Following with the release of the first pea aphid genome in 2010, the aphid community is gearing up for generating genome information for additional aphid species such as green peach aphid, green bug, Russian wheat aphid, potato and cotton aphid, and the pursuit of high-resolution comparative genomic and evolutionary analyses . However, a comprehensive view of the molecular profile of grain aphid, a diverged species from pea aphid with host plants adaptation mainly to cereal crops has not been documented so far. The alimentary canals of aphids play a crucial role in ingestion and digestion. This study compared two transcriptomic profiles of the alimentary canals of grain aphid, either with, or without the influence of feeding on wheat plants. 5490 unigenes were found to be differentially expressed, with 2918 genes up-regulated and 2572 genes down-regulated (See Additional file 1). While there were no significantly enriched cellular components showing differential expression, the genes involved in three molecular functions were found to be enriched upon feeding such as NADH dehydrogenase activity, oxidoreductase activity, and genes involved in eight biological processes were significantly enriched upon feeding on wheat plants such as generation of precursor metabolites and energy, oxidation-reduction process, energy derivation by oxidation of organic compounds and etc. (See Additional file 2). Furthermore, among the 2021 unigenes involved in ‘Metabolic pathways’, 420 showed differential expression, which accounted for 18.09% of total DEGs (2322) with pathway annotation (See Additional file 3). These results suggested that upon feeding on wheat plants, these diversified genes and/or pathways played important roles in nutrition ingestion and digestion in aphid. The obtained transcriptome profile of the alimentary canal of grain aphid upon feeding on wheat plants facilitates our understanding of the molecular mechanisms underlying feeding, ingestion and digestion.
For species without sufficient genomic information, transcriptome profiling through RNA-seq could provide a mass-screening approach to identify candidate genes for RNAi targeting for potential application in pest insect control . In this study, we chose the candidate RNAi targets based on comparison of the transcriptome profiling data of the alimentary canals of grain aphids pre- and post feeding on wheat plants. Among 5490 differentially expressed unigenes detected in grain aphid upon feeding, 16 candidate genes which were highly expressed (based on their RPKM values) in both Chuli and Duizhao, or only expressed in one treatment, were selected for the dsRNA artificial diet feeding assay (Table 2). Of these, 5 were ultimately identified as effective potential RNAi targets (Figure 6 and Figure 7). Successful and unsuccessful RNAi experiments have been also observed in a number of lepidopteran species . In lepidoptera, out of 130 genes used for the dsRNA artificial diet feeding analysis, only 38% were silenced at high levels while 48% and 14% of the genes failed to be silenced or were silenced at low levels, respectively. The reason for the low rate of silencing may be due to the efficiency of RNAi-mediated knockdown appears to depend on the identity and nature of the target gene. The type of gene to be silenced can significantly affect the outcome of an RNAi experiment . The susceptibility of different targets to RNAi effects also shows considerable variation in model species . Some targets have proved to be completely refractory to suppression as observed in most of the neuronal expressed genes in C. elegans. In this study, we observed the same phenomena and noticed that not all the dsRNAs of the candidate unigenes tested could lead to the knock-down of target genes and the developmental stunting or death of aphids. Among the selected 16 up- and down- regulated genes, only 5 of them were effective RNAi targets (Figure 6). As indicated in Table 2, except for unigenes 23028 and 29698 which only expressed in Chuli or Duizhao, respectively, the rest of RNAi target genes such as 21088, 21789 and 28469 were highly expressed in the alimentary canal in either Chuli or Duizhao or both treatments (Table 2). This suggested that the dsRNAs of highly expressed genes involved in ingestion and digestion might achieve more effective knock-down or silencing of the target genes and higher mortality of aphid.
Furthermore, for potential RNAi target selection in plant-mediated RNAi for aphid control, we need to keep in mind that the silencing must be highly specific for the intended target gene. The risk of unintended cross-species silencing would be a major biosafety concern in the future application of RNAi mediated aphid resistance. It is therefore obvious to select highly insect-specific genes with no good match to sequences in non-target organisms such as the donor plants for engineering, the natural enemies of the target pest or humans and animals that may consume the crop as food or feed. Of the five effective RNAi targets identified in this study, three unigenes 21789, 28469 and 29698 had no orthologs identified (Table 2), suggesting they were novel genes/transcripts identified in grain aphid for the first time, and could be potential RNAi target genes in managing aphid resistance in transgenic wheat plants. Therefore, massive screening and careful selection of the RNAi target genes would be essential for the future application of plant-mediated RNAi for aphid control.
In artificial feeding assays, the concentrations of dsRNAs may be a determing factor on the final RNAi effect, either on in vitro artificial feeding assay or in planta through plant-mediated RNAi for aphid control. The occurrence of RNAi effect either through feeding or injection depends on both the gene targeted and the insect species investigated. For example, a great variation exists among different lepidopteran species with respect to their sensitivity to systemic RNAi and variable levels of silencing can occur at very different concentrations of dsRNA. It is not true that exceeding the optimal concentration results in more silencing [25, 38, 39]. In a few species, including H. cecropia, Antheraea pernyi and M. sexta, high levels of silencing can be achieved by application of very low amounts of dsRNA (less than 10 ng per mg tissue in injection experiment) [40–42]. For the dsRNA feeding experiments, 15 insect species (representing 7 different orders), were investigated, and the amount of dsRNA applied varied from 5.4 ng/cm2 to 80 μg (no indication of the concentration) . In pea aphid, lethal effects were achieved with different concentrations of dsRNA or SiRNA used for injection or feeding. For example, each aphid was injected with 5 nl C002 SiRNA (10 μg/μl) led to knockdown of C002 gene . So far, dose–response relationships using lower concentration of dsRNA to establish the sensitivity to RNAi has not been reported in insects . In this study, by using SaC002 as a positive control, a clear dose–response relationship was established between the relative lower concentration of dsRNA and the aphid’s sensitivity to RNAi in artificial diet feeding bioassay in grain aphid (Figure 5). Based on this experiment, when dsRNAs were added at a concentration of 7.5 ng/μl, a lethal effect had been achieved for 5 potential RNAi target genes, with significant effects on mortality observed 4 d after feeding with dsRNAs targeting unigenes such as 23028, 29698 and 2 d for 28469, which performed even better than C002 dsRNA (Figure 6). The lethal efficacy of the relative low concentration of dsRNA (7.5 ng/μl) observed in this study will not only maintain the minimal risk of non-specific effects but also facilitate the application of plant-mediated RNAi silencing of these target genes for aphid control in agricultural practice.
Systemic RNAi encompasses both cell-autonomous and environmental RNAi in which the silencing signal is transported from the cell in which the dsRNA is applied or expressed to other cells or tissues . Small RNA pathways are highly conserved in animals including aphids [12, 43]. However, although orthologs of RNA-dependent RNA polymerase (RdRP) are present in nematodes, plants and higher animals, the presence of RdRP was never confirmed in insects [20, 28, 44]. Therefore, the absence of dsRNA amplification and RdRP in insects suggests that gene knockdown effects exhibited by injecting and/or feeding dsRNA to insects would be temporary, limited to cells that have taken up dsRNA and would require continuous input of dsRNA to persist, or in other words, systemic RNAi probably does not exist in insects [9, 45]. Nevertheless, systemic RNAi has been demonstrated in some insect species, such as Hyalophora cecropia and B. mori, in which injection of dsRNA into the pupa can result in phenotypic effects in developing embryos, indicating dsRNA uptake by the developing oocytes of the pupa, and knockdown of a gene expressed in adult antenna of light brown apple moth (Epiphyas postvittana) could be achieved through feeding dsRNA to larvae, demonstrating a persistence of the RNAi signal throughout the larval and adult stages and systemic spread of RNAi signal from the gut to the antennae [38, 46]. Successful knockdown of target genes through RNAi was also observed in pea and peach aphid [22–25], however, no direct evidence for the existence of systemic RNAi has ever been presented in aphid species. Given that MpC002 expression, which occurs predominantly in the salivary glands, is knocked down by up to 60% in peach aphid upon feeding on dsRNA transgenic tobacco plants, the silencing signal appears to spread between organs in aphid species . In this study, CTP labelled with Cy3 was added during the synthesis of dsRNA designed to unigene 23028 and the long-lasting RNAi effect was observed. The fluorescence signal were observed first in the mouthparts, and then centralized in the midgut and finally it spread through the whole body (Figure 8). BLAST analysis of the transcriptome data obtained in this study against the public databases revealed the presence of some RNAi core machinery elements such as Argonaute-2B, Dicer-1, SID-1 and also TAR RNA binding protein (TRBP), which function in assisting the RISC formation (data not shown). However, the mechanisms underpinning the spread of fluorescence signal still need to be further investigated, for example, the spread of fluorescence signal is through the aphid’s circulatory system or the in vivo amplification of siRNA, in which cells or tissues the target genes were silenced, and whether the proposed receptor mediated endocytosis or the transmembrane channel-mediated uptake are the mechanism leading to the persistence of RNAi effect.
Nevertheless, we here identified 5 novel and effective RNAi targets in grain aphid based on comparison of the transcriptome profiles of the alimentary canal of grain aphid upon feeding on wheat plant and presented the first evidence that fluorescent labelled dsRNA could be taken up through the digestive system and was not localized to the midgut (the site of dsRNA delivery) and temporally limited, could spread to the whole body tissues in grain aphid, then lead to a down-regulation/knock out of the target gene expression and finally to the development retarding and/or death of grain aphid. This laid a fundamental basis for future plant-mediated RNAi for aphid control in agriculture.