2.5 RNA-seq and transcriptome data processing
A. flavipes were obtained by overnight live-trapping in Samford Valley, QLD, Australia (27° 22′ S, 152° 52′ E). Animals were euthanised by CO2 and tissues frozen at -80 °C. Eight specimens collected in 2017 were sequenced by BGI Hong Kong. The RNA of the remainder [seven males and six females collected in 2018] was extracted by BGI Hong Kong and sequenced by BGI Qingdao. Only the samples collected during the 2018 breeding season in Samford Valley were used for differential gene expression analysis (the samples collected in 2017 were used for gene annotation only). Raw data was filtered using Flexbar v3.4.0 (Dodt, Roehr, Ahmed, & Dieterich, 2012; Roehr, Dieterich, & Reinert, 2017) with default settings (removes reads with any uncalled bases). BGISEQ-500 adapters were obtained from a comparative study of Illumina and BGI sequencing platforms (Mak et al., 2017). Any residual ribosomal RNA reads (the majority removed by poly(A) selection prior to sequencing library generation) were removed from the reads using SortMeRNA v2.1b (Kopylova, Noe, & Touzet, 2012) against the SILVA v119 ribosomal database (Quast et al., 2013). Tissue transcriptomes were de novo assembled using Trinity v2.8.4 (Grabherr et al., 2011; Haas et al., 2013; Henschel et al., 2012) with default parameters. The A. flavipes transcriptome assemblies were assessed using BUSCO (Seppey et al., 2019). Details of transcriptome sequencing data used in study are listed in Tables S2-S4 (also see Supplemental Methods).