title: L4
organism: Acyrthosiphon pisum
description: Aphids were reared on Vicia fabae plant inside regulated cabinets. Biological material was prepared at constant temperature of 18*C at 12h of light to induce the production of sexual morphs. To initiate the experiment, two groups of apterous L3 larvae were placed and plants. This corresponds to generation G0. When adults they were left to lay their offspring: one larva of the 1st stage (L1) was kept per adult per plant. This larva was selected among the 20 first born larvae. This is the generation G1. When these females reached the fourth larval stages (L4-G1), 24 h after their moult from the third stage, the five largest embryos were dissected, frozen in liquid nitrogen and kept at -80*C. Another batch of these females were kept till they reached the adult stage (VA-G1), and the five largest embryos from 24h old female were dissected, frozen in liquid nitrogen and kept at -80*C. Total RNA was extracted from these embryos. In each sample (L4-G1 and VA-G1), a total of about 100 embryos was used for RNA extraction. RNAs were extracted using the SV Total RNA Isolation kit (Promega, Madison, WI, USA). The integrity and quantity of RNAs were verified using a Bioanalyser 2100 (Agilent Tech. Inc., Palo Alto, CA, USA).