Vestigial mediated by miR-147b regulates wing development in the bird cherry-oat Rhopalosiphum padi CURRENT

Background: The wing polyphenism occurs under crowding and nutrition-deficiency conditions in most aphid species. Although the influence of environmental factors on wing polyphenism of aphids have been extensively investigated, molecular mechanisms underlining morph differentiation (i.e. wing development /degeneration) has been poorly understood. Results: We examined the expression levels of the twenty genes involved in wing patterning network, and only vestigial (vg ) showed significantly different expression levels in both whole-body and wall-body of third instar nymphs, with 5.4- and 16.14- fold higher in winged lines compared to wingless lines, respectively in Rhopalosiphum padi . Moreover, vg expressions were higher in winged aphids compared to wingless aphids at third, fourth instar nymphs and adults, and larger difference ratio were observed in third (21.38-fold) and fourth (20.91-fold) instar nymphs relative to adult (3.12-fold) between wing morphs. Suppression of vg using RNAi repressed the wing development of third winged morphs. Furthermore, dual luciferase reporter assay revealed that the miR-147 can target the vg mRNA, and modulation of miR-147b levels by microinjection of its mimics decreased vg expression levels and repressed wing development. Conclusions: Our findings suggest that vg is essential for wing development and that miR-147b modulates its expression. To our knowledge, our results provide an evidence that miRNA is involved in the regulation of wing morphs in aphids. morphs in outer morphology) in R. padi , and only vg showed significantly different expressions in both cases. The role of vg in wing development in R. padi was further investigated by vg RNAi. our results reveal that the expression of vg is regulated by miR-147b. These findings Cloning and sequence analysis of cDNA.

wing morphs. Suppression of vg using RNAi repressed the wing development of third winged morphs. Furthermore, dual luciferase reporter assay revealed that the miR-147 can target the vg mRNA, and modulation of miR-147b levels by microinjection of its mimics decreased vg expression levels and repressed wing development. Conclusions: Our findings suggest that vg is essential for wing development and that miR-147b modulates its expression. To our knowledge, our results provide an evidence that miRNA is involved in the regulation of wing morphs in aphids.

Background
Phenotypic plasticity is prevalent in organisms [1]. Polyphenism is an extreme case of phenotypic plasticity in which discrete multiple phenotypes are produced from the same genotype [2]. Most aphids exhibit wing polyphenism which winged and wingless morph are produced depending on environmental stimulus(e.g. population density and host nutrition) during the parthenogenetic generations [3]. The wingless morphs show adaptations to maximize reproduction allowing rapid colony growth. In contrast, the winged morphs engage in dispersal which enable them to seek out new habitats, mates, and food resources [4]. The bird cherry-oat aphid, Rhopalosiphum padi (L.), is one of the most globally abundant cereal aphid pests. In addition to directly feeding on plants, R. padi damages cereal crops by transmitting Barley yellow dwarf virus, which causes economically important crop losses [5,6]. R. padi, like most aphids, can produce wing polyphenism when experiencing the crowding and poor nutrition conditions [7,8]. It is easy to produce winged lines owing to its short life cycles and high reproductive rate [9].
Winged aphids are able to travel long distances and carry viruses in autumn which are considered as a major epidemiological factor for determining the disease incidence [10][11][12]. To date, the control of R. padi remains to rely on the application of chemical insecticides, which have leaded to insecticide resistance and environmental pollution [13].The fight abilities of winged forms and high fecundity of wingless forms have made aphids control more difficult. Therefore, understanding the molecular mechanisms of wing morphs is important for controlling R. padi effectively.
Generally, wing morphs include determination and differentiation processes that occur at completely different time during aphid development. Mostly, morph determination occurs during embryogenesis in the maternal ovarian cavity and morph differentiation (i.e. wing development/degeneration) occurs during postembryonic development [14]. Nowadays, the influence of external cues on wing dimorphism have been extensively investigated, especially environmentally regulated maternal hormone in aphids can mediate phenotype production of next generation in the wing-morph determination. Recently, molecular mechanisms of ecdysone signaling controlling wing morph determination was discovered in Acyrthosiphon pisum [ 15]. However, definitive molecular mechanisms in wing morph 4 differentiation have been poorly understood.
It is well established that wing development in aphids is the default developmental pathway. Specifically, all aphids are born with wing primordial, and they are degenerating by the second instar in the unwinged morph [16]. Gene networks underlying wing patterns have been well investigated in Drosophila melanogaster [17, 18], and principal wing development gene homologs are largely conserved across insects [17]. However, only in A. pisum, the expression levels of eleven genes involved in wing pattering were investigated between wing morphs, and one gene (i.e. apterous) was found to exhibit significantly high expression level. Unfortunately, the role of this gene in wing morphs was not further investigated. Therefore, the goal of the current study is to improve the understanding of whether some wing patterning genes contribute to wing development or degeneration in R. padi. Here, we depicted a gene network involved in major wing patterning events, including anterior-posterior (A-P) patterning genes such as engrailed (en), hedgehog (hh), decapentaplegic ( dpp), brinker ( brk), optomoter-blind ( omb), spalt- Also, our results reveal that the expression of vg is regulated by miR-147b. These findings 5 provide evidence that vg mediated by miR-147b regulates wing development in R. padi.

Expression profiles of wing patterning genes in wing morphs.
To determine which genes may be involved in wing differentiation during post-embryonic development in R. padi, we evaluated the expression levels of twenties known wing patterning genes (Fig. 1) between wingless and winged third instar nymphs using qRT-PCR. All genes had similar expression levels between wingless and winged whole bodies except for vg, in which expression was 5.4-fold higher in the whole bodies of winged aphids than in the wingless aphids (Fig. 2). Also, owing to wing bud extends from the body wall of thoracic part in winged line, these genes expression levels were also examined in body walls. Expression levels of vg, sal , omb and srf were 16.14-, 3.16-, 4.07-and 2.77fold higher in body walls of winged aphids relative to wingless aphids , respectively ( Fig.2C).

Expression patterns of vg in wing morphs
The expression patterns of vg were further determined in different tissues of the third instar nymphs and different developmental stages. The results showed that the expression levels of vg were the highest in the body wall of the third instar winged aphids (Fig. 3A) and the lowest in the body wall of the third instar wingless aphids (Fig. 3B). Wing development in the winged aphids is associated with various developmental stages, and the expression levels of vg were stable from the first to the second nymph stage, then increased sharply from the third nymph to the adult stage in the wingless morphs (Fig.   3C). In contrast, vg expression increased from the first to the third instar nymphs and then decreased in the adult stage in the winged morphs (Fig. 3D). Altogether, the highest expression of vg was found in the third instar nymphs, and it was 9.58-fold higher relative to the first instar nymphs, during winged nymph development.
Comparing the expression levels of vg between wingless and winged body walls with the development stages revealed interesting trends. The expression levels of vg was higher in winged aphids than in wingless aphids in the third and fourth instar nymphs as well as in adults, and higher difference ratios were observed in third (21.38-fold) and fourth (20.91fold) instar nymphs compared with the adult (3.12-fold) between wing morphs. However, the expression levels of vg had no significant difference in the first and second instar nymphs between wing morphs (Fig. 3E).

Conserved domains of vg and expression of VG protein.
We The VG protein contains the Vg_Tdu domain, which is highly conserved among holometabolous and hemimetabolous insects (Additional file 1).
To determine whether the VG protein had difference expression between wing morphs as vg mRNA did, we investigated the VG protein expression levels between wing morphs at third instar body walls. The result showed that there were higher levels of the protein in the body wall of winged aphids relative to wingless aphids (Fig. 3F).

RNAi knockdown of vg suppresses wing development.
RNAi experiments were performed to understand the relationship between wing development and vg gene expression. Third instar aphids of the winged lines were injected with dsRNA, and the mortality was 30 % (dsRNA) and 27% (dsEGFP) at 24 h following injection (n>100). In addition, at 24 h after injection with vg dsRNA, the mRNA levels of vg decreased significantly by 44% compared to control insects injected with dsEGFP ( Fig. 4A). After 48 h, 68% aphids injected by vg dsRNA (n~ 20) were under-developed wing compared to the dsEGFP control aphids, which were 100% normal wings ( Fig. 4B).

miR-147b putatively regulates the expression of vg.
To determine whether the differently expressed vg between wing morphs resulted from the vg DNA copy numbers, we investigated the vg DNA expression levels between body walls of third instar aphids. There was no significant difference in vg DNA expression levels between wing morphs (Additional file 2). miRNA predication showed a target site of miR-147b that was found in bases 877 to 899 of the ORF of vg with a high complementarity (Fig. 5A). The transcriptional levels of miR-147b in winged aphids were significantly lower than in wingless aphids ( To determine whether miR-147b can bind to vg, the predicted target sequences of vg were inserted into the pmirGLO vector to construct the recombinant vector pmirGLO-miR-147b. Firefly luciferase activity normalized against Renilla luciferase was significantly reduced when pmirGLO-miR-147b was co-transfected with the miR-147b agomir (mimic). However, the luciferase activity levels of the pmirGLOmiR-147b-mut construct were not dramatically affected by the miR-147b agomir compared with the unmutated constructs (Fig. 5C).
These results suggest that miR-147b may binds to the target sequence in the vg mRNA.

miR-147b can modulate wing development.
To verify whether the expression of vg is regulated by miR-147b, miR-147b agomir was injected into the winged third nymphs of R. padi, and we then examined the expressions 8 of miR-147b and vg after 24 h, respectively. The mortality was 28 % (miR-147b agomir) and 22% (agomir-NC) at 24 h following injection. Compared with control group, expression levels of vg was decreased by 47% after injection for 24 h. Wing development was dramatically repressed in the group injected with the miR-147b agomir, which exhibited two types of phenotypes at rates of 75% and 25% (n~20), respectively (Fig. 6D); however, wing development in the control group injected with the miRNA negative control was normal at rates of 100% after 48 h (Fig. 6C). These results demonstrated that miR-147b can affect vg expression and modulate wing development. shows higher expression levels in both the whole body and the body wall of winged lines, compared to wingless morphs, in R. padi (Fig.2). Importantly, the expression of vg shows a larger difference ratio between the two wing morphs, 16.14-fold for the body wall and 5.4fold for the whole body between the winged and wingless morphs, respectively (Fig.2). Vg expression levels were the highest in the body wall of the winged lines, while they were the lowest in the body wall of the wingless lines. These suggested vg may play a key role in wing development in aphids. The point was subsequently verified by showing that vg dsRNA can suppress wing development (Fig. 4). The higher expression levels of sal and srf were also found in the body wall of winged aphids compared to wingless aphids (Fig. 2C).

Discussion
This difference may be resulted from that vg regulate their expression because the sal 9 and srf is the downstream of vg in Drosophila (Fig 1) [30]. Also, Omb is expressed at higher levels in third nymphal instars of winged aphids relative to wingless aphids (Fig 2), Whether increased expression of omb results from higher levels of vg in winged aphids is unknown. The other sixteen genes had no significant morph effect in third nymphal instars (Fig. 2). Similar results were also observed by Brisson et al. (2010), who reported that the expression of en, hh, dpp, ubx, ap,wg, hth, and dll showed no significant differences between wing morphs of third instar nymphs in A. pisum, but did not examine expressions of vg, sal, omb, and srf Although the expression levels of en, hh, sal, wg, exd, and Ubx were found to be significantly different between macropterous (migratory) and brachypterous forms of Nilaparvata lugens [31], our study showed that there were no significant differences in the expressions of these genes between aphid morphs. There is the possibility that brachypterous N. lugens adults still have short wings, while wingless aphids have no wings because they degenerate by the second larval instar during development [31,32]. This suggests that different developmental divergence times likely require different molecular mechanisms.
All aphids are born with wing buds, and they degenerate in the unwinged morphs during the second instar. In contrast, the wing buds continue to develop in the winged morphs [17]. Our result showed the expression of vg was highest in wingless adult aphid during development, while the expression of the vg gene was highest in third instar nymphs of the winged aphids. Third instar nymphs showed the greatest difference ratios of vg expression (21.38-fold) during development compared to fourth instar nymph and adult between the wing morphs, while vg expression levels showed no significant difference in first and second instar nymphs (Fig. 3). It is worth noting that we were unable to distinguish winged from wingless first or second instar nymphs by external morphology.
The winged vs. wingless samples at these stages contain the opposite morph at 40% possibility (see methods). The different expression of vg between winged and wingless morphs at these two stages are likely underestimates of true differences.
Gene expression can be regulated by both transcriptional and post-transcriptional mechanisms. Transcriptional regulation is often determined by cis-elements located within a gene's promoter as well as by the epigenetic status of the gene and the adjacent DNA sequences [34]. The expression of vg during wing development may be regulated by wg, dpp, and su(h) interacting with vg enhancers in Drosophila [35,36] [28]. However, no differences in expression levels for wg, dpp, and su(h) between the wing morphs in our study (Fig. 2). Also, the DNA duplication of vg between the wing morphs in aphids showed no significant difference (Additional file 2). Therefore, we hypothesized that vg expression may be post-transcriptionally regulated by MicroRNAs (miRNAs). miRNAs are endogenous non-coding RNAs that post-transcriptionally regulate transcript levels and translational status of mRNA by degrading mRNA or terminating translation [37]. In addition, several reports have shown that mRNA-miRNA interactions may lead to the stabilization of mRNA [38]. miRNAs have been shown to regulate a variety of physiological and pathological processes throughout insect development including molting, metamorphosis, oogenesis, embryogenesis, behavior, and host-pathogen interactions [39]. However, few studies have investigated the potential role of miRNAs in wing polyphenism. Yang et al. (2014) found that miR-133 controls dopamine synthesis to control the production of solitarious versus gregarious forms in Locusta migratoria, however, direct evidence showing that miRNAs regulate wing development in aphids has yet to be reported. At present, there are few studies describing how miRNAs regulate expression of the vg gene. We used bioinformatics to predict that miR-147b could potentially regulate the expression of vg. In humans, miR-147b regulates some cellular effects including proliferation, migration, and 11 apoptosis [40]. Importantly, miR-147b is involved in endothelial barrier function and is a potent inducer of intestinal epithelial cell differentiation [41,42]. We found that vg expression was reduced and wing development was repressed after injecting the miR-147b mimic into winged lines at third instar nymph. This is consistent with the target experiments in which the co-transfection of miR-147b mimics with the corresponding target plasmids significantly decreased the relative luciferase activity. Our results provide direct evidence that miR-147b-meditated regulation of vg expression controls wing development in R. padi.
Although we determined here that vg plays an important role in wing development of R.
padi, wing polyphenism is involved in both initial determination and subsequent differentiation [43]. Physical contacts (tactile stimulation) caused by crowding (high density) or poor nutrition can increase aphid dispersal [4]. In Nilaparvata lugens, two insulin receptors regulate wing bud development by responding to an insulin-like peptide secreted by the brain, and produce long-winged or short-winged forms [44]. Recently, the molecular mechanisms of ecdysone signaling in the control of wing morph determination were also determined in A. pisum [ 15]. High density has no effect on the expression of either vg or miR-147b in third instar nymphs (Additional file 3). These suggest that the endocrine may regulate the miR-147b expression levels in wing morphs. Therefore, we propose a hypothesis to explain wing degeneration in wingless aphids that includes four processes; 1) environmental factors cause endocrine changes, 2) the increase in the hormone signal results in increased expression of miR-147b in wing primordia, 3) miR-147b negatively regulates expression of vg by binding to the mRNA, and 4) wing discs degenerate in the wingless lines owing to the lack of vg expression (Fig. 7). The opposite occurs in the winged lines, where vg is expressed at high levels in the wing primordia.

Conclusions 12
In summary, of the twenties genes involved in major wing patterning events, only vg shows significantly different expression levels between wingless and winged third nymphs.
The vg plays an important role in wing development confirmed by Vg RNAi. Also, vg transcription is regulated by miR-147b, which binds to its target sequence present in the vg mRNA. These results provide an evidence that vg mediated by miR-147b regulates the wing development in R. padi.

Insects and cell line culture.
Colony of Rhopalosiphum padi was collected from a wheat field at the Agricultural Experiment Station of China Agricultural University (N40°03', E116°28') in May 2005 [9].
The stock parthenogenetic colony was derived from a single apterous female from the colony and maintained > 10 generations at low density (~10 aphids per plate) to get rid of the telescoping effects of generations in which adult parthenogenetic aphids carry not only their daughters but also some of their granddaughters within them. The aphids were reared in plastic petri dishes containing wheat seedlings in a climate controlled chamber under the following conditions: a temperature of 22±1°C, relative humidity of 50±10 %, and a photoperiod of 16 h:8 h (day:night). Both wing morphs were induced by manipulating the adult density. Specifically, the stock parthenogenetic colony was divided into two groups. For the high-density [45] condition to induce the winged morph, >30 adult wingless aphids were reared on wheat seedlings in each plastic peri dish (9 cm diameter, 20 cm tall), and the induction ratio of winged aphids in next generation under HD conditions was 43.0% ± 17.4 % (n=300 ± 38.4). Under the low-density (LD) condition, only one wingless adult was reared on wheat seedlings, and 100% (n=63 ± 4.8) wingless aphids were induced. The aphids were reared in plastic petri dishes containing wheat seedlings in a climate controlled chamber under the following conditions: a temperature of 13 22±1°C, relative humidity of 50±10 %, and a photoperiod of 16 h:8 h (day:night). All of the wingless morphs used in our study were obtained from the LD condition, and the winged morphs were induced under HD conditions except for the effect of density on gene expression in which the wingless morphs from HD conditions are also used.
The mammalian HEK293T cell line was a gift from Institute of Microbiology, Chinese Academy of Sciences and maintained at 37°C under a 5% CO 2 atmosphere in DMEM highglucose medium (Gibco, Grand Island, USA) containing 10% fetal bovine serum (Gibco).

RNA extraction and cDNA synthesis.
Because the third instar is the earliest stage when the wing morphs can be distinguished by examining outer morphology and the body wall is the part where the wing buds extend.
To determine whether wing pattern genes were differently expressed between wing morphs, two types of aphid samples were prepared from third instar wingless and winged aphids for total RNA extraction: 1) whole bodies of twenty aphids, 2) various body parts (head, body wall and body cavity) of fifty aphids. Body parts were dissected from aphid under a binocular microscope. Specifically, we placed the aphid supine on a rubber tray, anchored it by carefully piercing the posterior edge of the abdomen, and used the dissecting knife cut its head as the head sample. Next, we peeled the venter of the abdomen off using the tip of another pin or knives and obtained the inside liquid tissues as the body cavity sample. The remaining part was washed in cold phosphate-buffered saline (PBS: 130 mM NaCl, 7 mM Na2HPO4•2H20, 3 mM NaH2PO4•2H2O; pH 7.0), then removed excess water using paper as the body wall sample. Here, the body wall was considered as enriching in tissues containing cells to develop wing in winged aphid. To investigate the expression levels of vg between wing morphs at different developmental stages, body walls of 20 aphids from each instar and adult in each wing morph were collected for RNA extraction.
14 Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. An additional DNaseI digestion was performed using RNase-Free DNaseI (Takara, Dalian, China). First-strand cDNA synthesis was carried out with a Reverse Transcription System (Takara) according to the manufacturer's instructions.
Small RNAs were isolated from aphids using the miRNeasy Mini Kit (Qiagen, Germany) following the manufacturer's protocol. First-strand cDNA was synthesized from 2 μg of total RNA using the miScript II RT kit (Qiagen) as directed by the manufacturer.
In order to determine if gene DNA levels contribute to the different expression levels in mRNA levels in wing morph, the genomic DNA was isolated from body wall of 20 third instar of wing morphs using DNAzol (MRC) according to the manufacturer's instructions.

Quantitative real-time PCR (qRT-PCR).
qRT-PCR was performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara, Japan). The cycling program for qRT-PCR assays for miRNA or mRNA was as follows: initial incubation at 50°C for 2 min and then at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s according to the manufacturer's protocol. Analysis of the qRT-PCR data was carried out using the 2 − ∆∆Ct method of relative quantification. As an endogenous control, the EF-1α and U6 snRNA transcripts were used to normalize the expression level of mRNA (or DNA) and miRNA, respectively [46,47]. RT-qPCR plates were set up with three cDNA biological replicates and two technical replicates of each biological replicate. Samples for three biological replicates were collected over at least two days and two plastic petri dishes for wheat aphid culture. All primers were designed based on information from a transcriptome library (PRJNA555831) of R. padi and were listed in additional file 4.

Cloning and sequence analysis of vg cDNA.
qRT-PCR results showed that vg expression levels were significantly higher in the winged aphids relative to wingless aphids, so we cloned and sequencing vg cDNA to examine its role in wing development. Specifically, total RNA from a mixed sample consisting of 60 aphids from various developmental stages and morphs was isolated as described above

RNA interference ( RNAi)
The specific primers containing a T7 polymerase promoter sequence were designed on E-RNAi (http://www.dkfz.de/signaling/e-rnai3/). The specific primers were used to amplify the fragments of vg using reverse transcription PCR (RT-PCR). A 486 bp fragment of vg was used as the template for dsRNA synthesis using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Wilmington, DE, USA) synthesis following the manufacturer's instructions. The dsRNA of enhanced green fluorescent protein (EGFP) was used as a control. All of the synthesized dsRNAs were dissolved in nuclease-free water and then quantified using a NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA), and stored at −20 °C until use.
dsRNA-vg of approximately 13.8 nL (1000 ng/μL) were injected into thorax segments of third instar winged aphids using a micro-injector (Nanoliter 2000 Injector, WPI Inc. Sarasota, FL, USA). Controls were injected with dsEGFP. More than 100 injected aphids were placed on wheat seedlings to recover and were then reared under laboratory conditions. A total of twenty injected aphids were randomly collected at 24 h postinjection for the subsequent detection of vg expression using qRT-PCR. The remaining insects were maintained for observation of their phenotypes and growth status. Photos were taken with a Leica M165C microscope (Leica Microsystems, Wetzlar, Germany) at 48 h after injection. All experiments were independently repeated at least three times.

miRNA target studies of vg
To determine whether R. padi miRNA could target vg, two commonly miRNA target prediction programs(miRanda (http://www.microrna.org/microrna/getDownloads.do) and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/welcome.html)) and one miRNA library of R. padi (PRJNA555833) were used. The predicted miRNAs were selected to investigate their expression levels between third instar wingless and winged aphids using RT-qPCR. A total of 20 aphids were used as a biological replicate, and three replicates were performed.

Dual luciferase reporter (DLR) assay
The agomir (mimic) of miR-147b was designed and synthesized by GenePharm Co. Ltd (Shanghai, China). The miRNA agomir is a dsRNA form from the miRNA and its complimentary sequence with a chemical modification. The negative control was designed based on a Caenorhabditis elegans miRNA with no similarity to insect miRNAs. Two 226-bp fragments containing the miR-147b predicted target sites and the mutated miR-147b target DNA sequence were amplified by PCR and inserted downstream of the luciferase gene in the pmirGLO vector (Promega, USA) between the PmeI and XhoI restriction sites to give the pmirGLO-miR-147b and pmirGLO-miR-147b-mut target constructs. The dual luciferase reporter (DLR) assay was performed as previously described [47]. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was compared to that of the control pmirGLO Vector. The mean of the relative luciferase expression ratio (firefly luciferase/Renilla luciferase) of the control was set to 1. For each transfection, the luciferase activity was averaged from five replicates.

Modulation of miRNA and the subsequent impacts on wing development.
Each aphid was injected with 13.8 nL of a 40 μM agomir solution, and the control was Additional file 3: Figure S3. Expression levels of vg and miR-147b in the body walls of third instar wingless nymph (3rdWL) from low-density (LD) and high density [45] conditions . 3rdWL-LD were obtained from a single wingless adult female that was reared on wheat seedlings, and 100% wingless aphids were produced. 3rdWL-HD were produced under conditions of crowding, where >30 adult wingless aphids were reared on wheat seedlings in plastic petri dishes, and the percentage of winged aphids was 43.0±17.4%.
Additional file 4: Table S1. Primers and nucleotides used in experiments.

Figures
26 Figure 1 The     The effect of miR-147b on wing development. The expression levels of miR-147b (A) and vg (B) in third instar nymph winged lines after injection of miR-147b agomir for 24 h, resepectively. Phenotypes of third nymphal winged aphid after injecting with agomir-NC (C) and miR-147b agomir (D) for 48h, D (i) and [45] phenotypes are at rates of 75% and 25%, respectively. *Significant difference according to Student's t-test (P < 0.05).