Review2 exposure temperatures were set as 10? and

Review2 Materials 117 and Methods118 2.1 Rearing and collection of T. chilonis119 Eggs of T. chilonis and the factitious host Corcyra cephalonica (Stainton) were120 originally obtained from Plant Protection Research Institute, Guangdong Academy of121 Agricultural Sciences, People’s Republic of China. The rearing condition was set as122 25±1?, 75±5% relative humidity and a photoperiod of 14:10 (light: dark). Before123 formal trial, a colony of T. chilonis was reared on irradiated C. cephalonica eggs for124 ten generations. In addition, the developmental stage was confirmed by external125 characteristics, and the development time for each stage was same as that in the126 description from Volkoff (Volkoff et al., 1995). Finally, T. chilonis reared on eggs for127 4 days (prepupae) and 9 days (pupae) were chosen as experimental subjects that had128 the strongest hardiness to thermal stress (Yuan, Feng, Li, Zhang, & Wang, 2013). In129 addition, exposure temperatures were set as 10? and 40?, which are close to the130 temperature thresholds of T. chilons (Haile et al., 2002; Nadeem, Muhammad,131 Muhammad, Sohail, & Muhammad, 2009). In the formal experimental stage, newly132 emerged T. chilons were exposed to a 50 mm × 20 mm cardboard container of133 approximately 200 irradiated C. cephalonica eggs for 12 h. Parasitized eggs were134 separately reared for 4 d (host eggs, gray) and 9 d (host eggs, black) (Figure 1),135 followed by respective exposures to 10? and 40? for 2 h. In addition, parasitized136 eggs reared at 25? were used as controls. After identification, newly dissected insects137 were quickly reserved in Trizol (Invitrogen, USA). For each treatment, three138 biological replications were performed with 50 heads of T. chilons. The specimens139 were immediately frozen in liquid nitrogen and then stored in a -80°C refrigerator for140 RNA extraction.141 2.2 cDNA library constructi