We generated an isogenic S. avenae population using a single wingless aphid. Aphids were raised on wheat seedlings at 22°C with a 16h/8h light/dark photoperiod. Winged aphids were obtained by manipulating aphid densities. Under low-density, one wingless aphid was reared on a joint-stage wheat to maintain the wingless morph. Under high-density, 80 wingless adult aphids were reared on one ripe wheat to induce the winged morph (6).
To investigate IFM degeneration, we collected the winged aphids every 24h from eclosion (0 day), to migration (5th day), to reproduction (8th day), until death was observed. For each timepoint, half of the aphids were collected for morphological, histological, and apoptosis examinations. The other half were dissected for qPCR following a freeze-drying procedure (13).
We examined the external morphology of aphid thorax using scanning electron microscope (SEM). Aphids were fixed in 3% glutaraldehyde for 24hrs and transferred to 1% osmic acid. Then aphids were saturated with ethanol, exchanged using isopentyl acetate, and dried in a Hitachi CO2 Critical Point Dryer. Aphids were then coated with gold in a sputter coater (Hitachi, IB-5) and imaged under a Hitachi S-570 SEM.
We examined the internal morphology of aphid thorax using histological staining. Aphids were fixed in 4% paraformaldehyde for 4hrs. The specimens were dehydrated in a serial of ethanol solutions (70%, 80%, 90%, 100%, 10 mins/each), cleared in xylene, and embedded in paraffin. Serial sections were cut and stained with hematoxylin and eosin for imaging using a Carl Zeiss Primo Star Microscope.
Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling (TUNEL) assay
To examine apoptosis, we performed a TUNEL assay using an in-situ apoptosis detection kit (Boster, China). Briefly, paraffin-embedded aphids were sliced into 5μm sections, which were rehydrated in xylene for 20 mins and a serial of ethanol solutions (100%, 90%, 80%, 70%, 10 mins/each). Then all specimens were permeabilized using Proteinase K (1:200) for 10 mins and quenched using 3% H2O2 for 10 mins. Quenched specimen were labeled with TdT Labeling Reaction Mix (TdT:DIG-dUTP:Buffer=1:1:18) for 2hrs at 25°C. After wash with 0.01M TBS buffer, specimens were incubated with anti-DIG antibody (1:100) for 30 mins. Specimens were then incubated with SABC (1:100) for 30 mins and with Diaminobenzidine for 15 mins, then counterstained with Hematoxylin for 3 mins. Finally, specimens were washed with 100% ethanol, 100% xylene, and mounted for imaging.
Previous study suggested that feeding induces JH secretion, which triggers IFM degeneration (5). Therefore, we examined the IFM degeneration on fasted aphid as a control. For fasting, winged aphids were transferred to water-soaked sponges. Every 6hrs, aphids were transferred to rearing plants for 2hrs to avoid death.
Differential-display reverse transcription-PCR (DDRT-PCR)
To identify differentially expressed genes pre-/post-migration, we performed tethered flight using 3-4 days post-eclosion aphids. For pre-migration, aphids were tethered (not flighted) and flash-frozen. For post-migration, aphids were tethered and flighted for 24hrs using a flight-mill program (14); flighted aphids were flash-frozen.
Total RNA was extracted from single aphid using Easy-Spin Total RNA Rapid Extraction Kit (Biomad) and reverse-transcribed by PrimeScriptTM 1st-Strand cDNA Synthesis Kit (Takara). For DDRT-PCR, we designed three one-base anchored oligo-dT 3’ primers and eight arbitrary 5’ primers (13-mers) according to the GenHunter RNAimage DD Kit (3x8 pairs) (Table S1). Each PCR reaction was composed of 10μl 2X PCR mix, 1μl 5’ primer, 1μl 3’ primer, 2μl cDNA, and 6μl ddH2O. The PCR program was 94°C for 1 min, followed by 30 cycles of 94°C for 30s, 48°C for 1 min and 72°C for 1 min, and a final extension step at 72°C for 5 min. Then 2μl PCR products of each reaction were used as template for a second round of PCR. The final PCR products were visualized using 6% SDS polyacrylamide gel electrophoresis (Fig S2). Differentially expressed genes were excised for sequencing, obtained sequences were annotated on NCBI.
RPS27a dynamic expression
The sequence of S. avenae RPS27a was obtained using a rapid amplification of cDNA ends method (3’-RACE). Partial S. avenae RPS27a sequence containing the start codon was amplified using Ub-F/Ub-R primers designed based on A. pisum RPS27a. The complete C-terminal of RPS27a was obtained using a RACE kit (Takara) with gene specific primers and the kit-provided outer/inner primers (Table S2). To check the conservation of RPS27a, the nucleotide and deduced amino acid sequences were aligned with homologous from other insects using DNAMAN (Fig S3).
We quantified the RPS27a expression during IFM degeneration using qPCR in tissues including head, thorax, and abdomen. Briefly total RNA was extracted from tissues pooled from ~10 aphids, then same amount of RNA (0.1 μg) for each sample was used for cDNA Synthesis. The qPCR reaction was 25μl containing 12.5μl 2*PCR SuperMix, 0.5μl Passive Reference DYE, 0.5μl of each primer, 1μl cDNA and ddH2O. The qPCR program was 2 min at 95°C followed by 45 cycles of 15s at 95°C, 15s at 55.5°C and 30s at 72°C, and then melt curve under 55-95°C 0.5°C + (80 cycles). To calculate gene expression, a credible standard curve was constructed using a series of 10x dilutions of a standard sample. The experiment was repeated three times and each experiment included 3-4 technical replicates for each sample.