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 . 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  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 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% CO2 atmosphere in DMEM high-glucose 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.
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 The 5’- and 3’-ends of the cDNA molecules were ampliﬁed using the rapid ampliﬁcation of cDNA ends method with the Gene-RACE Kit (Takara Biotechnology, Dalian, China) following the manufacturer’s instructions. BLAST searches for homologous sequences and the prediction of conserved regions were performed on the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Total proteins were extracted from 300 body walls of third instar nymphs by using 1× SDS-PAGE loading buffer (diluted by 1× PBS buffer, pH 7.5). A total of 30 μg of proteins was loaded onto an SDS-polyacrylamide gel. After elevtrophoresis under 100 V for 2 h, protein was transferred to polyvinylidene difluoride membranes (Millipore, USA) under 100 mA for 20-30 min. Blots was then blocked in TBST (0.1% Tween 20 in TBS, pH 8.0) and 5% nonfat powdered dry milk (w/v) for 2 h. The membranes were incubated with primary antibodies for 2 h. After the membrane was washed with TBST three times for 10 min each time, the membranes were incubated with HRP-conjugated secondary antibodies for 30 min. After three additional washes with TBS, immunolabeled bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma, USA). Protein bands were scanned (Bio-Rad, Hercules, CA, USA). The antibodies used in this study were purchased from Abiotech (Jinan, China). All was performed at room temperature.
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 post-injection 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 . HEK293T cells were cultured in a 24-well plate and transfected with the target plasmids and either the miRNA agomir or NC using the Calcium Phosphate Cell Transfection Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. Each well contained 0.2 μg plasmid DNA with 100 nM final concentration of the miRNA agomir. Luciferase assays were performed using the Dual-Glo® Luciferase Assay System (Promega) 24 h post-transfection. 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 injected with agomir-NC in third instar winged aphids. At 24 h post-injection, the twenty nymphs in each sample were collected for later detection of gene expression. The relative expression levels of vg and miR-147b were determined using qRT-PCR. The remaining insects were maintained for observation of their phenotypes after injection 48 h. All experiments were performed in triplicate.