BACKGROUND
aphids are major agricultural pests which cause significant yield losses of the crop plants each year by inflicting damage, both through the direct effects of feeding and by vectoring debilitating plant viruses  <cit> .
annual worldwide crop losses due to aphids are estimated at hundreds of millions of dollars  <cit> .
the aphid species infesting wheat in china include grain aphid , greenbug , rhopalosiphum padi linnaeus and metopolophium dirhodum walker.
of these, the grain aphid is the most dominant and destructive, affecting 65% of wheat production areas in china  <cit> .
grain aphid is also the major pest of wheat in europe and north america  <cit> .
for the last few decades, conventional breeding programs have been undertaken in an attempt to increase wheat aphid resistance worldwide  <cit> .
however, due to the lack of effective aphid resistant germplasm, the complexity of plant-aphid interactions and the rapid development of resistant pest biotypes, outbreak of aphids causing substantial losses of wheat continue to be reported regularly  <cit> .
breeders and growers are still struggling to find an efficient genetic strategy for aphid control in wheat  <cit> .
expression in transgenic plants of double strand  designed against insect target genes has been shown to give protection against pests through rna interference , opening the way for a new generation of insect-resistant crops  <cit> .
in the case of plant-mediated rnai for insect control, both cell-autonomous and non-cell-autonomous rnai are required for the persistence of rnai effect.
for cell-autonomous rnai, the silencing process is limited to the cell in which the dsrna is introduced, expressed and encompasses the rnai process within individual cells  <cit> .
the interfering effect of non-cell-autonomous rnai, can take place in tissues/cells different from the location of application or production of the dsrna.
there are two different kinds of non-cell-autonomous rnai: environmental rnai and systemic rnai.
environmental rnai describes all processes in which dsrna is taken up by a cell from the environment  <cit> .
systemic rnai refers to all processes in which the silencing signal is transported from the cell in which the dsrna is applied or expressed to other cells and tissues in which the silencing could take place  <cit> .
in multicellular organisms, environmental rnai can be followed by systemic rnai and non-cell-autonomous rnai will always be followed by cell-autonomous rnai  <cit> .
at least two mechanisms underlying rnai in insects have been described so far, that is, the transmembrane channel-mediated uptake mechanism and an alternative endocytosis-mediated uptake mechanism  <cit> .
in aphid, except the existence of sid- <dig> which is a multispan transmembrane protein mediating a systemic rnai effect, the uptake mechanism of dsrna remains to be determined  <cit> .
nevertheless, rnai-mediated knockdown of c <dig>  a gene strongly expressed in the salivary glands of a. pisum have led to death of aphids through direct injection of sirna into aphid hemolymph  <cit> .
v-atpase is a membrane-bound protein that acts as a proton pump to establish the ph gradient within the gut lumen of many insects.
knockdown of vatpase transcripts following feeding on vatpase dsrnas also led to significant mortality of a. pisum <cit> .
furthermore, injection of dsrna targeting genes encoding a calcium-binding protein calreticulin and a gut cathepsin, and feeding dsrna of a water-specific aquaporin gene in artificial diet assay led to the down-regulation and malfunction of these targeted genes in a. pisum, although the target gene expression knockdown didn’t exceed 50% and was transient, persisting for less than a week  <cit> .
so far, two cases of plant-mediated rnai for aphid control have also been reported.
silencing c <dig> gene and a gut-specific gene rack- <dig> in peach aphid resulted in the knock down of these two genes by up to 60% after feeding on transgenic tobacco and arabidopsis plants, with affected aphids producing less progeny  <cit> .
host-generated sirnas attenuated the expression of a serine proteinase gene in peach aphid, leading to a significant reduction in their fecundity and parthenogenetic population upon feeding on transgenic arabidopsis plants  <cit> .
these two cases exemplify the feasibility of plant-mediated rnai approach for aphid control in agriculture.
however, up to now, rnai and plant-mediated rnai for aphid control have not been documented in grain aphid nor has the systemic rnai effect been observed previously in aphid species.
illumina rna sequencing  enables the de novo reconstruction of the transcriptome for a non-model organism and also acts as a potentially effective way to identify rnai targets by mass-screening of candidate genes in organisms with insufficient genomic information  <cit> .
the midgut of hemipterans and most other insects is lined by the perimicrovillar membrane   <cit> .
hence, the midgut is the only portion of an insect’s body that has an active interface with the physical environment.
the cells of the midgut, which are responsible for nutrient absorption from the gut lumen, can take up dsrna and are one of the routes through which rnai could be achieved in insects  <cit> .
in this study, rna-seq was used to investigate the transcriptome profile of the alimentary canal of grain aphid upon feeding on wheat plants.
comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphid on wheat plants revealed insights into the involvement of diversified genes and/or pathways.
furthermore, the systemic rnai effect was investigated for the first time in grain aphid.
novel and effective rnai target genes were explored through a dsrna artificial diet feeding assay and candidate genes were identified that could act as potential targets for plant-mediated rnai control of aphids in wheat.
RESULTS
analyses of the differentially expressed genes in the alimentary canal of grain aphid after feeding on wheat plants revealed the involvement of diversified genes and/or pathways in ingestion and digestion
to obtain a global view of the transcriptome profiles of the alimentary canals of grain aphids upon feeding, the alimentary canals of pre- and post-feeding grain aphids which were derived from a single clonal lineage were dissected for rna isolation.
high-throughput rna-seq was performed by illumina rna sequencing in bgi-shenzhen.
after filtering out the adaptors and low quality sequences,  <dig>  and  <dig>  million clean reads with a total read-length over  <dig>  and  <dig>  billion nucleotides respectively, were obtained from the alimentary canals of grain aphids pre-feeding   and 6 h after feeding  .
these reads were subsequently assembled into contigs and then unigenes using soapdenovo software.
in total,  <dig>  and  <dig>  unigenes were obtained for chuli and duizhao with a mean length of  <dig> and 493 bp, respectively.
the unigenes from the two samples were then clustered using tgi clustering tools   <cit>  and further assembled with phrap assembly program , resulting in a total of  <dig>  unigenes with mean length of 664 bp .
of these unigenes,  <dig>   had ncbi non-redundant protein database  annotations as determined by blast search.
clusters of orthologous groups , gene orthology , and kyoto encyclopedia of genes and genomes  annotations were performed to better understand the functions of these unigenes .
the expression levels of the unigenes in post-feeding  and pre-feeding  grain aphids were estimated based on rpkm  analysis  <cit> .
in total,  <dig> genes were found to be differentially expressed, among which,  <dig> genes were up-regulated whereas  <dig> genes were down-regulated  .
besides, among the differentially expressed genes , there were  <dig> genes only expressed in chuli and  <dig> genes only expressed in duizhao .
for these degs, nr, cog, go, and kegg annotations were performed.
of these  <dig> degs, only  <dig> were annotated in at least one of the above public databases .
annotation details of these degs were as listed in additional file  <dig>  for example, only  <dig> had go annotations .
whereas there were no presence of significantly enriched cellular components, three molecular functions were found to be enriched at p <  <dig>  level, including nadh dehydrogenase activity, oxidoreductase activity, acting on nadh or nadph, quinone or similar compound as acceptor, and nadh dehydrogenase  activity.
eight biological processes were significantly enriched  including organ morphogenesis, organ development, generation of precursor metabolites and energy, imaginal disc development, cellular respiration, system development, oxidation-reduction process, energy derivation by oxidation of organic compounds.
the enriched kegg pathways upon feeding were as listed in additional file  <dig>  among them, of the  <dig> unigenes involved in ‘metabolic pathways’ ,  <dig> showed differentially expressed manner, accounting for  <dig> % of total degs  with pathway annotation.
analyses of  <dig> highly up-regulated and down-regulated genes in grain aphid upon feeding and characterization of their tissue-specific expression manner
to screen for potential rnai target genes in grain aphid,  <dig> candidate genes which were highly expressed  in both chuli and duizhao, or only expressed in one treatment, were selected for further confirmation of their actual expression levels in aphids after feeding on wheat plants and dsrna artificial diet feeding assay .
the qrt-pcr analysis confirmed the degs analysis results of these selected genes in grain aphid upon feeding on wheat plants, with the relative expression levels of  <dig> unigenes  altered very significantly after feeding , whereas that of another  <dig> unigenes  also changed significantly but had a lower probability value  .
furthermore, semi-quantitative rt-pcr analyses of different aphid tissues  showed that the selected unigenes were expressed in the tested tissues at different levels except for unigenes  <dig>   <dig> and  <dig> which could not be detected in head samples .
screening for potential rnai targets using an artificial feeding assay and the effects of dsrnas on aphid development and mortality
to screen for potential rnai target genes in plant-mediated rnai for aphid control in wheat, dsrnas of the  <dig> candidate rnai target genes listed above was synthesized in vitro and incorporated into an artificial diet.
c <dig> is a gland-specific gene first isolated from the pea aphid and knock-down of this gene led to the death of pea aphid and improved peach aphid resistance in transgenic tobacco plants  <cit> .
a homologue of c <dig> from grain aphid was identified and named as sac <dig> in this study .
then, a 284 bp dsrna was in vitro synthesized and used as a positive control to test the efficacy of other candidate rnai target genes in this study.
first, in order to test the efficacy of dsrna on the mortality of aphid and its specificity, the effects of different concentration of c <dig> dsrna and gfp dsrna added to the artificial diet on the mortality of third instars of grain aphid at different time points were investigated.
as shown in figure  <dig>  compared with blank artificial diet without any dsrna , the gfp dsrna in the artificial diet had no effect on the mortality of aphids, whereas the mortality of aphids correlated with both increased concentration of c <dig> dsrna and feeding time, reaching a very significant level at  <dig> d for the concentration of 5 ng/μl, and  <dig> and/or  <dig> d for  <dig>  ng/μl.
therefore, we used the  <dig>  ng/μl concentration of dsrna to investigate the mortality of the third instars grain aphids feeding on artificial diet added with dsrna of  <dig> selected candidate rnai target genes.
as indicated in figure  <dig> and figure  <dig>  the dsrna of gfp control had no effect on the mortality of aphid, indicating the lethality mentioned above was caused by sequence-specific effect of the dsrnas rather than by the physical or chemical characteristics of dsrnas per se.
when  <dig>  ng/μl dsrna was added to the artificial diet,  <dig> d later, the mortality levels of aphids fed with dsrnas of c <dig> and unigenes  <dig>   <dig>   <dig>   <dig> and  <dig> were between 60% and 90% after correction.
these data were statistically significant when compared with control treatments without dsrna or with gfp dsrna, and led to even higher mortality and developmental stunting than the positive control c <dig> at day  <dig>  the dsrnas targeting the rest of the selected unigenes such as  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> also led to significant mortality either at day  <dig> or  <dig>  however, no big differences on mortality were found in the treatments with dsrna targeting unigenes  <dig>   <dig>   <dig>   <dig> and  <dig>  in addition, except for unigenes  <dig> and  <dig> which were predicted to encode orthologs of a cytochrome c oxidase subunit viic precursor and a zinc finger protein in pea aphid, respectively, kegg pathway analysis and blastx against the nr and apdidbase database  didn’t reveal any orthologs of  <dig>   <dig> and  <dig> unigenes, suggesting they were novel or newly annotated genes/transcripts identified in grain aphid.
to investigate the correlation of larval mortality with the relative expression level of the respective target gene and to verify that administration of dsrna does not affect the expression of household genes such as actin, five candidate rnai target genes including the unigene  <dig> which displayed highly up-regulated profile upon feeding on wheat plants  and other four unigenes  <dig>   <dig>   <dig> and  <dig>  were selected to detect their relative expression levels upon feeding on artificial diet added with the respective dsrnas at different time point by using the aphid actin gene as internal control.
the dsrnas were supplied to the artificial diet at concentration of  <dig>  ng/μl.
qrt-pcrs were performed using the total rna from the surviving instars at different time points after feeding on the artificial diet.
the relative expression levels of  <dig> rnai target genes were monitored at indicated time point in an  <dig> d period.
as indicated in figure  <dig>  the expression of unigenes  <dig>   <dig> and  <dig> was knocked down significantly after dsrna feeding at day  <dig>  with the latter two genes completely silenced at day  <dig> .
the expression levels of unigenes  <dig> and  <dig> increased 2 days after dsrna feeding, and decreased in the following days, with that of unigene  <dig> knocked down significantly at day  <dig> too .
these results indicated that the mortality and developmental stunting caused by feeding these dsrnas was due to the down-regulation and even silencing of the respective target gene.
combined with the results of mortality analysis, we would like to propose that unigenes  <dig>   <dig>   <dig>   <dig> and  <dig> would be good rnai targets for grain aphid control through plant-mediated rnai strategy in agricultural practice.
spread of fluorescence in the whole aphid body after feeding with labelled dsrna
for the artificial feeding assay, the mid-gut is the primary target organ.
thus it would be interesting to know if the silencing signal would spread from midgut to other tissues in aphid, causing systemic rnai.
to investigate the systemic long-lasting rnai effect, ctp labelled with cy <dig> was added during the dsrna synthesizing of unigene  <dig>  after removing the residual dna and single strand rna by dnase and rnase a digestion, the dsrna was purified by megacleartm columns  and supplied to the artificial diet at a concentration of  <dig>  ng/μl.
the fluorescence signals were observed in a 24 h period.
as indicated in figure  <dig>  2 h later, the fluorescence signal was mainly observed in the mouthparts, indicating the start of dsrna feeding .
three to four hours later, strong fluorescent signals were observed, mainly in the midgut with some also observed in the intestine part of the digestion system .
in the time period of dsrna artificial diet feeding experiment, namely  <dig>   <dig> or 24 h, the fluorescent signal spread around the whole body tissues .
this result showed the evidence for the first time that fluorescent labelled  <dig> dsrna could be taken up through the digestive system and was not localized to the midgut  and temporally limited.
it demonstrates that dsrna could penetrate the perimicrovillar membrane and 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 .
discussion
following with the release of the first pea aphid genome in  <dig>  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  <cit> .
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.
<dig> unigenes were found to be differentially expressed, with  <dig> genes up-regulated and  <dig> genes down-regulated .
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.
.
furthermore, among the  <dig> unigenes involved in ‘metabolic pathways’,  <dig> showed differential expression, which accounted for  <dig> % of total degs  with pathway annotation .
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  <cit> .
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  <dig> differentially expressed unigenes detected in grain aphid upon feeding,  <dig> candidate genes which were highly expressed  in both chuli and duizhao, or only expressed in one treatment, were selected for the dsrna artificial diet feeding assay .
of these,  <dig> were ultimately identified as effective potential rnai targets .
successful and unsuccessful rnai experiments have been also observed in a number of lepidopteran species  <cit> .
in lepidoptera, out of  <dig> 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  <cit> .
the susceptibility of different targets to rnai effects also shows considerable variation in model species  <cit> .
some targets have proved to be completely refractory to suppression as observed in most of the neuronal expressed genes in c. elegans <cit> .
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  <dig> up- and down- regulated genes, only  <dig> of them were effective rnai targets .
as indicated in table  <dig>  except for unigenes  <dig> and  <dig> which only expressed in chuli or duizhao, respectively, the rest of rnai target genes such as  <dig>   <dig> and  <dig> were highly expressed in the alimentary canal in either chuli or duizhao or both treatments .
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  <dig>   <dig> and  <dig> had no orthologs identified , 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  <cit> .
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   <cit> .
for the dsrna feeding experiments,  <dig> insect species , were investigated, and the amount of dsrna applied varied from  <dig>  ng/cm <dig> to  <dig> μg   <cit> .
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 c <dig> sirna  led to knockdown of c <dig> gene  <cit> .
so far, dose–response relationships using lower concentration of dsrna to establish the sensitivity to rnai has not been reported in insects  <cit> .
in this study, by using sac <dig> 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 .
based on this experiment, when dsrnas were added at a concentration of  <dig>  ng/μl, a lethal effect had been achieved for  <dig> potential rnai target genes, with significant effects on mortality observed  <dig> d after feeding with dsrnas targeting unigenes such as  <dig>   <dig> and  <dig> d for  <dig>  which performed even better than c <dig> dsrna .
the lethal efficacy of the relative low concentration of dsrna  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  <cit> .
small rna pathways are highly conserved in animals including aphids  <cit> .
however, although orthologs of rna-dependent rna polymerase  are present in nematodes, plants and higher animals, the presence of rdrp was never confirmed in insects  <cit> .
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  <cit> .
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  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  <cit> .
successful knockdown of target genes through rnai was also observed in pea and peach aphid  <cit> , however, no direct evidence for the existence of systemic rnai has ever been presented in aphid species.
given that mpc <dig> 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  <cit> .
in this study, ctp labelled with cy <dig> was added during the synthesis of dsrna designed to unigene  <dig> 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 .
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- <dig>  sid- <dig> and also tar rna binding protein , which function in assisting the risc formation .
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  <dig> 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  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.
CONCLUSIONS
in this study, we performed transcriptome assembly and gene expression analysis of the alimentary canals of pre- and post-feeding grain aphids on wheat plants using illumina’s rna sequencing.
we obtained  <dig>  unigenes which were expressed in grain aphid alimentary canal.
furthermore, of  <dig> unigenes differentially expressed upon feeding, diverse genes and/or pathways involved were identified.
moreover, among the selected  <dig> up- or down-regulated unigenes for dsrna artificial diet feeding test, the dsrnas of  <dig> unigenes led to higher mortality and developmental stunting due to the down-regulation of the target gene expression.
finally, by adding the fluorescent labelled dsrna into the artificial diet, the spread of fluorescent signal in the whole body was observed.
the obtained transcriptome profile of the alimentary canal of grain aphid upon feeding on wheat plants would facilitate our understanding the molecular mechanism underlying feeding, ingestion and digestion.
furthermore, the identified  <dig> novel and effective rnai target genes would provide a fundamental basis for aphid control in wheat through plant mediated rnai strategy.

 aphids are major agricultural pests which cause significant yield losses of the crop plants each year by inflicting damage, both through the direct effects of feeding and by vectoring debilitating plant viruses. annual worldwide crop losses due to aphids are estimated at hundreds of millions of dollars. the aphid species infesting wheat in china include grain aphid, greenbug, rhopalosiphum padi linnaeus and metopolophium dirhodum walker. of these, the grain aphid is the most dominant and destructive, affecting 65% of wheat production areas in china. grain aphid is also the major pest of wheat in europe and north america. for the last few decades, conventional breeding programs have been undertaken in an attempt to increase wheat aphid resistance worldwide. however, due to the lack of effective aphid resistant germplasm, the complexity of plant-aphid interactions and the rapid development of resistant pest biotypes, outbreak of aphids causing substantial losses of wheat continue to be reported regularly. breeders and growers are still struggling to find an efficient genetic strategy for aphid control in wheat. expression in transgenic plants of double strand designed against insect target genes has been shown to give protection against pests through rna interference, opening the way for a new generation of insect-resistant crops. in the case of plant-mediated rnai for insect control, both cell-autonomous and non-cell-autonomous rnai are required for the persistence of rnai effect. for cell-autonomous rnai, the silencing process is limited to the cell in which the dsrna is introduced, expressed and encompasses the rnai process within individual cells. the interfering effect of non-cell-autonomous rnai, can take place in tissues/cells different from the location of application or production of the dsrna. there are two different kinds of non-cell-autonomous rnai: environmental rnai and systemic rnai. environmental rnai describes all processes in which dsrna is taken up by a cell from the environment. systemic rnai refers to all processes in which the silencing signal is transported from the cell in which the dsrna is applied or expressed to other cells and tissues in which the silencing could take place. in multicellular organisms, environmental rnai can be followed by systemic rnai and non-cell-autonomous rnai will always be followed by cell-autonomous rnai. at least two mechanisms underlying rnai in insects have been described so far, that is, the transmembrane channel-mediated uptake mechanism and an alternative endocytosis-mediated uptake mechanism. in aphid, except the existence of sid- which is a multispan transmembrane protein mediating a systemic rnai effect, the uptake mechanism of dsrna remains to be determined. nevertheless, rnai-mediated knockdown of c a gene strongly expressed in the salivary glands of a. pisum have led to death of aphids through direct injection of sirna into aphid hemolymph. v-atpase is a membrane-bound protein that acts as a proton pump to establish the ph gradient within the gut lumen of many insects. knockdown of vatpase transcripts following feeding on vatpase dsrnas also led to significant mortality of a. pisum. furthermore, injection of dsrna targeting genes encoding a calcium-binding protein calreticulin and a gut cathepsin, and feeding dsrna of a water-specific aquaporin gene in artificial diet assay led to the down-regulation and malfunction of these targeted genes in a. pisum, although the target gene expression knockdown didn’t exceed 50% and was transient, persisting for less than a week. so far, two cases of plant-mediated rnai for aphid control have also been reported. silencing c gene and a gut-specific gene rack- in peach aphid resulted in the knock down of these two genes by up to 60% after feeding on transgenic tobacco and arabidopsis plants, with affected aphids producing less progeny. host-generated sirnas attenuated the expression of a serine proteinase gene in peach aphid, leading to a significant reduction in their fecundity and parthenogenetic population upon feeding on transgenic arabidopsis plants. these two cases exemplify the feasibility of plant-mediated rnai approach for aphid control in agriculture. however, up to now, rnai and plant-mediated rnai for aphid control have not been documented in grain aphid nor has the systemic rnai effect been observed previously in aphid species. illumina rna sequencing enables the de novo reconstruction of the transcriptome for a non-model organism and also acts as a potentially effective way to identify rnai targets by mass-screening of candidate genes in organisms with insufficient genomic information. the midgut of hemipterans and most other insects is lined by the perimicrovillar membrane. hence, the midgut is the only portion of an insect’s body that has an active interface with the physical environment. the cells of the midgut, which are responsible for nutrient absorption from the gut lumen, can take up dsrna and are one of the routes through which rnai could be achieved in insects. in this study, rna-seq was used to investigate the transcriptome profile of the alimentary canal of grain aphid upon feeding on wheat plants. comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphid on wheat plants revealed insights into the involvement of diversified genes and/or pathways. furthermore, the systemic rnai effect was investigated for the first time in grain aphid. novel and effective rnai target genes were explored through a dsrna artificial diet feeding assay and candidate genes were identified that could act as potential targets for plant-mediated rnai control of aphids in wheat.  analyses of the differentially expressed genes in the alimentary canal of grain aphid after feeding on wheat plants revealed the involvement of diversified genes and/or pathways in ingestion and digestion to obtain a global view of the transcriptome profiles of the alimentary canals of grain aphids upon feeding, the alimentary canals of pre- and post-feeding grain aphids which were derived from a single clonal lineage were dissected for rna isolation. high-throughput rna-seq was performed by illumina rna sequencing in bgi-shenzhen. after filtering out the adaptors and low quality sequences, and million clean reads with a total read-length over and billion nucleotides respectively, were obtained from the alimentary canals of grain aphids pre-feeding and 6 h after feeding. these reads were subsequently assembled into contigs and then unigenes using soapdenovo software. in total, and unigenes were obtained for chuli and duizhao with a mean length of and 493 bp, respectively. the unigenes from the two samples were then clustered using tgi clustering tools and further assembled with phrap assembly program, resulting in a total of unigenes with mean length of 664 bp. of these unigenes, had ncbi non-redundant protein database annotations as determined by blast search. clusters of orthologous groups, gene orthology, and kyoto encyclopedia of genes and genomes annotations were performed to better understand the functions of these unigenes. the expression levels of the unigenes in post-feeding and pre-feeding grain aphids were estimated based on rpkm analysis. in total, genes were found to be differentially expressed, among which, genes were up-regulated whereas genes were down-regulated. besides, among the differentially expressed genes, there were genes only expressed in chuli and genes only expressed in duizhao. for these degs, nr, cog, go, and kegg annotations were performed. of these degs, only were annotated in at least one of the above public databases. annotation details of these degs were as listed in additional file for example, only had go annotations. whereas there were no presence of significantly enriched cellular components, three molecular functions were found to be enriched at p <  level, including nadh dehydrogenase activity, oxidoreductase activity, acting on nadh or nadph, quinone or similar compound as acceptor, and nadh dehydrogenase activity. eight biological processes were significantly enriched including organ morphogenesis, organ development, generation of precursor metabolites and energy, imaginal disc development, cellular respiration, system development, oxidation-reduction process, energy derivation by oxidation of organic compounds. the enriched kegg pathways upon feeding were as listed in additional file among them, of the unigenes involved in ‘metabolic pathways’, showed differentially expressed manner, accounting for % of total degs with pathway annotation. analyses of highly up-regulated and down-regulated genes in grain aphid upon feeding and characterization of their tissue-specific expression manner to screen for potential rnai target genes in grain aphid, candidate genes which were highly expressed in both chuli and duizhao, or only expressed in one treatment, were selected for further confirmation of their actual expression levels in aphids after feeding on wheat plants and dsrna artificial diet feeding assay. the qrt-pcr analysis confirmed the degs analysis results of these selected genes in grain aphid upon feeding on wheat plants, with the relative expression levels of unigenes altered very significantly after feeding, whereas that of another unigenes also changed significantly but had a lower probability value. furthermore, semi-quantitative rt-pcr analyses of different aphid tissues showed that the selected unigenes were expressed in the tested tissues at different levels except for unigenes and which could not be detected in head samples. screening for potential rnai targets using an artificial feeding assay and the effects of dsrnas on aphid development and mortality to screen for potential rnai target genes in plant-mediated rnai for aphid control in wheat, dsrnas of the candidate rnai target genes listed above was synthesized in vitro and incorporated into an artificial diet. c is a gland-specific gene first isolated from the pea aphid and knock-down of this gene led to the death of pea aphid and improved peach aphid resistance in transgenic tobacco plants. a homologue of c from grain aphid was identified and named as sac in this study. then, a 284 bp dsrna was in vitro synthesized and used as a positive control to test the efficacy of other candidate rnai target genes in this study. first, in order to test the efficacy of dsrna on the mortality of aphid and its specificity, the effects of different concentration of c dsrna and gfp dsrna added to the artificial diet on the mortality of third instars of grain aphid at different time points were investigated. as shown in figure  compared with blank artificial diet without any dsrna, the gfp dsrna in the artificial diet had no effect on the mortality of aphids, whereas the mortality of aphids correlated with both increased concentration of c dsrna and feeding time, reaching a very significant level at d for the concentration of 5 ng/μl, and and/or d for  ng/μl. therefore, we used the  ng/μl concentration of dsrna to investigate the mortality of the third instars grain aphids feeding on artificial diet added with dsrna of selected candidate rnai target genes. as indicated in figure  and figure  the dsrna of gfp control had no effect on the mortality of aphid, indicating the lethality mentioned above was caused by sequence-specific effect of the dsrnas rather than by the physical or chemical characteristics of dsrnas per se. when  ng/μl dsrna was added to the artificial diet, d later, the mortality levels of aphids fed with dsrnas of c and unigenes and were between 60% and 90% after correction. these data were statistically significant when compared with control treatments without dsrna or with gfp dsrna, and led to even higher mortality and developmental stunting than the positive control c at day the dsrnas targeting the rest of the selected unigenes such as and also led to significant mortality either at day or however, no big differences on mortality were found in the treatments with dsrna targeting unigenes and in addition, except for unigenes and which were predicted to encode orthologs of a cytochrome c oxidase subunit viic precursor and a zinc finger protein in pea aphid, respectively, kegg pathway analysis and blastx against the nr and apdidbase database didn’t reveal any orthologs of and unigenes, suggesting they were novel or newly annotated genes/transcripts identified in grain aphid. to investigate the correlation of larval mortality with the relative expression level of the respective target gene and to verify that administration of dsrna does not affect the expression of household genes such as actin, five candidate rnai target genes including the unigene which displayed highly up-regulated profile upon feeding on wheat plants and other four unigenes and were selected to detect their relative expression levels upon feeding on artificial diet added with the respective dsrnas at different time point by using the aphid actin gene as internal control. the dsrnas were supplied to the artificial diet at concentration of  ng/μl. qrt-pcrs were performed using the total rna from the surviving instars at different time points after feeding on the artificial diet. the relative expression levels of rnai target genes were monitored at indicated time point in an d period. as indicated in figure  the expression of unigenes and was knocked down significantly after dsrna feeding at day with the latter two genes completely silenced at day. the expression levels of unigenes and increased 2 days after dsrna feeding, and decreased in the following days, with that of unigene knocked down significantly at day too. these results indicated that the mortality and developmental stunting caused by feeding these dsrnas was due to the down-regulation and even silencing of the respective target gene. combined with the results of mortality analysis, we would like to propose that unigenes and would be good rnai targets for grain aphid control through plant-mediated rnai strategy in agricultural practice. spread of fluorescence in the whole aphid body after feeding with labelled dsrna for the artificial feeding assay, the mid-gut is the primary target organ. thus it would be interesting to know if the silencing signal would spread from midgut to other tissues in aphid, causing systemic rnai. to investigate the systemic long-lasting rnai effect, ctp labelled with cy was added during the dsrna synthesizing of unigene after removing the residual dna and single strand rna by dnase and rnase a digestion, the dsrna was purified by megacleartm columns and supplied to the artificial diet at a concentration of  ng/μl. the fluorescence signals were observed in a 24 h period. as indicated in figure  2 h later, the fluorescence signal was mainly observed in the mouthparts, indicating the start of dsrna feeding. three to four hours later, strong fluorescent signals were observed, mainly in the midgut with some also observed in the intestine part of the digestion system. in the time period of dsrna artificial diet feeding experiment, namely or 24 h, the fluorescent signal spread around the whole body tissues. this result showed the evidence for the first time that fluorescent labelled dsrna could be taken up through the digestive system and was not localized to the midgut and temporally limited. it demonstrates that dsrna could penetrate the perimicrovillar membrane and 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. discussion following with the release of the first pea aphid genome in 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. unigenes were found to be differentially expressed, with genes up-regulated and genes down-regulated. 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.. furthermore, among the unigenes involved in ‘metabolic pathways’, showed differential expression, which accounted for % of total degs with pathway annotation. 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 differentially expressed unigenes detected in grain aphid upon feeding, candidate genes which were highly expressed in both chuli and duizhao, or only expressed in one treatment, were selected for the dsrna artificial diet feeding assay. of these, were ultimately identified as effective potential rnai targets. successful and unsuccessful rnai experiments have been also observed in a number of lepidopteran species. in lepidoptera, out of 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 up- and down- regulated genes, only of them were effective rnai targets. as indicated in table  except for unigenes and which only expressed in chuli or duizhao, respectively, the rest of rnai target genes such as and were highly expressed in the alimentary canal in either chuli or duizhao or both treatments. 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 and had no orthologs identified, 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. 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. for the dsrna feeding experiments, insect species, were investigated, and the amount of dsrna applied varied from  ng/cm to μg. 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 c sirna led to knockdown of c 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 sac 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. based on this experiment, when dsrnas were added at a concentration of  ng/μl, a lethal effect had been achieved for potential rnai target genes, with significant effects on mortality observed d after feeding with dsrnas targeting unigenes such as and d for which performed even better than c dsrna. the lethal efficacy of the relative low concentration of dsrna 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. however, although orthologs of rna-dependent rna polymerase are present in nematodes, plants and higher animals, the presence of rdrp was never confirmed in insects. 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. 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 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. successful knockdown of target genes through rnai was also observed in pea and peach aphid, however, no direct evidence for the existence of systemic rnai has ever been presented in aphid species. given that mpc 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 cy was added during the synthesis of dsrna designed to unigene 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. 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- sid- and also tar rna binding protein, which function in assisting the risc formation. 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 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 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.  in this study, we performed transcriptome assembly and gene expression analysis of the alimentary canals of pre- and post-feeding grain aphids on wheat plants using illumina’s rna sequencing. we obtained unigenes which were expressed in grain aphid alimentary canal. furthermore, of unigenes differentially expressed upon feeding, diverse genes and/or pathways involved were identified. moreover, among the selected up- or down-regulated unigenes for dsrna artificial diet feeding test, the dsrnas of unigenes led to higher mortality and developmental stunting due to the down-regulation of the target gene expression. finally, by adding the fluorescent labelled dsrna into the artificial diet, the spread of fluorescent signal in the whole body was observed. the obtained transcriptome profile of the alimentary canal of grain aphid upon feeding on wheat plants would facilitate our understanding the molecular mechanism underlying feeding, ingestion and digestion. furthermore, the identified novel and effective rnai target genes would provide a fundamental basis for aphid control in wheat through plant mediated rnai strategy. 
furthermore, selection of aphid-specific rnai target genes would be essential for utilizing a plant-mediated rnai strategy to control aphids via a non-toxic mode of action. this finding would provide a fundamental basis for aphid control in wheat through plant mediated rnai strategy. finally, by adding fluorescently labelled dsrna into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed. furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our understanding of the molecular mechanisms underlying feeding, ingestion and digestion. the grain aphid is a major agricultural pest which causes significant yield losses of wheat in china, europe and north america annually. in this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using illumina rna sequencing. grain aphid alimentary canaltranscriptome profiledouble strand rna artificial feeding assayrna interference aphid control transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. however, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the rnai targets is a challenging task that as far as we are aware, has never been documented previously. of these, unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. furthermore, five novel and effective potential rnai target genes were identified in grain aphid for the first time.
 the grain aphid is a major agricultural pest which causes significant yield losses of wheat in china, europe and north america annually. transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. furthermore, selection of aphid-specific rnai target genes would be essential for utilizing a plant-mediated rnai strategy to control aphids via a non-toxic mode of action. however, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the rnai targets is a challenging task that as far as we are aware, has never been documented previously.  in this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using illumina rna sequencing. the transcriptome profiling generated unigenes with an average length of 664 bp. furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. based on the rpkm values of these unigenes, of them that were significantly up or down-regulated upon feeding were selected for dsrna artificial feeding assay. of these, unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. finally, by adding fluorescently labelled dsrna into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed.  comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our understanding of the molecular mechanisms underlying feeding, ingestion and digestion. furthermore, five novel and effective potential rnai target genes were identified in grain aphid for the first time. this finding would provide a fundamental basis for aphid control in wheat through plant mediated rnai strategy. grain aphid alimentary canaltranscriptome profiledouble strand rna artificial feeding assayrna interference aphid control 