Iq5 optical system software v2

Total RNA was isolated from cells with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was reverse-transcribed using the iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and realtime quantitative PCR were run in triplicate for each sample on a Bio-Rad My Cycler iQ5 (Bio-Rad). Primer sequences were obtained from Stabvida (Costa da Caparica, Portugal) and thoroughly tested. The resulting RT product was expanded using the Syber Green Supermix (Bio-Rad). The results were then normalized to the expression of a housekeeping gene Srp72. After amplification, a threshold was set for each gene and cycle threshold-values (Ct-values) were calculated for all samples. Gene expression changes were analysed using the built-in iQ5 Optical system software v2.1 (Bio-Rad). The complete list of primers used is given in Table 1.

We used the JHAMT protein sequences from the following species: A. aegypti melanogaster (AAF46666) and S. gregaria (HQ634704). Sequences were aligned using the iterative refinement method G-INS-i from the program MAFFT v. 7 (Katoh & Standley, 2016) . The alignment was analyzed using the RAxML-HPC BlackBox tool of the CIPRES Science Gateway v. 3.1 (Miller et al., 2010) . The data sets were bootstrapped for 100 replicates.
RNA extraction, cDNA synthesis and real-time PCR analyses cDNAs were synthetized using as previously described (Abrisqueta et al., 2017) . The absence of genomic contamination was confirmed using a control without reverse transcription. Gene expression was analyzed by quantitative real-time PCR using iQ SYBR Green supermix in an iQ cycler and iQ single color detection system and the iQ5 optical system software v. 2.0 (Bio-Rad Laboratories, Hercules, CA, USA), as previously described (Maestro et al., 2010) . Primers used to amplify total BgJHAMT as well as the different 3'-UTR isoforms can be found in Table S1. Primers used to amplify vitellogenin (BgVg) and BgActin 5C (used as a reference) have been already reported (Süren-Castillo et al., 2012) . The primers efficiencies were validated by constructing serial cDNA dilutions standard curves. Results were expressed as copies of a specific mRNA per copies of BgActin 5C.

The corpora allata and fat body mRNA levels of the different studied genes were analysed using quantitative real-time PCR. cDNAs were synthesized from total RNA as previously described (Abrisqueta et al., 2014; Maestro et al., 2009) . In the case of fat bodies, 500 ng of total RNA was used, whereas for corpora allata, the whole RNA from one pair of glands was used. The absence of genomic contamination was confirmed using a control without reverse transcription. The cDNA levels were quantified using iQ SYBR Green supermix in an iQ cycler and iQ single colour detection system and the iQ5 optical system software v. 2.0 (Bio-Rad Laboratories, Hercules, California), as previously described (Maestro et al., 2010) . Primer sequences to amplify BgS6K can be found in Table S1. Primers used to amplify HMG-CoA synthase-1 and -2, HMG-CoA reductase, vitellogenin (BgVg) and BgActin 5C (used as a reference) have been already reported (Maestro et al., 2010; Suren-Castillo et al., 2012) . The total reaction volume was 20 µL. All reactions were run in duplicate or triplicate. Results were expressed as copies of a specific mRNA per copies of BgActin 5C.

The expression levels of the different genes studied were analyzed using quantitative real-time PCR (qRT-PCR) in cDNA prepared from different tissues. cDNA was synthesized from total RNA as described above. The absence of genomic contamination was confirmed using a control without reverse transcription. cDNA amplifications of OKA, OKB, Vg, 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMG-CoA synthase-1), juvenile hormone acid methyltransferase (JHAMT) and actin 5C were performed in duplicate or triplicate, in a 20 ll final volume (primers detailed in Supplementary Table 1). cDNA levels were quantified using iQ SYBR Green supermix (Bio-Rad) in an iQ cycler and iQ single colour detection system (Bio-Rad). The schedule used for the amplifying reaction was as follows: (i) 95 °C for 3 min, (ii) 95 °C for 10 s; (iii) 57 °C for 1 min; (iv) steps (i) and (ii) were repeated for 50 cycles. Real-time data was collected through the iQ5 optical system software v.2.0 (BioRad). For quantification of soluble proteins, ovaries were dissected and placed at À80 °C until their use. Total soluble proteins were extracted by ultrasonication and centrifugation in NaCl 0.4 M solution and quantified according to Bradford (Bradford, 1976) .