dsRNA Injection and Gene Expression Analysis

We injected dsRNA (1 µl of 2.5 µg µL^-1) into 3^rd and 4^th abdominal segment of M. separata pupa (7 days old) using Eppendorf microinjection system TransferMan NK2. The injected pupae were kept under control conditions of 70% ± 5% relative humidity and 25 ± 1°C temperature until eclosion. RNAi had three treatments as follows: (i) non-injected (control), (ii) dsGFP-treated and (iii) dsMsepCSP5 treated. After eclosion, moths were kept into individual cages for each treatment. For RNAi analysis, three adults of M. separata from each sex were taken from each treatment at 24, 48, 72, 96, and 120 h of eclosion. The RT-qPCR for expression profiling was conducted under the concurrent conditions as described above.

The Y-tube bioassay (olfactometer) was also performed to examine gene knockdown effects after RNAi, and the procedure was same as described above. Three treatments (control, dsGFP and dsMsepCSP5) were tested in the bioassay for 72 h old moth of both sexes. For post RNAi bioassay, M. separata (30 female and 30 male) were used for each tested volatiles. 1-octen-3-ol and 3-pentanol were used for post RNAi behavioral bioassay and selection was based on the significant attraction of moths to the tested volatiles before RNAi.