Aphid rearing, dissection and extraction
Clonal descendants from the sequenced Acyrthosiphon pisum LSR1 clone were reared on Vicia fabae at 18°C and at low density (5 individuals per plant) to prevent the production of winged morphs. Parthenogenesis was maintained under long photoperiod (16 h). In our conditions, the shortening of the photoperiod (12 h) requires three generations to induce the production of sexual morphs. The first asexual generation was reared under long photoperiod and transferred to short photoperiod (12 h) after the third instar moult. Adult asexual virginoparae females produced the second asexual generation, named sexuparae. Adult sexuparae, reared in short photoperiods, produced the third generation of sexual females oviparous, males, and asexual viviparous females.
Aphid sexuparae were synchronised at the fourth instar moult, during a 6 hour window. 24 h after, 400 ng of kinoprene (Sigma Aldrich) diluted in 50 μL of acetone were ectopically applied on sexuparae abdomens of 100 females. The progeny of treated aphids was collected daily for 5 days. When they reached adulthood, collected aphids were dissected and their reproductive type was determined according to the type of ovaries they contained within their abdomen: sexual ovaries that are only made of haploid eggs, ambiphasic ovaries that contain a mixture of eggs and embryos, and asexual ovaries that are only constituted of embryos at different stages of development. Ectopic applications of 50 μL of acetone were performed as a negative control.
Sampling, RNA isolation and microarray hybridization
Synchronized sexuparae were randomly divided into 2 batches and treated with kinoprene or acetone (as a control) 24 h after fourth instar moult (Additional file 4). 25 treated sexuparae were collected 24, 48, and 72 hours after kinoprene or acetone application. For each condition, the 5 most developed embryos were isolated from each of the 25 treated sexuparae by dissection, pooled together, frozen into liquid nitrogen and stored at -80°C. These most developed embryos within treated sexuparae collected 24, 48, and 72 hours after treatment correspond to the developemental stage 18, 19 or 20 respectively. This procedure was repeated 5 times to generate as much independent biological replicates. Total RNAs were isolated from each sample by using the RNeasy Mini kit (Qiagen) according to manufacturer's instructions. RNA quality was checked on Bioanalyser (Agilent) and quantified on Nanodrop (Thermo scientific). For each sample, 20 μg of total RNAs were sent to the NimbleGen expression array platform (Roche). Double stranded cDNA synthesis and Cy3 end-labelling were performed by NimbleGen.
Microarrays design and analysis
Custom microarrays were constructed on NimbleGen (Roche) 385K 4-plex (4 × 72,000 probes). 24,011 transcripts were represented by 3 60-mers oligonucleotides probes (MIAMExpress, http://www.ebi.ac.uk/miamexpress/, microarray: INRA-BF2I_A.pisum_Nimblegen-ACYPI_4x72k_v1; Array express accession: A-MEXP-1999). Hybridization and scanning were performed by NimbleGen, providing the final raw data file (experiment: Aphid_embryo, ArrayExpress accession: E-MEXP-3481). The limma 2.16 package  in R 2.9.2  was used for statistical analyses. For each transcript the average of the 3 probe signals was considered. Median normalization between microarrays was performed. A design matrix incorporating effects of treatment K (kinoprene) or A (acetone) and developing stage (18, 19, 20) was constructed. Variance was adjusted for Bayesian fitting of the model. Differential expression was determined with a two-way ANOVA considering the effect of time and treatment between 3 contrasts: 'A18 versus K18', 'A19 versus K19' and 'A20 versus K20'. Significance of differential expression was assigned with a 10% false discovery rate (FDR) . Annotation of differentially expressed transcripts was curated using pea aphid RNA-seq data (published in SRA of NCBI), the software CAP3  and Blastx on NCBI non-redundant protein sequence data bank. Functional annotations were performed using the Gene Ontology (http://www.geneontology.org/).
Riboprobe synthesis for in situ hybridization
Templates for synthesis of riboprobes were obtained from full-length cDNAs collection (ACYPI003103, ACYPI010052) or amplified by RT-PCR and cloned (Additional file 5). Total RNAs were extracted from parthenogenetic virginoparae females with RNeasy plant kit (Qiagen). DNA contaminations were removed by a treatment with RQ1 RNase-free DNAse (Promega). First strand cDNAs were produced from 1 μg of total RNAs by using random primer 9 (New England BioLabs) and SuperScript® III Reverse Transcriptase (Invitrogen) following the supplier's instructions. cDNAs were used as a matrix for PCR amplification with specific primers (Additional file 5). Amplified fragments were cloned into the StrataClone PCR Cloning Vector pSC-A-amp/kan (StrataClone) or pENTR Directional TOPO (Invitrogen) and sequenced (Genoscreen). Linear PCR products were amplified from cloned sequence with universal primers M13 and used as a matrix for synthesis of sense and antisense riboprobes by using digoxigenin-labelled dNTPs and the appropriate RNA polymerase (T3/T7/SP6) supplied in the DIG RNA labelling kit (Roche). Remaining DNA was removed with RQ1 RNase-free DNAse treatment (Promega) and riboprobes were purified with the RNeasy mini kit (Qiagen). Riboprobe quality and quantity were checked on Nanodrop (Thermo scientific).
Whole mount in situ hybridization and microscopy
Whole mount in situ hybridization was performed on ovaries of sexual or asexual nymphs. Ovaries were dissected and fixed in 4% paraformaldehyde in 1× PBS at room temperature (RT) for 30 min. Ovaries were washed in 50% methanol for 30 min, then dehydrated and stored in methanol at -20°C. They were rehydrated in graded methanol/PTw (1× PBS, 0.2% Tween-20) solutions (70%; 50%; 30%, 10 min each), post fixated in 4% paraformaldehyde in 1× PBS for 20 min and washed 3 times for 5 min in PTw. Ovaries were washed for 45 min in 1% SDS, 0.5% Tween-20, 50 mM Tris-HCl (pH 7,5), 1 mM EDTA (pH 8), 150 mM NaCl and 5 times for 5 min with PTw. Pre-hybridization was performed 10 min in hyb-wash solution (5× SSC, 50% formamide, 0.1% Tween-20) and 1 hour at 65°C in hyb solution (0.3% SDS, 5× SSC, 50% formamide, 100 μg/mL heparin, 0.1% Tween-20, 100 μg/mL yeast RNA, 10 mM DTT). Hybridization was performed overnight at 65°C with 500 ng/mL of denatured sense or antisense RNA probe diluted in hyb solution. Nonspecific hybridizations were washed off twice at 65°C in hyb-wash solution for 30 min, 3 times in 50% hyb-wash solution/PTw 30 min and 3 times in PTw for 10 min at RT. Ovaries were incubated in blocking solution (0.2% BSA in PTw) for 5 min and 1 h at RT, and bound digoxigenin labelled probes were detected overnight at 4°C with anti-DIG-alkaline phosphatase (AP) Fab fragments (Roche) diluted 1:2000 in blocking solution. Ovaries were washed 4 times 20 min and twice for 1 h in blocking solution at RT. After 3 washes in AP reaction buffer (100 mM Tris (pH 9,5), 100 mM NaCl, 5 mM MgCl2, 0.2% Tween-20), signal was revealed with 4 μl NitroBlue Tetrazolium/5-Bromo-4-Chloro-3-Indolyl Phosphate (NBT/BCIP) Stock Solution (Roche)/ml AP reaction buffer. Finally, ovaries were rinsed at least 3 times for 5 min in PTw, dehydrated 5 min in methanol, and rinsed twice again 5 min in PTw, before mounting in 70% glycerol in PBS. Samples were photographed with a microscope Nikon 90i connected to a Nikon type DS-Ri1 camera or with Olympus BX61 connected to Nikon DS-Fi1 camera.
For the fluorescence protocol, the same procedure was followed until hybridization step. Then TSA™ Biotin System (Perkin Elmer, Waltham, USA) was used for detection as follows. After overnight hybridization of the probes, nonspecific probes were washed off twice at 65°C in hyb-wash solution for 30 min, once in 50% hyb-wash solution/TNT buffer (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.05% Tween-20) for 30 min at RT, and 3 times in TNT buffer for 10 min at RT. Ovaries were blocked with the TSA™ blocking reagent, for 30 min at RT. Bound digoxigenin labelled probes were detected with HRP conjugated anti-DIG (Boehringer-Mannheim) (1:250) diluted in TNT buffer at 4°C for overnight incubation. Ovaries were washed four times in TNT buffer for 15 min. The TSA™ Biotin System Amplification was used by incubation 20 min into a diluted solution of the Biotinyl Tyramide (Amplification Reagent) in 1× Amplification Diluent (1:50). HRP catalyzes the formation of TSA free radicals, which form covalent bounds to tyrosine residues proximal to HRP. They were then washed 3 times 15 min in TNT buffer, and 4 times 20 min in PTw. Ovaries were incubated 2 h into Alexa 594-conjugated Streptavidin (Invitrogen) diluted in TNT buffer (1:500) and rinsed into PTw before performing a nuclear stain with TO-PRO3® (1:1000 in PTw) for 30 min. Finally, ovaries were washed 3 times in PTw before mounting. Images were acquired and processed using an Olympus FV1000 confocal microscope.
Phalloidin and propidium iodide staining
Oviparous ovaries were dissected and fixed as previously described. Samples were washed 3 times in PTw, followed by PTw for 30 min and 2% Normal Goat Serum (NGS)/BPTw for 60 min. Samples were then incubated overnight at 4°C with Alexa Fluor 488 phalloidin (Invitrogen) at a concentration of 1:200 to visualize F-actin. The next day samples were washed 4 times in PTw, and placed in 20 μg/mL RNaseA (QIAGEN)/PTw for 3 h at RT. Samples were washed twice in PTw, and incubated with 10 μg/mL propidium iodide (Invitrogen) in 20 μg/mL RNaseA/PTw for 60 min at RT to visualize DNA. Samples were washed 4 times in PTw and mounted in Vectashield (Vector Laboratories). Images were acquired and processed using an Olympus FV1000 confocal microscope.