ferguson, J., Palli, S.R., 2011. Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Manag. Sci. 67, 175e182.
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Review
Recent advances in RNA interference research in insects: Implications for future insect pest management strategies Liuqi Gu, Douglas C. Knipple* Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Received in revised form 2 October 2012 Accepted 3 October 2012
The manipulation of the posttranscriptional gene silencing phenomenon known as RNA interference (RNAi), demonstrated more than a decade ago in the genetic model systems Caenorhabditis elegans Maupas (Fire et al., 1998) and Drosophila melanogaster Meigen (Kennerdell and Carthew, 1998), has provided a powerful reverse genetic tool for the elucidation of gene function. Since its discovery, myriad reports have been published describing efforts to apply RNAi approaches in insect species lacking well developed genetics or characterized genomes. Here we review recent progress in this area, focusing in particular on several recent landmark studies that demonstrate the potential practical value of this gene silencing technique for the development of new tools for the management of insect pests of agriculture. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: RNA interference Systemic RNAi High throughput screening Microbial insecticide Transgenic crop plants
1. Mechanism of RNAi The introduction of exogenous double-stranded RNA (dsRNA) into the cells of diverse eukaryotic organisms has been shown to induce rapid and sustained degradation of mRNAs containing sequences complementary to the dsRNA (Mello and Conte, 2004). This evolutionarily conserved post-transcriptional gene silencing mechanism is hypothesized to represent an active organismal response against viral infection and mobilized transposable elements, as well as playing a role in developmentally regulated translational suppression (Ding, 2010). The RNAi pathway in the cell is initiated by an RNase III enzyme called Dicer, which processes dsRNAs into short (21e25 nucleotide) small interfering RNAs (siRNAs) (Elbashir et al., 2001). These siRNAs become incorporated into a protein complex known as the RNA induced silencing complex (RISC). Once formed, the RISC is guided to a specific mRNA that is complementary to one of the strands of the siRNA causing its degradation. Argonaute protein is the major component in the RISC and mediates target recognition and cleavage (Hammond et al., 2001).
* Corresponding author. Department of Entomology, Cornell University, New York State Agricultural Experiment Station, 630 W. North Street, Geneva, New York 14456-1371, USA. Tel.: þ1 315 787 2363; fax: þ1 315 787 2326. E-mail address: [email protected] (D.C. Knipple). 0261-2194/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2012.10.004
Three types of RNAi response can be defined according to Whangbo and Hunter (2008): cell autonomous, environmental and systemic, with the latter two also referred together as non-cell autonomous RNAi. In cell autonomous RNAi the silencing effect is encompassed within the cells where dsRNA is constitutively expressed or exogenously introduced whereas in environmental RNAi silencing signal is directly picked up by cells from the immediate environment, such as gut or hemocoel. If the silencing signal spreads to neighboring cells from an epicenter of cells, then systemic RNAi is triggered. Notably, silencing signal can be takenup and passed-on even among cells where the target genes are absent (Winston et al., 2007). With regard to the effort to apply RNAi to pest management, the focus has been on non-cell autonomous RNAi. Two types of dsRNA uptake mechanisms have been identified. In Caenorhabditis elegans Maupas, the best characterized animal for RNAi, two transmembrane proteins involved in the dsRNA uptake in non-cell autonomous RNAi were identified. SID-1 (Systemic RNAi Defective) is essential and sufficient to mediate systemic spreading of RNAi signal while SID-2 is gut-specific and mainly facilitates environmental RNAi in cooperation with SID-1 (Feinberg and Hunter, 2003; McEwan et al., 2012; Winston et al., 2002, 2007). The second dsRNA uptake mechanism involves a receptormediated endocytosis pathway specific for environmental RNAi. It was first discovered in Drosophila S2 cells and later shown to also play a role in worms indicating its evolutionary conservation (Jose and Hunter, 2007; Saleh et al., 2006; Ulvila et al., 2006).
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2. Variable sensitivity of different insect species to systemic RNAi While C. elegans demonstrated a very strong RNAi response, among the thirty or so insect species in which the RNAi phenomenon has been investigated thus far, sensitivity to systemic RNAi has been found to vary considerably, with successful suppression of gene expression presumed to depend on intrinsic properties of species, as well as the genes and tissues being targeted (reviewed in Bellés (2010)). In several less derived insect species, systemic RNAi responses are quite robust, even persisting into subsequent generations via germ line transmission (Bucher et al., 2002; Liu and Kaufman, 2004a, b; Lynch and Desplan, 2006; Mito et al., 2008; Ronco et al., 2008). In contrast, some of the more derived dipteran and lepidopteran species that have been examined appear to be refractory to systemic RNAi. Responses to injected dsRNA in the Lepidoptera have been found to be particularly variable (reviewed in Terenius et al. (2011)). Among several proposed contributing factors in the susceptibility of insect species to RNAi, the stability of dsRNA after entering into the insect has been highlighted by a few recent studies. DNA/ RNAse non-specific activity distinct from that of dicer has been reported in several lepidopteran species (Allen and Walker, 2012; Arimatsu et al., 2007; Garbutt et al., 2012; Liu et al., 2012). These extracellular enzymes are secreted into various tissues and digest dsRNA. This at least partially explains the observation that in Drosophila melanogaster Meigan and lepidopterans, hemocytes are in general much easier to target for RNAi than other tissues, since dsRNAs are usually directly injected into hemolymph. More intriguingly, one study showed that for an RNAi-insensitive insect, Manduca sexta Linnaeus, exogenous dsRNA was subject to rapid degradation in hemolymph whilst for Blattella germanica Linnaeus, a phylogenetically more basal species known to be highly susceptible to RNAi, dsRNA persisted much longer (Garbutt et al., 2012). In C. elegans, RNA-dependent RNA polymerase (RdRP) amplifies the primary siRNA forming secondary dsRNAs that feed back into the front end of the RNAi pathway (Sijen et al., 2001). However, core RNAi machineries for siRNA production are not involved in systemic spreading of RNAi, and siRNA amplification is not necessary for the systemic RNAi effect (Tomoyasu et al., 2008). In general, the core RNAi machineries are conserved among all insects species examined, while RdRP homologs have never been identified, even in those showing robust systemic RNAi (Tomoyasu et al., 2008). Nonetheless, RdRP-like activity via alternative enzymes has been reported in Drosophila cells (Lipardi et al., 2005). SID-1 is a dsRNA-selective dsRNA-gated channel (Shih and Hunter, 2011) and its role in dsRNA uptake is the key to systemic spreading of RNAi in C. elegans. In insects, the presence of SID-1-like (SIL) proteins appears to vary phylogenetically, e.g., being notably absent in Dipterans (Tomoyasu et al., 2008). However, several studies cast doubts on their roles in dsRNA uptake. First, sensitivity to RNAi is not always associated with the presence of sil. For instance, the silkmoth Bombyx mori Linnaeus possesses three sid-1 orthologs but is not susceptible to experimental RNAi. However, when ectopically expressed in Bombyx cells, C. elegans SID-1 could aid dsRNA uptake thereby greatly enhance the cells’ sensitivity to RNAi (Kobayashi et al., 2012). Second, for those species with robust systemic RNAi that also possess sils, the sils are actually dispensable with regard to the RNAi effect (Luo et al., 2012; Tomoyasu et al., 2008). Further, insect sils appear to share more similarity in sequence with C. elegans tag-130, which is not involved in RNAi (Tomoyasu et al., 2008). Therefore, insects amenable to systemic RNAi must possess alternative mechanism(s) for the systemic spreading of RNAi signal.
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3. Potential of RNAi-based approaches for crop protection In insects other than D. melanogaster, research on RNAi has largely focused on the non-cell autonomous (environmental and systemic) RNAi response. Until recently, most investigations of RNAi in insects have involved delivery of in vitro synthesized dsRNAs into embryos or the hemocoel by microinjection. This method of dsRNA delivery has provided a powerful reverse genetic tool for investigating gene function in species lacking well developed genetics as well as a means to evaluate the relative sensitivity of a given species to systemic RNAi. However, microinjection is obviously not a useful means to deliver dsRNA for pest control. The potential utility of RNAi for insect pest control was suggested by two studies published in 2006 demonstrating that RNAi can be elicited in insects by oral administration of dsRNA (Araujo et al., 2006; Turner et al., 2006). In the former study, second instar nymphs of the triatomine bug Rhodnius prolixus Stål were fed dsRNAs targeting transcripts encoding the anticoagulant hemebinding protein nitrophorin 2, resulting in significantly reduced levels of anticoagulant activity in salivary glands. In the latter study third instar larvae of the light brown apple moth Epiphyas postvittana Walker were fed dsRNAs targeting transcripts encoding a larval gut enzyme and a pheromone binding protein (PBP) in adult antennae, resulting in reduced levels of both transcripts in the tissues in which they are normally expressed. The fact that PBP transcript levels were significantly reduced in adult moths demonstrated that the ingested dsRNA was not only taken up by larval midgut cells, but also was transported to cells in the eye/antennal disc, where it persisted for at least 18 days from third larval instar through adult eclosion. These two studies demonstrated that administration of dsRNA via oral route can also induce systemic RNAi. Subsequently, several studies have been published that corroborate the general utility of direct feeding of in vitro synthesized dsRNA to elicit RNAi in a variety of pest species covering a broad spectrum of different orders. Investigations in the mosquito Aedes aegypti Linnaeus provided the first demonstration that RNAi can be induced in insects by topical application of dsRNA (Pridgeon et al., 2008). In this study, expression of an inhibitor of apoptosis protein 1 gene (AaeIAP1) was suppressed by applying dsRNA diluted in acetone to the dorsal thorax of adult females producing significant mortality. Subsequently, the topical application of dsRNA was also demonstrated in the Asian corn borer Ostrinia furnacalis Guenée (Wang et al., 2011). In this study, RNAi was induced by spraying an aqueous solution of dsRNA directly onto larvae leading to developmental stunting or death. It was further shown that eggs soaked in dsRNA solutions had significantly decreased rates of hatching relative to control treatments and that fluorescently labeled dsRNA delivered to eggs persisted in larvae to reach gut, hemocytes and silk fiber. The demonstration that topical application of dsRNA could induce RNAi was quite unexpected, since it previously had been thought that oral administration was the only possible way to deliver dsRNAs to target tissues, other than injection, as the insect midgut is not protected by chitin. Assuming that the chitinous exoskeleton of the insect does, in fact, present an impervious barrier to exogenous dsRNA delivery, the induction of RNAi by topical application of dsRNA reported here could be explained by passage to interior tissues via the tracheal system. In most RNAi studies of nonmodel insects, RNAi reagents are produced through in vitro enzymatic reverse transcription or chemical synthesis. However, this is impractical for field application for pest control because of its high cost. An alternative way of inducing RNAi is to express the dsRNA in vivo via vector constructs harboring segments of target gene sequence. Recently, three such systems, mediated respectively by bacteria (Li et al., 2011; Tian
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et al., 2009; Zhu et al., 2011), host plant (Baum et al., 2007; Kumar et al., 2012; Mao et al., 2007; Pitino et al., 2011; Zha et al., 2011) and host plant virus (Kumar et al., 2012), have been successfully implemented to target the expression of insect genes. These studies employing transgene mediated RNAi represent significant progress toward developing RNAi approaches for pest management. In the first system, dsRNAs of the targeted insect genes are expressed from a plasmid with T7 promoters in inverted orientation flanking the inserted partial cDNA sequence of the target gene in an Escherichia coli Migula strain. Thus, dsRNA is produced in the bacterial cells by a process similar to in vitro synthesis. The ingestion of such bacteria expressing dsRNA has been shown to produce robust RNAi responses at both transcriptional and phenotypic levels in Spodoptera frugiperda J. E. Smith (Tian et al., 2009), Bactrocera dorsalis Hendel (Li et al., 2011), and Leptinotarsa decemlineata Say (Zhu et al., 2011). Notably, all three of these investigations showed gene silencing effects induced in tissues beyond the gut, i.e., systemic RNAi. These dsRNA expressing bacteria could potentially serve as novel biological insecticides. However, multiple applications might still be required in order to achieve effective control of insect pests. Thus, the idea of developing transgenic plants capable of inducing RNAi in insect pests has drawn considerable attention in recent years. In this system, the host plants are transformed via Agrobacterium tumefaciens Smith & Townsend with vectors carrying inverted repeats of target insect gene sequences, which when transcribed form hairpin RNAs (hpRNAs) that are functionally equivalent to linear dsRNAs. So far, this approach has been shown to effectively induce RNAi resulting in mortality in the western corn rootworm Diabrotica virgifera LeConte (Baum et al., 2007), the cotton bollworm Helicoverpa armigera Hübner (Mao et al., 2007), the tobacco hornworm M. sexta (Kumar et al., 2012) and two phloem sap feeders, the brown planthopper Nilaparvata lugens Stål (Zha et al., 2011) and the green peach aphid Myzus persicae Sulzer (Pitino et al., 2011). Notably, all five studies focused on direct silencing of gut specific genes, i.e., environmental RNAi, although the latter study also showed that the expression of a gene expressed in salivary gland but not in gut was also effectively suppressed, suggesting systemic RNAi. Although these in planta expressed insect hpRNAs were able to reduce transcript levels of targeted genes to a certain extent, the level of induction of lethal phenotypes they produced were generally lower than those obtained in the bacteria based system. The most dramatic outcome was observed in transgenic corn plants expressing hpRNA that targeted the A subunit of V-ATPase, an integral membrane proton pump expressed in the D. virgifera midgut, resulting in significant mortality and concomitant reduction in feeding damage by this pest (Baum et al., 2007). It has been observed that hpRNA expressed in planta are at least partially processed into siRNA by plant Dicer before being ingested by insects (Pitino et al., 2011; Zha et al., 2011). In both these studies, siRNAs were detected in the phloem sap of the transgenic plants. Intriguingly, knocking out the host plant dicer genes resulted in substantially higher levels of long hpRNA and exhibited more profound silencing effect in the targeted insect compared to wild-type plants, indicating that originally longer hpRNA promotes more efficient RNAi in insect than siRNA readily processed by the host plant (Kumar et al., 2012; Mao et al., 2007). Although the development of pest resistant RNAi transgenic plants seems quite promising, the production of stable transgenic lines is a time-consuming and laborious process. In plants, virusinduced gene silencing (VIGS) provides a more simple and efficient alternative to knockdown of endogenous gene expression. In VIGS, a recombinant virus is used as the silencing vector to target a host plant gene by way of triggering the antiviral RNA-silencing
pathway with little induction of viral disease symptoms in the host plant. Quite a few plant viruses have been used to develop VIGS vectors in a wide range of host plant species (Bachan and Dinesh-Kumar, 2012). A recent study provides the first instance of using VIGS to silence genes in an herbivore of the host plant (Kumar et al., 2012). In this system, Nicotiana attenuata Torr. ex S. Watson was inoculated with tobacco rattle virus as the silencing vector to target three midgut expressed cytochrome P450 (CYP) genes in M. sexta. For comparison, they also carried out plant mediated RNAi and demonstrated that the VIGS based approach gives comparable silencing effect in term of specificity and robustness. Whereas VIGS provides a more rapid process than constructing RNAi transgenic plants, its silencing effect is transient since genetic material does not integrate into the plant genome. Thus, VIGS enables high throughput screening of potential targets in insect pests, as well as permitting the investigation of a multitude of ecological questions at the molecular level (Bachan and Dinesh-Kumar, 2012). 4. High selectivity of RNAi permits species-specific gene silencing The high selectivity of RNAi is conferred by the nucleotide sequence identity of the dsRNA to its target sequence. This selectivity was elegantly demonstrated in a study in which dsRNA was made to target the expression of the E subunit of the V-ATPase midgut-expressed proton pump in D. melanogaster, M. sexta, Tribolium castaneum Herbst, and Acyrthosiphon pisum Harris (Whyard et al., 2009). Feeding of each unique dsRNA to the four species resulted in the selective killing of only the species from which the sequence of the dsRNA was matched to its target. This investigation further demonstrated that, by designing dsRNA targeting the variable 30 untranslated regions of g-tubulin genes of four Drosophila species, even orthologous genes of closely related species with high sequence identity in their coding regions can be selectively silenced by RNAi. High intraspecific specificity of RNAi was also shown using dsRNAs designed to silence three CYP genes in M. sexta (Kumar et al., 2012). In this study no off-target effect was observed even in genes sharing the highest sequence similarity with the targets. These investigations compellingly demonstrate that the RNAi response can be exploited to devise species-specific insect pest control strategies through careful target sequence selection and design of dsRNA. 5. Streamlining of high throughput target selection and screening As noted above, the variable effectiveness of dsRNAs to inhibit target gene expression in insects has been attributed not only to the relative sensitivity of a given species to systemic RNAi, but also to intrinsic properties of specific genes and gene products as well as the tissues in which they are expressed. Until recently, most experimental work attempting to identify genes of insect pest species that might be suitable candidates for future RNAi-based control strategies has involved injection of dsRNA targeting the expression of individual genes of known function. This approach is labor intensive and inefficient, insofar as it requires preexisting genomic or cDNA libraries that are not available for most nonmodel organisms, and the vast majority of potential targets are not considered. A recent landmark paper establishes methodological advances that address the above limitations (Wang et al., 2011). In this investigation, RNAseq, Illumina’s second-generation sequencing technology, was used in combination with 30 digital gene expression tag (DGE-tag) technology to characterize the expression profiles of several tens of thousands of unique tagged sequences at
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each of four developmental stages (embryonic, larval, pupal and adult) of the Asian corn borer O. furnacalis. This methodological approach is not biased toward genes of known function and is highly comprehensive. In this investigation about 1000 developmental stage-specific unique tagged sequences corresponding to expressed genes were identified at each developmental stage and their relative levels of expression measured. Remarkably, of ten abundantly expressed, larval stage-specific sequences tested for the ability of their corresponding dsRNAs to induce an RNAi response, nine produced high levels of mortality and developmental stunting following spraying of dsRNA onto newly hatched O. furnacalis larvae. This work establishes a paradigm for efficiently identifying suitable targets in pest insects for RNAi-based pest control, by combining high throughput genome-wide searching for candidate target genes and screening for optimal ones with bioassays. As a consequence of the greatly reduced cost of second generation sequencing, more transcriptomes are becoming available for a broad spectrum of species at different developmental stages and in different tissues. Although currently good quality full genome sequences are available for only a few pests of agricultural significance (such as the red flour beetle T. castaneum and the pea aphid A. pisum), in 2011 the i5k initiative was launched with the objective of sequencing the genomes of 5000 insect and related arthropod species over the following 5 years (http://arthropodgenomes.org/ wiki/i5K). This transformative project is intended to cover all insect species known to be important to worldwide agriculture, food safety, medicine, and energy production. Comprehensive genomic information will not only facilitate the selection of the most desirable targets, but will also ensure the specificity and maximal effectiveness of RNAi reagents. For example, the open source software NEXT-RNAi facilitates the automated design of dsRNAs to maximize silencing efficiency and minimize off-target effects (Horn et al., 2010). Meanwhile, genomic information will permit cross-referencing among ecologically interacting species such as predators and natural enemies in order to avoid off-target effects. Furthermore, the above mentioned methods of RNAi administration, including topical application of dsRNA, bacteria or plant virus based RNAi systems, are all amenable to streamlined high throughput screening. For RNAi screening in plants, transient transformation based on agro-infiltration of leaf discs can also be used to evaluate the system before investing the time to construct a stable transgenic line (Pitino et al., 2011). The efficient construction of transgenic RNAi plant lines has been facilitated by the development of hpRNA-expressing vectors, such as the widely used GATEWAY system including the pHELLSGATE and pIPK vectors (Helliwell and Waterhouse, 2005; Waterhouse and Helliwell, 2003; Wielopolska et al., 2005). More recently, a newly developed approach, pRNAi-GG, allows the building of an hpRNA expression construct from a single PCR product of the gene of interest by one-tube restriction-ligation and one-step transformation, further improving the cloning efficiency (Yan et al., 2012). 6. Concluding remarks The results of the recent research summarized in this review point to the tremendous potential of using RNAi approaches to develop novel management tools for the control of insect pests of agriculture. Because the core RNAi machinery is present in all insects, it is theoretically possible to devise RNAi-based management strategies for virtually any pest species by disrupting the expression of essential genes. Importantly, it appears that even for those insect species lacking a systemic RNAi response, genes expressed in the midgut are susceptible to silencing by ingested dsRNA. Future research and discovery efforts aimed at developing
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novel RNAi-based crop protection strategies should focus on identifying additional gene targets in this tissue, particularly for species lacking systemic RNAi. For insect pest species with systemic RNAi, the recent advances in high throughput screening approaches (Wang et al., 2011) permit the efficient identification of a wide variety of potential candidate target genes, the expression of which can be disrupted by RNAi to produce diverse physiological, developmental, reproductive and homeostatic effects. The enormous inventory of genes with various functions and expression profiles that can be targeted in species with systemic RNAi makes it feasible to explore the usefulness of RNAi-induced phenotypic effects other than direct mortality and developmental stunting, such as increased susceptibility to insecticides (Mao et al., 2007), disruption of host seeking behavior (Zhao et al., 2011) and infertility (Pitino et al., 2011), potentially enabling the development of multi-dimensional management strategies. A desirable feature of RNAi approaches for crop protection is the exquisite selectivity of RNAi based on the sequence identity of the dsRNA with the sequence of its target transcript. This selectivity can be exploited to devise RNAi-based pest management strategies that have no effect on non-target species, thus permitting their integration into existing integrated pest management programs. Optimization of pest management strategies based on RNAi must take into consideration potential pitfalls and limitations, most notably, the ability of a pest species to develop resistance to an RNAi-based control agent. It has been suggested that the ability of a dsRNA to produce a useful phenotypic effect could be overcome by sequence polymorphisms in the target gene of a pest population (Gordon and Waterhouse, 2007). It is therefore important to evaluate the extent of sequence polymorphism in specific target genes in pest populations and to design dsRNAs that act on large stretches of target gene sequence before investing in the development and deployment of dsRNA agents targeting their expression. It is also possible that another biochemical pathway or a paralogous gene with partially overlapping function could compensate for the loss of function of an RNAi-induced phenotype (Price and Gatehouse, 2008). The potential to develop this type of resistance can be minimized by careful design of dsRNAs targeting the expression of well understood target genes. It has also been reported that continuous feeding of dsRNA over several days induced up-regulation of some targeted genes in B. dorsalis (Li et al., 2011). Although the expression of other genes examined in the latter study were effectively suppressed by their corresponding dsRNAs, it would be desirable to conduct further investigations to elucidate the mechanism underlying the observed over-expression to determine whether it reflects intrinsic properties of these particular genes or a more general compensatory response. Other strategies to minimize the possibility of resistance development and to enhance the efficacy of RNAi-based control strategies include deployment of combinations of dsRNAs targeting different genes and the use of more than one delivery method, such as a combination of a microbial insecticide and transgenic host plants expressing insect pest dsRNAs. Although our understanding of RNAi in insects is still limited, with many knowledge gaps, recent advances suggest the exceptional promise this field holds for developing a new generation of management tools for the control of agricultural pests. References Allen, M.L., Walker, W.B., 2012. Saliva of Lygus lineolaris digests double stranded ribonucleic acids. J. Insect Physiol. 58, 391e396. Araujo, R.N., Santos, A., Pinto, F.S., Gontijo, N.F., Lehane, M.J., Pereira, M.H., 2006. RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochem. Mol. Biol. 36, 683e693.
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