Transgenic crops have emerged as an effective tool for global insect pest management. Plants expressing cry-toxin encoding genes from the bacterium Bacillus thuringienesis (Bt) have achieved tremendous success for managing various lepidopteran and coleopteran pests [1,2]. However, Bt-based transgenic crops are not effective against hemipteran crop pests, which include aphids, leafhoppers, and whiteflies [3–5]. The inefficacy of Bt toxins to hemipteran insects has been attributed to various factors [6–11] but the exact cause remains unclear, thus providing difficult challenges for managing this group of economically important crop pests.

Employing RNA interference (RNAi) offers an alternative method for hemipteran pest control, and is expected to achieve the same level of success as Bt-based transgenic crops [12]. RNAi is based on the introduction of dsRNA which, after processing, binds to the mRNA transcript of the target gene [13]. For RNAi-based crops to be successful, the target insect must express the RNAi pathway as well as a systemic response that leads to dsRNA processing and mRNA degradation in several tissues, not just the midgut where dsRNA uptake occurs [14]. In the absence of proper genetic machinery or systemic RNAi, the resulting knockdown will have either no effect or be localized (which may or may not cause mortality).

Two parallel and closely related RNA pathways exist in eukaryotes: RNAi (also referred to as small interfering, siRNA) and micro-RNA (miRNA) pathways [15]. Both plants and invertebrates use the RNAi pathway as an innate immune system against viruses [16–18]. Alternatively, the miRNA pathway is triggered by small non-coding RNAs (ncRNAs) that regulate gene expression (reviewed in [19]). Despite their different roles in cellular processes, similar proteins are used. The proteins Dicer1, Loquacious and Ago1 are involved in the miRNA pathway whereas Dicer2, R2d2 and Ago2 are involved in the RNAi pathway. Previous investigations of the miRNA pathway genes in the pea aphid (Acyrthosiphon pisum) revealed the presence of duplications as well as positive selection and rapid evolutionary divergence [20]. While no duplications were found in RNAi pathway genes, no molecular or comparative genomic characterization was provided. In terms of RNAi-based control, the sequence characterization and gene expression of the RNAi pathway genes in aphids is necessary.

In insects, the red flour beetle, Tribolium castaneum, has emerged as the model for systemic RNAi [21,22]. However, unlike T. castaneum, Drosophila melanogaster is not very sensitive to systemic RNAi. This difference could possibly due to the gene called systemic RNA interference deficient-1 (Sid1) [23,24]. While the T. casteneum genome encodes for three Sid1 like genes, Sid1 homologs are absent in D. melanogaster. The Sid1-encoded protein is responsible for spreading the amplified signal for RNAi [25]. Studies on insect Sid1 genes are preliminary, although homologs can be found in other insect taxa [26–28]. Development of RNAi-based insect control, especially for non-model insects such as aphids, can be expedited through the understanding of RNAi pathway, including evidence for a systemic response. For example, the absence of Sid1, or its weak levels of expression in multiple tissues, would indicate a lack of systemic RNAi.

A. glycines is a major pest of soybean throughout soybean-growing regions in North America [29], causing yield losses as high as 40%. Soybean producers have adopted regular scouting and insecticidal sprays as part of their management practices, which eventually have led to a significant economic impact on soybean production [30]. Several soybean genes have been identified that provide some level of resistance to A. glycines[31]. However, A. glycines has already overcome this resistance [32], threatening the utility and sustainability of this management tactic. Novel strategies are needed to control this pest, including RNAi-based insect resistance.

Our goal, therefore, was to provide a more comprehensive characterization of the aphid RNAi machinery, including comparisons of sequence evolution and gene expression across multiple tissues. Characterization and understanding of the RNAi machinery will help develop and improve robust RNAi methods for gene knockout in A. glycines. In the current study, we have identified and characterized genes encoding the major components of RNAi machinery, specifically Dcr2, Ago2, and R2d2 from a transcriptome database [33]. Using comparative genomic, molecular evolution and phylogenetic approaches, we determined evolutionary relationships among the hemipteran RNAi genes and those from other insect orders. We also characterized Sid1, which could possibly be involved in a systemic response for RNAi-mediated gene knockdown. Additionally, we compared the expression of these genes in multiple developmental stages and tissues to evaluate the RNAi pathway activity.

In aphids, there is an evidence of extensive gene duplications and positive selection in the miRNA pathway genes, Dcr-1 and Ago-1[20]. In the current study, we did not find any evidence of duplication for RNAi pathway genes (as also reported by [20]), but did observe strong evidence for conservation and purifying selection. The strict selection and conservation seen with RNAi pathway genes may be related to their function in immune response, specifically virus defense. Aphids lack genes involved in the IMD pathway (a signaling pathway in immune response) that functions against viruses [39,40], and bacteria [41] in other insects. In fact, the analysis of A. pisum genome has revealed a highly reduced immune system in this insect [41,42], an observation which might extend to other aphids. The role of conservation of RNAi pathway genes is further supported by the ubiquitous expression of these genes in several tissues and developmental stages, which indicate the maintenance of extensively active RNAi pathway (Figure 6). Most aphids, including A. glycines, are efficient vectors and are constantly exposed to several plant viruses [43]. As a result of this constant exposure, the RNAi machinery may remain active for virus defense. Further research is needed to fully explain the role of RNAi in virus defense.

Although our sequence homology (Figures 1 and 2) and phylogenetic analyses (Figure 5) suggested conservation in major RNAi genes, this may not correlate with a robust RNAi phenotype in aphids. Previous studies reported only a transient reduction of gene expression in A. pisum midgut following dsRNA injection and feeding, respectively [44,45]. In another study, the injection of siRNAs for coo2, a gene encoding an aphid salivary protein, resulted in robust knockdown of expression and a lethal phenotype in A. pisum[46]. However, a similar response was not observed when coo2 dsRNA was fed to the green peach aphid, M. persicae, through plants [47]. This variation could either be due to a difference in delivery methods or different functions of coo2 among aphid species. Future studies to knockdown multiple genes in multiple species will better provide the definitive evidence on the RNAi response in aphids. Preliminary attempts in our laboratory using injection have led to significant mortality in control individuals, probably due to the small size of A. glycines (about 10 times smaller than the pea aphid). Feeding dsRNA through artificial diet or plants offers the best option for RNAi in this insect. Moreover, RNAi studies through feeding would translate more efficiently in the field for A. glycines control using transgenic plants expressing RNAi [48,49]. Although no such feeding assay yet exists, our characterization and gene expression analysis shows that RNAi machinery would unlikely be a limitation.

RNAi experiments in aphid species have been successful [44–47] but it is unknown if these studies led to a systemic response. Among insects that have been investigated so far, Sid1 has been found in highly diverse insect lineages such as Orthoptera, Phthiraptera, Hemiptera, Coleoptera, Lepidoptera, and Hymenoptera but is absent only in Diptera. In most of these lineages, RNAi has been demonstrated [50]. The presence of Sid1 in various aphid species [51] tends to support the occurrence of systemic RNAi. The absence of Sid1 in Diptera is surprising, given our results showing strong conservation and evidence for purifying selection with this gene. Further research into the molecular evolution of Sid1 should focus on a wide sampling of insect orders, as well as within Diptera, to determine when and how the loss of this gene occurred.

Development and success of RNAi-based transgenic crops for pest control is absolutely dependent upon a robust and systemic RNAi response in target insects. Characterization and comparison of insects’ RNAi machinery coupled with knowledge on their RNAi response may explain the molecular basis of a robust and systemic RNAi. This, in turn, will help to develop methods to derive a robust and systemic RNAi-mediated knockdown in insects. Our study will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America.

We have characterized the core components of the RNAi pathway in A. glycines, an economically important aphid species. Through comparative genetic analyses among several insect taxa, we show that these components exhibit much conservation across functionally important regions. Additionally, we have identified a putative Sid1 like cDNA in A. glycines, suggesting the presence of a functioning systemic RNAi pathway in this insect, which is a critical first step towards the development of RNAi-based functional genetic assay or novel insect control strategies.