2.7 ∣ Phylogenetic reconstruction
The phylogenetic tree was reconstructed by using shared single-copy genes. Protein sequences for these single-copy genes were aligned using MUSCLE, then protein sequence alignments were transformed back to CDS alignments. CDS alignments of single-copy genes were then concatenated into a super matrix. Based on this super matrix, A phylogenetic tree was constructed using the ML (maximum likelihood) algorithm as implemented in RAxML software (version 7.2.3) and in MEGA Version 5 software (Alexandros & Stamatakis, 2006; Tamura et al., 2011).
2.8 ∣ RNA sequencing and transcriptome assembly
Total RNA was extracted, separately, from female nymphs of first instar (FF), female nymphs of second instar (SF), , male first instar larvae (FM), and male second instar larvae (SM) using the TRIzol kit (Life Technologies, Camarillo, USA) following the procedure provided. A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Libraries were constructed and sequenced according to the Illumina protocol. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structures were ligated to prepare for hybridization. To select cDNA fragments of preferentially, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μlUSER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated.