Insect and culture conditions. Clones of red and green morphs of A. pisum were established from single virginiparous females. Samples were collected in 2017 from same Alfalfa plant Medicago sativa in field, Lanzhou, China, and reared on the fava bean Vicia faba in the laboratory. All plants and A. pisum cultures were reared in an artificial climate incubator at 20 ± 1℃, 70 ± 10% relative humidity, with a photoperiod of 16 h L: 8 h D. Mature A. pisum were put on a fava bean leaf for 12 h and the resulting neonate nymphs, 0-12 h old, were used for experiments throughout this study.
RNA isolation and first-strand cDNA synthesis. Total RNA was isolated using TRizol reagent (BBI Life Sciences, Shanghai, China) following the manufacturer’s instructions. The total quantity of extracted RNA was assessed using a micro-volume UV spectrophotometer (Quawell Q5000, Quawell, USA). The RNA integrity was confirmed further by 1% formaldehyde agarose gel electrophoresis. Total RNA was dissolved in 50 mL DEPC-water and stored at -80℃. The first-strand cDNA was synthesized using a First-Strand cDNA Synthesis kit (BioTeke, Beijing, China) and stored at -20℃ for subsequent experiments.
Cloning of TPS and TRE cDNAs. The primer sets, TPS-F/R of ApTPS and TRE-F/R of ApTRE, were designed using the primer software Primer 5.0 (Premier Biosoft, Palo Alto, CA, USA) based on the TPS gene sequence (GENBANK accession: XM_001943581.5) and the TRE gene sequence (GENBANK accession: XM_003245847.4) of A. pisum. The primers of the green fluorescent protein gene (GFP, pET28a-EGFP, Miaolingbio, Wuhan, China) were referenced from Yang et al.16. These primers are listed in Table S1. The components of the PCR reaction mixture included 1.0 mL of the template (1 ng/mL), 12.5 mL 2× Power Tap PCR MasterMix (BioTeke, Beijing, China), 1.0 mL of each primer (10 mmol/mL), and 9.5 mL Rnase-free H2O concentration for a final volume of 20 mL. The PCR reaction conditions were pre-denatured at 95℃ for 5 min, followed by 35 cycles of 95℃/45 s for denature 55℃/45 s for annealing and 72℃/1 min for extension, and then 10 min at 72℃ for a final extension. PCR products were subjected to 1.0% agarose gel electrophoresis and purified by DNA gel extraction kit (BioTeke, Beijing, China). The purified DNA was ligated into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced by Tsing Ke Biological Technology (Tsing Ke Biological Technology, Beijing, China) using the dideoxynucleotide method. The lengths of the resulting ApTPS, ApTRE, and GFP genes were 421 bp, 416 bp, and 688 bp, respectively.
dsRNA synthesis. Three pairs of primers (dsTPS-F/R, dsTRE-F/R and dsGFP-F/R), with the T7 RNA promoter sequence flanking the 5’-end of each gene, were designed and synthesized (Tab. S1), and used to make the templates for in vitro dsRNA transcription via PCR. The dsRNAs were synthesized using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Wilmington, DE, USA) according to the manufacturer’s protocol40. The size of the dsRNA products was confirmed by electrophoresis on a 1.5% agarose gel and the concentration was assessed using a micro-volume UV spectrophotometer.
dsRNA and high sugars diet treatments. The artificial diet bioassay was performed according to the following procedure41. A liquid artificial diet was prepared as described previously42,43, filtered through a 2 mm membrane, dispensed in 1.0 mL aliquots, and stored at -20℃ before assays. The testing diets were prepared by adding either each of dsRNA (dsTPS, dsTRE and dsGFP) or each of sugar (trehalose and glucose) to the 1.0 mL artificial diet for a final concentration of 400 ng/mL (dsRNA) and 100 mg/mL (sugar). The diet containing nuclease-free water was used as control of the high sugar diet treatments and diet containing dsGFP was used as control of the RNAi treatments. There was a total of 6 treatments including two controls for either red or green A. pisum.
Glass vials (2.5 cm in diameter) were sterilized for the aphid artificial double-membrane feeding assay and one opening was completely sealed with parafilm. Seventy microliters of the testing diet were placed on the parafilm and covered with parafilm. So the testing diet was sandwiched between two layers of the parafilm membrane at one opening of the glass vials44. The control group was fed with only the artificial diet without dsRNA or sugars.
Fifteen 3-day-old A. pisum were introduced into one vial, and the vial was closed with a piece of sterilized gauze as one of bioassays. The artificial diet was replaced every other day to prevent dsRNA degradation. After 4 days, all surviving A. pisum were transferred to fresh bean leaf discs.
Quantification of gene expression levels after RNAi treatments. Seven A. pisum were collected from fresh bean leaf discs at 24 h and 48 h after the 4-day treatment with the testing diet containing each of dsRNAs. A. pisum were immediately frozen in liquid nitrogen and three replicates were carried for each treatment. Total RNA was isolated from the seven pooled whole A. pisum bodies. The first-strand cDNA was synthesized from total RNA using a First-Strand cDNA Synthesis kit (BioTeke, Beijing, China). The RT-qPCR analysis was carried out in 96-well 0.1-mL block plates using a QuantStudioTM 5 system (Thermo Scientific, Wilmington, DE, USA). Each reaction contained 1.0 mL of the cDNA template, 10.0 mL 2×Plus SYBR real-time PCR mixture (BioTeke, Beijing, China), 0.5 mL of each primer (10 mmol/mL), 8 mL EDPC-ddH2O, and 0.5 mL 50 × ROX Reference Dye concentration for a final volume of 20 mL. The RT-qPCR reaction conditions were pre-denatured at 94℃ for 2 min, by 40 cycles of 94℃/15 s, and 55-62℃/30 s for annealing. After each reaction, a melting curve analysis (denatured at 95℃ for 15 s, annealed at 60℃ for 1 min, and denatured at 95℃ for 15 s) was conducted to ensure consistency and specificity of the amplified product. Three biological replicates and three technical replicates were set for each treatment in the RT-qPCR analysis. Quantification of the transcript level was conducted according to the method45, and the ribosomal protein L27 gene (rpL27) was used as a reference gene46.
Trehalose and glucose content assays after high sugar diet treatments. Ten A. pisum were collected from fresh bean leaf discs at 24 h and 48 h after the 4-day treatment with the testing diet containing each of sugars. A. pisum were immediately frozen in liquid nitrogen and three replicates were carried for each treatment. The trehalose content assay was conducted according to the method described by Yang et al.16. Briefly, ten whole A. pisum bodies were ground in phosphate-buffered saline (PBS: 130 mM NaCl; 7 mM Na2HPO4·2H2O; 3 mM NaH2PO4·2H2O; pH 7.0), and then a 25 mL of tissue was taken and uniformly mixed with 25 mL of 1% sulfuric acid. The mixture was incubated at 90℃ for 10 min and placed in ice for 3 min, and then 25 mL of 30% potassium hydroxide solution was added into the sample and mixed uniformly. The resultant mixture was incubated at 90℃ for 10 min and then in ice for 3 min. Finally, 500 mL of 0.2% anthrone reagent was added to the sample and incubated at 90℃ for 10 min and then in ice for 3 min. The trehalose content was assayed by measuring the absorbance of the final reaction mixture at 630 nm. The glucose content was determined using the glucose assay kit (Solarbio Biochemical Assay Division, Beijing, China) according to the manufacturer’s protocols.
Evaluation of A. pisum feeding behavior. The probing behaviour was evaluated with the electrical penetration graph (EPG) using an 8-channel DC-EPG device (Wageningen University, the Netherlands). Eight plants were placed in a faraday cage, and wingless A. pisum were placed on the abaxial side of the second fully expanded leaf from the top. Before exposure A. pisum to the plant, a 6 to 8 cm long gold wire (diameter 18 mm) was conductively glued (water-based silver glue) to A. pisum dorsum as the recording electrode. The other end of the gold wire was attached to a 3 cm long copper wire (diameter 0.2 mm) which was connected to the first head stage on the DC-EPG amplifier with the setting of 1 Giga-Ohm input resistance and 50×gain. The reference electrode was inserted into the soil and connected to the plant voltage output of the DC-EPG device. A. pisum from each treatment were randomly distributed during recording. For each treatment, only the A. pisum that showed activities in an 8 h recording period were considered as valid replicates.
The EPG signal was recorded by the Stylet+d software and the EPG waveforms were recognized and labeled using Stylet+av01.30 software (EPG Systems, Wageningen, Netherlands). The EPG parameters were calculated for each A. pisum treatment using the Excel workbook for automatic parameter calculation of EPG data 4.4.347,48 and then the means and standard errors of the mean (SEM) were calculated for each treatment at 24 h and 48 h on fresh bean leaf discs after 4-day treatments.
Survival and reproduction assays. A. pisum were reared on fresh bean leaf discs after the treatments in an artificial climate incubator at 20 ± 1℃, 70 ± 10% relative humidity, with a photoperiod of 16 h L: 8 h D. Survival and reproduction assays were conducted for the control and treated A. pisum. The daily numbers of adult A. pisum deaths and newborn nymphs per adult A. pisum were recorded until they no longer produced nymphs, once per day starting from the first day after the treatments.
Multiple nonlinear regression. The 48 h trehalose and glucose content data of the A. pisum obtained in 2.7 and the percentage of EPG waveforms data of the A. pisum obtained in 2.8. The relationshops between the percentage of EPG waveforms under each treatment (z) and the corresponding physiological trehalose content (x) and glucose content (y) were then analyzed as by nonlinear curve fitting with the softwave 1stOpt 15.0 (7D-Doft High Technology lnc, China), where a1, a2, a3, a4, and a5: coefficient; b: constant.
Statistical analysis. All statistical analyses were performed using 1stOpt 15.0, SPSS 19.0, and Origin 8.5 were used to construct the histograms. The RT-qPCR and sugar data were analyzed by Student’s t-test. The EPG data (Tab. S2) and the total reproduction data (Fig. S2) were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. The survival data were subjected to a Kaplan-Meier survival log-rank analysis (Fig. S1)49. A p-value <0.05 was considered statistically signiﬁcant.