title: Acyrthosiphon pisum clover race OP1_5_12  parthenogenetic female
organism: Acyrthosiphon pisum
description: The four obligately parthenogenetic (OP) Acyrthosiphon pisum lineages (5_12, Mn_tr_06, T_tr_04 and Ta_tr_36) were collected in clover fields in the west of France and the four cyclically parthenogenetic (CP) A. pisum lineages (Ar_tr_17, Ed_tr_12, Mab_tr_02 and Vm_tr_08) were collected in clover fields the east of France. To produce sexual morphs, the eight lineages were reared under sex-inducing conditions (see Le Trionnaire et al. 2009, 10.1186/1471-2164-10-456). Briefly, one one-day old larva was placed on a Vicia faba plant stored at 18°C with a 16:8 L:D photoperiod regime for one week. Then, the one-week-old individual was moved to short-day conditions (12:12 L:D regime at 18°C). Six days later, four one-day old larvae (i.e. the second generation) were placed on a new plant and maintained under short-day conditions. Once adults, the four individuals produce sexual morphs (males and oviparous sexual females in CP lineages, males only in OP lineages). They were transferred to a new plant every day. We isolated sexual females from males prior to sexual maturation (at fourth larval stage) to ensure that sexual morphs were virgin. We collected adult individuals two to three days after their imaginal molting. Parthenogenetic females were obtained by rearing the eight lineages under a 16:8 L:D photoperiod regime at 18°C. One one-day-old larva of each lineage was deposited on a Vicia faba. Once adult, the female starts to lay offspring. We waited for 6 offspring to be laid, which takes one day, and the female was then removed from the plant. These individuals were collected two to three days after their imaginal molt. All individuals were flash-frozen in liquid nitrogen and stored at -80°C. We pooled six to ten males per replicate, three sexual females per replicate and two parthenogenetic females per replicate. Two biological replicates per morph and lineage were generated. We performed a total of RNA extractions using the SV Total RNA Isolation System (Promega) following manufacturer's instructions. The quality of the RNA samples was checked on Bioanalyzer (Agilent) and quantified on Nanodrop (Thermo Scientific). The RNA samples were subsequently sent to the NGS platform Institut Curie (Paris, France) for library preparation (Illumina stranded mRNA Ligation) and for 100 bp RNA paired-end sequencing (Illumina NovaSeq6000).